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A Nonconventional Archaeal Fluorinase Identified by In Silico Mining for Enhanced Fluorine Biocatalysis

  • Isabel Pardo
    Isabel Pardo
    The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
    More by Isabel Pardo
  • , 
  • David Bednar
    David Bednar
    Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, 601 77 Brno, Czech Republic
    International Clinical Research Centre, St. Anne’s University Hospital, 656 91 Brno, Czech Republic
    More by David Bednar
  • , 
  • Patricia Calero
    Patricia Calero
    The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
    More by Patricia Calero
  • , 
  • Daniel C. Volke
    Daniel C. Volke
    The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
    More by Daniel C. Volke
  • , 
  • Jiří Damborský
    Jiří Damborský
    Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, 601 77 Brno, Czech Republic
    International Clinical Research Centre, St. Anne’s University Hospital, 656 91 Brno, Czech Republic
    More by Jiří Damborský
  • , and 
  • Pablo I. Nikel*
    Pablo I. Nikel
    The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
    *Email: [email protected]
    More by Pablo I. Nikel
    Orcidhttps://orcid.org/0000-0002-9313-7481
Cite this: ACS Catal. 2022, 12, 11, 6570–6577
Publication Date (Web):May 19, 2022
https://doi.org/10.1021/acscatal.2c01184

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Abstract

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Fluorinases, the only enzymes known to catalyze the transfer of fluorine to an organic molecule, are essential catalysts for the biological synthesis of valuable organofluorines. However, the few fluorinases identified so far have low turnover rates that hamper biotechnological applications. Here, we isolated and characterized putative fluorinases retrieved from systematic in silico mining and identified a nonconventional archaeal enzyme from Methanosaeta sp. that mediates the fastest SN2 fluorination rate reported to date. Furthermore, we demonstrate enhanced production of fluoronucleotides in vivo in a bacterial host engineered with this archaeal fluorinase, paving the way toward synthetic metabolism for efficient biohalogenation.

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Fluorinated organic compounds (organofluorines), containing at least one fluorine (F) atom, are chemicals of enormous industrial interest (1,2)─as evidenced by their increasing prevalence in pharmaceuticals (almost one-third of the pharma molecules in the market contain F) and agrochemicals. (3−5) The unique physicochemical properties of F endow organofluorines with advantageous properties with respect to their nonfluorinated counterparts, e.g. increased chemical stability or improved bioavailability. (6) However, the abundance of human-made organofluorines contrasts with their relative scarcity in Nature. (7,8) 5′-Fluoro-5′-deoxyadenosine (5′-FDA) synthase, or fluorinase (FlA), is the only one enzyme known to naturally catalyze the formation of the C–F bond, which requires a high activation energy for desolvation of the fluoride ion (F–). This enzyme, originally identified in Streptomyces cattleya, (9,10) catalyzes the SN2 transfer of F– to the C5′ of the essential methyl donor S-adenosyl-l-methionine (SAM), thereby generating 5′-FDA and l-methionine (l-Met) as products (11) (step I in Scheme 1). Since the discovery of FlA in 2003, only six other fluorinases have been reported in the literature, all of them sourced from actinomycetes. (12−14) A chlorinase, catalyzing 5′-chloro-5′-deoxyadenosine (5′-ClDA) synthesis and closely related to FlAs, has also been identified in the marine actinomycete Salinispora tropica (15) (step II in Scheme 1). FlA from S. cattleya is capable of catalyzing the chlorination reaction as well, albeit much less efficiently than fluorination. (16) Conversely, SalL, the chlorinase of S. tropica, cannot catalyze the formation of C–F bonds. This activity difference has been attributed to the presence of a 23-residue loop, present in all known FlAs but absent in SalL. (17) It was hypothesized that this loop, located near the catalytic site, could influence halide specificity by modifying the architecture of the binding pocket. (18)

Scheme 1

Scheme 1. Fluorometabolite Biosynthesis Pathways and Reactions Catalyzed by Fluorinase/Chlorinasea
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aReactions catalyzed by fluorinase/chlorinase are indicated in gray: (I) forward fluorination reaction, (II) forward chlorination reaction, and (III) reverse chlorination reaction. The common step in fluorometabolite biosynthetic pathways is shaded in orange. The canonical fluoroacetate and 4-fluoro-l-threonine biosynthetic pathway are show in purple. The 5′-fluoro-5′-deoxy-d-ribose biosynthetic route is indicated in light blue. Compound abbreviations (blue): 5′-ClDA, 5′-chloro-5′-deoxyadenosine; SAM, S-adenosyl-l-methionine; 5′-FDA, 5′-fluoro-5′-deoxyadenosine; 5-FDRP, 5′-fluoro-5′-deoxy-d-ribose 1-phosphate; 5-FDRulP, 5-fluoro-5-deoxy-d-ribulose 1-phosphate; FAld, fluoroacetaldehyde; FAc, fluoroacetate; 4-FT, 4-fluoro-l-threonine; 5-FDR, 5′-fluoro-5′-deoxy-d-ribose; 5-FHPA, 5-fluoro-2,3,4-trihydroxypentanoic acid. Enzyme abbreviations (black bold): FlA, fluorinase; FlB, 5′-fluoro-5′-deoxyadenosine phosphorylase; FlIso, 5-fluoro-5-deoxy-d-ribose 1-phosphate isomerase; FlFT, 4-fluoro-l-threonine transaldolase; FdrA, 5-fluoro-5-deoxy-d-ribose 1-phosphate phosphoesterase; and FdrC, 5-fluoro-5-deoxy-d-ribose dehydrogenase.

Considering the environmentally harsh conditions currently required for the chemical synthesis of organofluorines, FlAs are promising biocatalysts for “green” production (19) of new-to-Nature, bioderived organofluorines and for the implementation of synthetic metabolism with fluorinated intermediates in living cells. (20−22) However, all known FlAs are poor biocatalysts, (23) with turnover rates <1 min–1. So far, the handful of protein engineering efforts aimed at the improvement of FlA activity have had limited success. (24−26) Furthermore, these studies mostly relied on employing surrogate substrates, for example, 5′-ClDA, to select for enzyme variants with improved transhalogenation activity (25) (see steps III and I in Scheme 1). This strategy hampers the applicability of FlAs in a consolidated, whole-cell bioprocess where only F– and an appropriate carbon substrate would be supplied as feedstock to support de novo biofluorination. (27)

Genome-wide databases are a rich source of potentially valuable enzymes, (28) yet their continuous, exponential expansion makes the selection of catalytically attractive candidates challenging. The EnzymeMiner platform (29) has been recently developed to address this issue as an interactive Web site (https://loschmidt.chemi.muni.cz/enzymeminer). This user-friendly bioinformatic tool searches through databases upon submitting a sequence of at least one representative member of the target enzyme family, together with the identification of essential (i.e., catalytic) residues. EnzymeMiner conducts multiple database searches and accompanying calculations, which provide a set of hits and their systematic annotation based on protein solubility, possible extremophilicity, domain structures, and other structural information. These collected and calculated annotations provide users with key information needed for the selection of the most promising sequences for gene synthesis, small-scale protein expression, purification, and functional characterization. (30)

With the goal of expanding the FlA toolset for the biological production of organofluorines in engineered bacterial cell factories, here we describe the systematic screening, in vitro characterization, and in vivo implementation of hitherto unknown FlAs retrieved from genome databases. First, in an effort to identify “Nature’s best” biocatalyst, the fluorinase from Streptomyces sp. MA37 (FlAMA37) was used as the query sequence (UniProt W0W999), and the amino acid residues D16, Y77, S158, D210 and N215 were specified as essential based on their implication in catalysis and substrate binding in EnzymeMiner (Figure 1a). We selected this enzyme since it is one of the most efficient fluorinases reported in the literature thus far, and it has been used as template for directed evolution experiments. (12,24)

Figure 1

Figure 1. Putative fluorinases identified by genome mining. (a) Residues specified as essential for the EnzymeMiner search, based on the crystal structure of FlAMA37 (PDB ID 5B6I). The SAM substrate is shown as a ball-and-stick representation. (b) Phylogenetic tree of retrieved fluorinase sequences obtained using the MEGAX software, (31) inferred using the Neighbor-Joining method with a bootstrap of 10 000 iterations. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Sequences sourced from Actinomycetes are highlighted as blue squares. Enzymes previously characterized in the literature are indicated in blue bold font. (c-e) 3D structures for FlAMA37 (c), wild-type SalLStro (d, PDB ID 6RYZ) and FlAPtaU1 (e, modeled with the SWISS-MODEL Alignment Mode tool using the FlAScat crystal structure PDB ID 2 V7 V as template). The loop hypothesized to differentiate fluorinases from chlorinases is circled in a dashed gray line. Two chains from the homotrimer for each structure are shown as cartoon and surface representations, respectively.

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After curing out redundant sequences, 16 unique candidates were obtained (Table 1 and Figure 1b). Some of the retrieved amino acid sequences were found to be missing several N-terminal residues, which were added after manually curating the deposited genome sequences where the fluorinase genes had been predicted (Table S1). Out of the 16 sequences retrieved, five corresponded to fluorinases reported in the literature (thus serving as an internal quality control of the prediction routine), while nine corresponded to new putative fluorinases. Another two sequences corresponded to a site-directed mutagenesis variant of the chlorinase from Salinispora tropica CNB-440 (SalL; carrying point substitutions Y70T and G131S) (15) and a putative chlorinase from the archaea Methanosaeta sp. PtaU1.Bin055 (FlAPtaU1). Both of these sequences lack the 23-residue loop previously hypothesized to differentiate fluorinases from chlorinases (Figure 1c–e). Notably, only four of all the retrieved sequences were not sourced from Actinobacteria. These include the putative enzymes from a Chloroflexi bacterium (Chloroflexi), Peptococcaceae bacterium CEB3 (Clostridia), Thermosulforhabdus norvegica (Deltaproteobacteria), and Methanosaeta sp. PtaU1.Bin055 (Methanomicrobia). Phylogenetic analysis of the 16S rRNA sequences of the fluorinase-encoding organisms gave a similar result to that obtained when using the fluorinase amino acid sequences, except that, expectedly, S. tropica groups together with the other Actinomycetes, in a clade separate from the one formed by Streptomyces sp. (Figure S1 and Table S2).

Table 1. Putative Fluorinases Retrieved from EnzymeMiner Analysis Using FlAMA37 as the Query
nameorganism and referenceID (%)a
FlAMA37Streptomyces sp. MA37 (12)query
FlAScatStreptomyces cattleya1087.6%
FlASxinStreptomyces xinghaiensis1386.0%
FlASAJ15Streptomyces sp. SAJ1585.0%
FlAN902Actinoplanes sp. N902–1091280.7%
FlAAmzaActinopolyspora mzabensis1478.9%
FlAAbarAmycolatopsis bartoniae79.1%
FlACA12Amycolatopsis sp. CA-12877278.6%
FlAAN11Goodfellowiella sp. AN11030577.7%
FlANbra2Nocardia brasiliensis IFM 1084775.7%
FlANbra3Nocardia brasiliensis NCTC 1129475.3%
FlANbra1Nocardia brasiliensis ATCC 7003581275.3%
FlACbacChloroflexi bacterium69.3%
FlAPbacPeptococcaceae bacterium CEB364.8%
FlATnorThermodesulforhabdus norvegica54.5%
FlAPtaU1Methanosaeta sp. PtaU1.Bin05549.5%
SalLStroSalinispora tropica CNB-4401535.6%
a

Sequence identity. References to known FlAs are indicated.

The genomic context of the different flA genes was likewise examined (Table S3). As reported for the fluorination gene clusters of Streptomyces sp. MA37, N. brasiliensis, Actinoplanes sp. N902–109, and S. xinghaiensis, all Actinomycetes harbor gene clusters resembling that of S. cattleya, the most studied source of fl genes described to date (23,32) (Figure 2). The genes flB (encoding a 5′-FDA phosphorylase), flG (encoding a response regulator), flH (encoding a putative cation:H+ antiporter), and flI (encoding a S-adenosyl-L-homocysteinase) were highly conserved in all actinomycetes. Most of them also presented the genes flIso (5-fluoro-5-deoxy-d-ribose 1-phosphate isomerase) and flFT (4-fluoro-l-threonine transaldolase), involved in the synthesis of fluoroacetate and 4-fluoro-l-threonine. These are the two canonical end fluorometabolites described thus far. (1) Also, genes encoding a prolyl-tRNA synthetase-associated protein and an EamA family transporter were usually found in proximity to flFT. In S. cattleya, the products of these genes (termed fthB and fthC, respectively) play a role in detoxification by deacylation of 4-fluoro-l-threoninyl-tRNA and export of 4-fluoro-l-threonine. (33) Interestingly, Amycolatopsis bartoniae and Goodfellowiella sp. AN110305 lacked either flIso and flFT orthologues within the fl cluster, presenting, instead, orthologues to the fdr genes from Streptomyces sp. MA37. The genes are probably involved in the biosynthesis of 5-fluoro-2,3,4-trihydroxypentanoic acid via the fluorosugar intermediate 5-fluoro-5-deoxy-d-ribose. (34) Further biochemical activities encoded in these gene clusters include phosphoesterases, short chain dehydrogenases, dihydroxyacid dehydratases and cyclases, suggesting that the main fluorinated compounds produced by these microorganisms could be different from the canonical fluorometabolites fluoroacetate and 4-fluoro-l-threonine. Similar activities seem to be also encoded by genes in the vicinity of flA in Chloroflexi bacterium and salL in S. tropica. (35) Other genes widely distributed among the different actinomycotal clusters encoded activities related to SAM synthesis (i.e., SAM synthetase) and S-adenosyl-l-homocysteine degradation (i.e., S-adenosyl-L-homocysteinase), a competitive inhibitor of fluorinase activity. (10) As indicated above, the latter gene (flI) was present in all actinomycotal clusters. Since SAM and S-adenosyl-l-homocysteine are involved in essential cellular reactions, it is likely that these enzymes modulate the levels of these compounds during secondary metabolism, when organofluorines are actively produced. (36) Further analysis of the genes found in these fl clusters will provide clues as to what activities are needed to establish robust and efficient biofluorination pathways in heterologous hosts. This prospect is particularly exciting at the light of the need of novel organofluorine biosynthesis enzymes that could be sourced from environmental microbes. (1)

Figure 2

Figure 2. Fluorination gene clusters in actinomycetes. For clarity, the clusters are drawn centered on flA (identified as A) in the sense orientation. Numbers under dashed lines indicate the distance between open reading frames (ORFs) found in the same sequence entry; ORFs in separate entries are not connected by a line. Italicized letters indicate orthologues to the corresponding fl genes from S. cattleya. J′ indicates duplicate flJ copies (encoding DUF190 domain-containing protein). FT* is a truncated pseudogene homologous to flFT. Orthologues to fdr genes from Streptomyces sp. MA37 are indicated as white blocks with blue outlines. ORFs outlined in black represent genes with other/unknown functions. MFS, major facilitator superfamily; HTH, helix-turn-helix.

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Next, the coding sequences of all FlA candidates were codon-optimized for production in Escherichia coli as N-terminal His-tag fusions (flAMA37, flAScat and flASxin had been previously codon-optimized for expression in Gram-negative hosts; (27) see also Tables S4 and S5). SalLStro was not included in this experimental set since it is reportedly inactive on F–. (15) The expression of the 16 candidate genes was initially evaluated in 96-well microtiter plate cultures. FlATnor, FlAAmza, and FlAPbac could not be obtained as soluble enzymes and were not included in further analyses. Moreover, very faint bands of the expected size were observed in SDS-PAGE of E. coli extracts producing either FlATnor or FlAAmza, suggesting limited expression levels or poor translation (Figure S2). Therefore, we proceeded to obtain the remaining 13 candidates in medium-scale shaken-flask cultures for His-tag purification and activity assays. The purified enzymes were incubated in the presence of increasing SAM concentrations for 1 h, after which 5′-FDA was measured by HPLC. 5′-FDA synthase activity could be detected for 12 out of the 13 candidates (Figure S3). The protein concentration was normalized for these assays, although the enzymes were recovered with varying degrees of purity due to differences in solubility─typical of proteins from high-G+C-content species when produced in a Gram-negative host. (37) Notably, the enzyme from Methanosaeta sp. (FlAPtaU1, predicted to be a chlorinase), was one of the top performers. FlASAJ15 also had high 5′-FDA synthase activity in vitro. These two enzymes had specific activities comparable to those of FlAMA37 and FlASxin, with the highest catalytic efficiencies on SAM-dependent SN2 fluorination reported to date.

FlAPtaU1 and FlASAJ15 were selected for large-scale shaken-flask production and a more detailed biochemical characterization. Steady-state kinetics assays with 1 μM of the purified protein, varying concentrations of SAM (1.5–800 μM) and 75 mM KF revealed that both of these enzymes presented higher turnover rates (kcat) than FlAMA37 and FlASxin (Figure 3a and Table 2). In particular, the kcat of FlAPtaU1 was 2.6-fold larger than that of FlAMA37. Surprisingly, KMSAM values were consistently <10 μM, much lower than what had been previously reported in the literature for fluorinases. (12−14) Notably, previous studies used high enzyme concentrations (>10 μM), which impedes reaching a steady state of the reaction for substrate concentrations below 10 μM. We also used a KF concentration that ensures F– saturation without causing any inhibitory effect (previous studies have used KF concentrations >200 mM).

Table 2. Michaelis–Menten Kinetic Constants of Selected Fluorinasesa
fluorinaseKMSAM (μM)kcat (min–1)kcat/KMSAM (mM–1 min–1)
FlAMA374.42 ± 0.580.16 ± 0.0136.36 ± 4.82
FlASxin3.76 ± 0.150.22 ± 0.0158.63 ± 2.63
FlASAJ159.62 ± 1.430.34 ± 0.0135.81 ± 5.43
FlAPtaU16.99 ± 1.060.41 ± 0.0157.54 ± 8.85
a

Assays conducted in 50 mM HEPES, pH = 7.8, with 75 mM KF and varying SAM concentrations incubated at 37 °C. Average and standard deviations are given for triplicate independent measurements.

Figure 3

Figure 3. Biochemical characterization and residue conservation of selected fluorinases. (a) Steady-state fluorination assays using increasing SAM concentrations. Reactions were carried out at 37 °C in 50 mM HEPES buffer, pH = 7.8, with 75 mM KF. Dotted lines show fits to the Michaelis–Menten equation (R2 > 0.95 in all cases). (b) End-point (1 h) transhalogenation assays with increasing 5′-ClDA concentrations. Reactions were carried out at 37 °C in 50 mM HEPES buffer, pH = 7.8, with 75 mM KF and 1 mM l-Met. Error bars represent standard deviations from triplicate independent assays. Symbols and color codes are kept in both panels. Simplified schematics for the corresponding reactions are shown above each panel. (c–f) Variable residues in the substrate binding pocket of FlAMA37 (c), FlASAJ15 (d), FlAPtaU1 (e), and SalLStro (f). Residues that differ from those of FlAMA37 are labeled in bold font, whereas conserved residues are labeled in italics. FlASxin residues are identical with those of FlAMA37. The SAM substrate is shown in ball-and-stick representation.

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To gain insight on the structural factors that could determine these differences in fluorination activity, we inspected the predicted crystal structures of FlAMA37, FlASxin, FlASAJ15, FlAPtaU1, and SalLStro. Examination of the amino acid residues potentially interacting with SAM (at distances <5 Å) revealed important variations between the substrate binding pocket of FlAPtaU1 and that of the other fluorinases known to date (Figure 3c–f). The alterations could be mapped near to the adenyl moiety of SAM, and involve the substitution of a conserved proline for an arginine residue and an RNAA motif for YYGG. This motif is found in the C-terminal domain of other fluorinases, which is more variable than the N-terminal domain and is presumably also involved in hexamer formation (38) (Figure S4). Interestingly, the catalytic features found in FlAPtaU1 do not resemble those of the SalLStro chlorinase, which would place FlAPtaU1 in a different functional group of SN2 halogenases. Evaluating the effect of these amino acid differences in fluorinase activity will be of interest for enzyme engineering efforts.

Since FlAPtaU1 was predicted to be a chlorinase, we evaluated whether it was also active in SN2-dependent addition of Cl– to SAM. Unexpectedly, no 5′-ClDA accumulation could be detected in enzymatic reactions in which KF was replaced by KCl─in contrast to what has been reported for SalLStro. (15) Previous studies have shown that FlAScat can also catalyze the chlorination reaction. (16) However, this feature requires the simultaneous removal of l-Met or 5′-ClDA, the reaction products, since the reverse dehalogenation reaction is favored. We could observe transhalogenation on 5′-ClDA (i.e., 5′-FDA production in the presence of l-Met and F–, steps III and I in Scheme 1; Figure 3b). Again, FlAPtaU1 catalytically outperformed all other fluorinases, with a 3-fold higher Vmax value. Although we cannot rule out that FlAPtaU1 could also execute de novo chlorination, the 23-residue loop reportedly found in “conventional” fluorinases is not essential for the activity toward F–.

With this background, we tested the biosynthesis of fluorometabolites in vivo by engineering selected fluorinases in the bacterial platform Pseudomonas putida, a robust chassis for engineering complex chemistries using synthetic biology tools. (39−43) We have designed a fluoride-responsive genetic circuit that enabled biofluorination in this Gram-negative host. (27) Here, this system was adapted to express either flAPtaU1 or flASAJ15, the best-performing fluorinases according to the kinetic parameters in Table 2. FlAMA37 and FlASxin were included in control experiments, as we have previously used them for engineering in vivo fluorination. (27) Upon inducing gene expression with NaF (which is also the substrate of the reaction of interest) and producing the fluorinases for 20 h at 30 °C, 5′-FDA biosynthesis was determined by LC-MS to evaluate de novo fluorination activity (Figure 4a). Production of 5′-FDA by engineered P. putida could be detected in all cases (Figure 4b). Notably, the 5′-FDA content, indicative of in vivo biofluorination, was 12-fold higher in cells expressing flAPtaU1 with respect to any other fluorinase gene. Fluorination activity in cell-free extracts of P. putida incubated for 20 h at 30 °C in the presence of exogenously added 200 μM SAM and 5 mM NaF was similar for the fluorinases tested (Figure S5), with a higher activity detected in cell-free extracts carrying FlAPtaU1, the Archaeal fluorinase. In the cell-free extract assay, the final 5′-FDA concentrations detected were within the ranges previously reported. (27,38) Interestingly, no other fluorometabolites than 5′-FDA could be detected in these assays.

Figure 4

Figure 4. Engineering in vivo biofluorination in P. putida. (a) Schematic representation of the fluoride-responsive genetic circuit based on the T7 phage RNA polymerase (T7RNAP) (17) and workflow for the biofluorination assay. Expression of the different fluorinase genes was induced when the cultures reached an OD600 = 0.4–0.6 by adding NaF at 15 mM. Next, following an incubation at 30 °C for 20 h, aliquots were taken for metabolite extraction and quantification by LC-MS. Further details are provided in the Supporting Information. (b) Quantification of the intracellular 5′-FDA content in engineered P. putida expressing the different fluorinase genes. In this case, the intracellular 5′-FDA concentration is normalized by the cell dry weight (CDW). Black dots show individual values from six independent biological replicates, and the error bars represent standard deviations. Asterisks indicate significant differences with p-values <0.1 (*) or <0.05 (**) for a two-sample, one-sided Welch’s t-test.

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In conclusion, out of the 10 newly identified enzymes, the nonconventional FlA from the archaea Methanosaeta sp. PtaU1.Bin055 (FlAPtaU1) was found to present turnover rates superior to those of all FlAs reported to date. Surprisingly, this enzyme lacks the loop that was so far hypothesized to be a differentiating feature between fluorinases and chlorinases, challenging the hypothesis that this loop is required for activity toward F–. Engineering this nonconventional fluorinase in P. putida mediated the highest in vivo production of 5′-FDA described to date─and, for that matter, the highest fluorometabolite levels reported for any biological system, either natural or engineered. This work highlights the importance of systematic and efficient biocatalyst selection across the ever-expanding genomic databases, followed by careful characterization in vitro and cell factory engineering in vivo. This study also expands the known sequence diversity for fluorinase enzymes, helping in the identification of other nonintuitive sequence features. Interestingly, when the mining run was repeated with either FlAMA37 or FlAPtaU1 as query, the number of putative fluorinase sequences retrieved (24 hits) was essentially the same as obtained with the enzyme from S. cattleya as the template. These features will be useful for predicting protein function(s) from genomic databases annotations. Additionally, this fundamental knowledge will inform future engineering endeavors of fluorinases by rational and semirational design. Taken together, our results open avenues for the implementation of neo-metabolic pathways to incorporate F atoms in bacterial hosts by synthetic biology approaches.

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  • Corresponding Author
  • Authors
    • Isabel Pardo - The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark;  Present Address: Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas Margarita Salas, CSIC, 28040 Madrid, Spain
    • David Bednar - Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, 601 77 Brno, Czech Republic;  International Clinical Research Centre, St. Anne’s University Hospital, 656 91 Brno, Czech Republic
    • Patricia Calero - The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
    • Daniel C. Volke - The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kongens Lyngby, Denmark
    • Jiří Damborský - Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, 601 77 Brno, Czech Republic;  International Clinical Research Centre, St. Anne’s University Hospital, 656 91 Brno, Czech Republic
  • Author Contributions

    I.P. performed most of the experimental work and phylogenetic analysis, interpreted the data, and wrote the manuscript draft. P.C. and C.D.V. performed experimental work and contributed to manuscript writing. D.B. and J.D. performed in silico work and contributed to manuscript writing. P.I.N. acquired funding, conceptualized the study, supervised the work and finalized the manuscript. All authors have approved the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

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This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No. 814418 (SinFonia), the Czech Ministry of Education (INBIO CZ.02.1.01/0.0/0.0/16_026/0008451), and the Czech Grant Agency (DB 20-15915Y).

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This article references 43 other publications.

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    A review. The pressing need for novel bioprodn. approaches faces a limitation in the no. and type of mols. accessed through synthetic biol. Halogenation is widely used for tuning physicochem. properties of mols. and polymers, but traditional halogenation chem. often lacks specificity and generates harmful byproducts. Here, we pose that deploying synthetic metab. tailored for biohalogenation represents an unique opportunity towards economically attractive and environmentally friendly organohalide prodn. On this background, we discuss growth-coupled selection of functional metabolic modules that harness the rich repertoire of biosynthetic and biodegrdn. capabilities of environmental bacteria for in vivo biohalogenation. By rationally combining these approaches, the chem. landscape of living cells can accommodate bioprodn. of added-value organohalides which, as of today, are obtained by traditional chem.
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    Walker, M. C.; Chang, M. C. Y. Natural and Engineered Biosynthesis of Fluorinated Natural Products. Chem. Soc. Rev. 2014, 43 (18), 6527– 6536,  DOI: 10.1039/C4CS00027G
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    Natural and engineered biosynthesis of fluorinated natural products
    Walker, Mark C.; Chang, Michelle C. Y.
    Chemical Society Reviews (2014), 43 (18), 6527-6536CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Both natural products and synthetic organofluorines play important roles in the discovery and design of pharmaceuticals. The combination of these two classes of mols. has the potential to be useful in the ongoing search for new bioactive compds. but our ability to produce site-selectively fluorinated natural products remains limited by challenges in compatibility between their high structural complexity and current methods for fluorination. Living systems provide an alternative route to chem. fluorination and could enable the prodn. of organofluorine natural products through synthetic biol. approaches. While the identification of biogenic organofluorines has been limited, the study of the native organisms and enzymes that utilize these compds. can help to guide efforts to engineer the incorporation of this unusual element into complex pharmacol. active natural products. This review covers recent advances in understanding both natural and engineered prodn. of organofluorine natural products.
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  3. 3
    Harsanyi, A.; Sandford, G. Organofluorine Chemistry: Applications, Sources and Sustainability. Green Chem. 2015, 17 (4), 2081– 2086,  DOI: 10.1039/C4GC02166E
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    Organofluorine chemistry: applications, sources and sustainability
    Harsanyi, Antal; Sandford, Graham
    Green Chemistry (2015), 17 (4), 2081-2086CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
    A review. Fluorine is an essential element for life in the developed world that impacts hugely on the general public because many pharmaceuticals, agrochems., anesthetics, materials and air conditioning materials owe their important properties to the presence of fluorine atoms within their structures. All fluorine atoms used in org. chem. are ultimately sourced from a mined raw material, fluorspar (CaF2), but, given current usage and global reserve ests., there is only sufficient fluorspar available for a further 100 years. New large scale raw material sources of fluorine are available but must be sufficiently developed for the benefits of fluorinated systems to continue in the long term.
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  4. 4
    Inoue, M.; Sumii, Y.; Shibata, N. Contribution of Organofluorine Compounds to Pharmaceuticals. ACS Omega 2020, 5 (19), 10633– 10640,  DOI: 10.1021/acsomega.0c00830
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    Contribution of Organofluorine Compounds to Pharmaceuticals
    Inoue, Munenori; Sumii, Yuji; Shibata, Norio
    ACS Omega (2020), 5 (19), 10633-10640CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
    A review. Inspired by the success of fluorinated corticosteroids in the 1950s and fluoroquinolones in the 1980s, fluorine-contg. pharmaceuticals, which are also known as fluoro-pharmaceuticals, have been attracting attention for more than half of a century. Presently, about 20% of the com. pharmaceuticals are fluoro-pharmaceuticals. In this mini-review, we analyze the prevalence of fluoro-pharmaceuticals in the market and categorize them into several groups based on the chemotype of the fluoro-functional groups, their therapeutic purpose, and the presence of heterocycles and/or chirality to highlight the structural motifs, patterns, and promising trends in fluorine-based drug design. Our database contains 340 fluoro-pharmaceuticals, from the first fluoro-pharmaceutical, Florinef, to the latest fluoro-pharmaceuticals registered in 2019 and drugs that have been withdrawn. The names and chem. structures of all the 340 fluorinated drugs discussed are provided in the Supporting Information.
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    Ogawa, Y.; Tokunaga, E.; Kobayashi, O.; Hirai, K.; Shibata, N. Current Contributions of Organofluorine Compounds to the Agrochemical Industry. iScience 2020, 23 (9), 101467,  DOI: 10.1016/j.isci.2020.101467
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    Current Contributions of Organofluorine Compounds to the Agrochemical Industry
    Ogawa, Yuta; Tokunaga, Etsuko; Kobayashi, Osamu; Hirai, Kenji; Shibata, Norio
    iScience (2020), 23 (9), 101467CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
    A review. Currently, more than 1,200 agrochems. are listed and many of these are regularly used by farmers to generate the food supply to support the expanding global population. However, resistance to pesticides is an ever more frequently occurring phenomenon, and thus, a continuous supply of novel agrochems. with high efficiency, selectivity, and low toxicity is required. Moreover, the demand for a more sustainable society, by reducing the risk chems. pose to human health and by minimizing their environmental footprint, renders the development of novel agrochems. an ever more challenging undertaking. In the last two decades, fluoro-chems. have been assocd. with significant advances in the agrochem. development process. We herein analyze the contribution that organofluorine compds. make to the agrochem. industry. Our database covers 424 fluoro-agrochems. and is subdivided into several categories including chemotypes, mode of action, heterocycles, and chirality. This in-depth anal. reveals the unique relationship between fluorine and agrochems.
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    O’Hagan, D. Understanding Organofluorine Chemistry. An Introduction to the C–F Bond. Chem. Soc. Rev. 2008, 37, 308– 319,  DOI: 10.1039/B711844A
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    Understanding organofluorine chemistry. An introduction to the C-F bond
    O'Hagan, David
    Chemical Society Reviews (2008), 37 (2), 308-319CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    A review. Fluorine is the most electroneg. element in the periodic table. When bound to carbon it forms the strongest bonds in org. chem. and this makes fluorine substitution attractive for the development of pharmaceuticals and a wide range of specialty materials. Although highly polarized, the C-F bond gains stability from the resultant electrostatic attraction between the polarized Cδ+ and Fδ- atoms. This polarity suppresses lone pair donation from fluorine and in general fluorine is a weak coordinator. However, the C-F bond has interesting properties which can be understood either in terms of electrostatic/dipole interactions or by considering stereoelectronic interactions with neighboring bonds or lone pairs. In this tutorial review these fundamental aspects of the C-F bond are explored to rationalize the geometry, conformation and reactivity of individual organofluorine compds.
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  7. 7
    Carvalho, M. F.; Oliveira, R. S. Natural Production of Fluorinated Compounds and Biotechnological Prospects of the Fluorinase Enzyme. Crit. Rev. Biotechnol. 2017, 37 (7), 880– 897,  DOI: 10.1080/07388551.2016.1267109
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    7
    Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme
    Carvalho, Maria F.; Oliveira, Rui S.
    Critical Reviews in Biotechnology (2017), 37 (7), 880-897CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)
    Fluorinated compds. are finding increasing uses in several applications. They are employed in almost all areas of modern society. These compds. are all produced by chem. synthesis and their abundance highly contrasts with fluorinated mols. of natural origin. To date, only some plants and a handful of actinomycetes species are known to produce a small no. of fluorinated compds. that include fluoroacetate (FA), some ω-fluorinated fatty acids, nucleocidin, 4-fluorothreonine (4-FT), and the more recently identified (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid. This largely differs from other naturally produced halogenated compds., which totals more than 5000. The mechanisms underlying biol. fluorination have been uncovered after discovering the first actinomycete species, Streptomyces cattleya, that is capable of producing FA and 4-FT, and a fluorinase has been identified as the enzyme responsible for the formation of the C-F bond. The discovery of this enzyme has opened new perspectives for the biotechnol. prodn. of fluorinated compds. and many advancements have been achieved in its application mainly as a biocatalyst for the synthesis of [18F]-labeled radiotracers for medical imaging. Natural fluorinated compds. may also be derived from abiogenic sources, such as volcanoes and rocks, though their concns. and prodn. mechanisms are not well known. This review provides an outlook of what is currently known about fluorinated compds. with natural origin. The paucity of these compds. and the biol. mechanisms responsible for their prodn. are addressed. Due to its relevance, special emphasis is given to the discovery, characterization and biotechnol. potential of the unique fluorinase enzyme.
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  8. 8
    Deng, H.; O’Hagan, D.; Schaffrath, C. Fluorometabolite Biosynthesis and the Fluorinase from Streptomyces cattleya. Nat. Prod. Rep. 2004, 21 (6), 773– 784,  DOI: 10.1039/b415087m
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    Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleya
    Deng, Hai; O'Hagan, David; Schaffrath, Christoph
    Natural Product Reports (2004), 21 (6), 773-784CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
    A review. This review outlines the recent developments in uncovering the enzymes and intermediates involved in fluorometabolite biosynthesis in the bacterium Streptomyces cattleya. A particular emphasis is placed on the purifn. and characterization of the fluorinase, the C-F bond forming enzyme which initiates the biosynthesis. Nature has hardly developed a biochem. around fluorine, yet fluorinated orgs. are important com. entities, therefore a biotransformation from inorg. to org. fluorine is novel and of contemporary interest.
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  9. 9
    O’Hagan, D.; Schaffrath, C.; Cobb, S. L.; Hamilton, J. T.; Murphy, C. D. Biochemistry: Biosynthesis of an Organofluorine Molecule. Nature 2002, 416 (6878), 279,  DOI: 10.1038/416279a
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    9
    Biochemistry: Biosynthesis of an organofluorine molecule
    O'Hagan, David; Schaffrath, Christoph; Cobb, Steven L.; Hamilton, John T. G.; Murphy, Cormac D.
    Nature (London, United Kingdom) (2002), 416 (6878), 279CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    An enzymic reaction that occurs in the bacterium Streptomyces cattleya and which catalyzes the conversion of fluoride ion and S-adenosylmethionine (SAM) to 5'-fluoro-5'-deoxyadenosine is described. This is the first fluorinase enzyme to be identified, a discovery that opens up a new biotechnol. opportunity for the prepn. of organofluorine compds. The crude cell-free protein prepn. from S. cattleya cells was able to mediate the biotransformation of SAM and fluoride ion all the way to fluoroacetate, suggesting that this organism also contains the necessary enzyme activities to convert 5'-FDA to fluoroacetate. The fluorination reaction appears to involve a nucleophilic attack by fluoride ion at the C-5' carbon of SAM, generating 5'-FDA and concomitantly displacing L-methionine.
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  10. 10
    Schaffrath, C.; Deng, H.; O’Hagan, D. Isolation and Characterisation of 5′-Fluorodeoxyadenosine Synthase, a Fluorination Enzyme from Streptomyces cattleya. FEBS Lett. 2003, 547 (1–3), 111– 114,  DOI: 10.1016/S0014-5793(03)00688-4
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    10
    Isolation and characterization of 5'-fluorodeoxyadenosine synthase, a fluorination enzyme from Streptomyces cattleya
    Schaffrath, Christoph; Deng, Hai; O'Hagan, David
    FEBS Letters (2003), 547 (1-3), 111-114CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)
    5'-Fluorodeoxyadenosine synthase/fluorinase (I), a C-F bond-forming enzyme, was purified from S. cattleya and characterized. I mediates a reaction between F- and S-adenosyl-L-methionine (SAM) to generate 5'-fluoro-5'-deoxyadenosine. The mol. wt. of the monomeric protein was shown to be 32.2 kDa by electrospray mass spectrometry. The kinetic parameters for SAM (Km = 0.42 mM, Vmax = 1.28 U/mg) and F- (Km = 8.56 mM, Vmax = 1.59 U/mg) were evaluated. Cl- was also studied up to 10 mM concn. but was not found to be a substrate or inhibitor of I. Both S-adenosyl-L-homocysteine (SAH) and sinefungin were explored as inhibitors of the enzyme. SAH emerged as a potent competitive inhibitor (Ki = 29 μM), whereas sinefungin was only weakly inhibitory. The N-terminal sequence (24 residues) of I was detd.
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  11. 11
    Zhu, X.; Robinson, D. A.; McEwan, A. R.; O’Hagan, D.; Naismith, J. H. Mechanism of Enzymatic Fluorination in Streptomyces cattleya. J. Am. Chem. Soc. 2007, 129 (47), 14597– 14604,  DOI: 10.1021/ja0731569
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    11
    Mechanism of Enzymatic Fluorination in Streptomyces cattleya
    Zhu, Xiaofeng; Robinson, David A.; McEwan, Andrew R.; O'Hagan, David; Naismith, James H.
    Journal of the American Chemical Society (2007), 129 (47), 14597-14604CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-L-methionine (SAM) with the concomitant displacement of L-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the mol. mechanism remained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, and isothermal calorimetry (ITC) expts. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallog.; however, using chloride ion (also a substrate) with a mutant of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the pos. charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a mol. mechanism for FDAS in which fluoride weakly assocs. with the enzyme exchanging two water mols. for protein ligation. The binding of SAM expels remaining water assocd. with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating L-methionine and 5'-FDA. L-Methionine then dissocs. from the enzyme followed by 5'-FDA.
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  12. 12
    Deng, H.; Ma, L.; Bandaranayaka, N.; Qin, Z.; Mann, G.; Kyeremeh, K.; Yu, Y.; Shepherd, T.; Naismith, J. H.; O’Hagan, D. Identification of Fluorinases from Streptomyces sp Ma37, Nocardia brasiliensis, and Actinoplanes sp N902–109 by Genome Mining. ChemBioChem. 2014, 15 (3), 364– 368,  DOI: 10.1002/cbic.201300732
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    12
    Identification of Fluorinases from Streptomyces sp MA37, Nocardia brasiliensis, and Actinoplanes sp N902-109 by Genome Mining
    Deng, Hai; Ma, Long; Bandaranayaka, Nouchali; Qin, Zhiwei; Mann, Greg; Kyeremeh, Kwaku; Yu, Yi; Shepherd, Thomas; Naismith, James H.; O'Hagan, David
    ChemBioChem (2014), 15 (3), 364-368CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The fluorinase is an enzyme that catalyzes the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterized. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, the authors report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homol. (80-87% identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and are fluorination enzymes. An x-ray crystallog. study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnol.
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  13. 13
    Ma, L.; Li, Y.; Meng, L.; Deng, H.; Li, Y.; Zhang, Q.; Diao, A. Biological Fluorination from the Sea: Discovery of a SAM-Dependent Nucleophilic Fluorinating Enzyme from the Marine-Derived Bacterium Streptomyces xinghaiensis NRRL B24674. RSC Adv. 2016, 6, 27047– 27051,  DOI: 10.1039/C6RA00100A
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    13
    Biological fluorination from the sea: discovery of a SAM-dependent nucleophilic fluorinating enzyme from the marine-derived bacterium Streptomyces xinghaiensis NRRL B24674
    Ma, Long; Li, Yufeng; Meng, Lingpei; Deng, Hai; Li, Yuyin; Zhang, Qiang; Diao, Aipo
    RSC Advances (2016), 6 (32), 27047-27051CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    The authors have discovered and characterised a fluorinating enzyme from marine-derived Streptomyces xinghaiensis. It is able to install a fluorine atom into S-adenosyl-L-methionine to form 5'-fluorodeoxy adenosine. Apparently, this is the first fluorinase unveiled from a marine source.
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  14. 14
    Sooklal, S. A.; de Koning, C.; Brady, D.; Rumbold, K. Identification and Characterisation of a Fluorinase from Actinopolyspora mzabensis. Protein Expr. Purif. 2020, 166, 105508,  DOI: 10.1016/j.pep.2019.105508
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    14
    Identification and characterisation of a fluorinase from Actinopolyspora mzabensis
    Sooklal, Selisha Ann; De Koning, Charles; Brady, Dean; Rumbold, Karl
    Protein Expression and Purification (2020), 166 (), 105508CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
    The incorporation of fluorine has been shown to improve the biophys. and bioactive properties of several org. compds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyze a C-F bond, hence, have vast potential to be applied as biocatalysts in the prepn. of fine chems. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of sol. protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biol. active form. Following hydrophobic interaction chromatog., >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An addnl. gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temp. was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ± 0.03 μM min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.
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  15. 15
    Eustáquio, A. S.; Pojer, F.; Noel, J. P.; Moore, B. S. Discovery and Characterization of a Marine Bacterial SAM-Dependent Chlorinase. Nat. Chem. Biol. 2008, 4 (1), 69– 74,  DOI: 10.1038/nchembio.2007.56
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    15
    Discovery and characterization of a marine bacterial SAM-dependent chlorinase
    Eustaquio, Alessandra S.; Pojer, Florence; Noel, Joseph P.; Moore, Bradley S.
    Nature Chemical Biology (2008), 4 (1), 69-74CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    Halogen atom incorporation into a scaffold of bioactive compds. often amplifies biol. activity, as is the case for the anticancer agent salinosporamide A, a chlorinated natural product from the marine bacterium Salinispora tropica. Significant effort in understanding enzymic chlorination shows that oxidative routes predominate to form reactive electrophilic or radical chlorine species. Here we report the genetic, biochem. and structural characterization of the chlorinase SalL, which halogenates S-adenosyl-L-methionine with chloride to generate 5'-chloro-5'-deoxyadenosine and L-methionine in a rarely obsd. nucleophilic substitution strategy analogous to that of Streptomyces cattleya fluorinase. Further metabolic tailoring produces a halogenated polyketide synthase substrate specific for salinosporamide A biosynthesis. SalL also accepts bromide and iodide as substrates, but not fluoride. High-resoln. crystal structures of SalL and active site mutants complexed with substrates and products support the SN2 nucleophilic substitution mechanism and further illuminate halide specificity in this newly discovered halogenase family.
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    Deng, H.; Cobb, S. L.; McEwan, A. R.; McGlinchey, R. P.; Naismith, J. H.; O’Hagan, D.; Robinson, D. A.; Spencer, J. B. The Fluorinase from Streptomyces cattleya is Also a Chlorinase. Angew. Chem., Int. Ed. Engl. 2006, 45 (5), 759– 762,  DOI: 10.1002/anie.200503582
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    16
    The fluorinase from Streptomyces cattleya is also a chlorinase
    Deng Hai; Cobb Steven L; McEwan Andrew R; McGlinchey Ryan P; Naismith James H; O'Hagan David; Robinson David A; Spencer Jonathan B
    Angewandte Chemie (International ed. in English) (2006), 45 (5), 759-62 ISSN:1433-7851.
    There is no expanded citation for this reference.
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    Deng, H.; O’Hagan, D. The Fluorinase, the Chlorinase and the Duf-62 Enzymes. Curr. Opin. Chem. Biol. 2008, 12 (5), 582– 592,  DOI: 10.1016/j.cbpa.2008.06.036
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    17
    The fluorinase, the chlorinase and the duf-62 enzymes
    Deng, Hai; O'Hagan, David
    Current Opinion in Chemical Biology (2008), 12 (5), 582-592CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    A review. The fluorinase from Streptomyces cattleya and chlorinase from Salinispora tropica have a commonality in that they mediate nucleophilic reactions of their resp. halide ions to the C-5' carbon of S-adenosyl-L-methionine (SAM). These enzyme reactions fall into the relatively small group of SN2 substitution reactions found in enzymol. These enzymes have some homol. to a larger class of proteins expressed by the duf-62 gene, of which around 200 representatives have been sequenced and deposited in databases. The duf-62 genes express a protein which mediates a hydrolytic cleavage of SAM to generate adenosine and L-methionine. Superficially this enzyme operates very similarly to the halogenases in that water/hydroxide replaces the halide ion. However structural examn. of the duf-62 gene product reveals a very different organization of the active site suggesting a novel mechanism for water activation.
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    Pereira, P. R. M.; Araújo, J. O.; Silva, J. R. A.; Alves, C. N.; Lameira, J.; Lima, A. H. Exploring Chloride Selectivity and Halogenase Regioselectivity of the Sall Enzyme through Quantum Mechanical/Molecular Mechanical Modeling. J. Chem. Inf. Model. 2020, 60 (2), 738– 746,  DOI: 10.1021/acs.jcim.9b01079
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    18
    Exploring Chloride Selectivity and Halogenase Regioselectivity of the SalL Enzyme through Quantum Mechanical/Molecular Mechanical Modeling
    Pereira, Paulo R. M.; Araujo, Jessica de O.; Silva, Jose Rogerio A.; Alves, Claudio N.; Lameira, Jeronimo; Lima, Anderson H.
    Journal of Chemical Information and Modeling (2020), 60 (2), 738-746CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
    The catalytic mechanism of SalL chlorinase has been investigated by combining quantum mech./mol. mech. (QM/MM) techniques and umbrella sampling simulations to compute free energy profiles. Our results shed light on the interesting fact that the substitution of chloride with fluorine in SalL chlorinase leads to a loss of halogenase activity. The potential of mean force based on DFTB3/MM anal. shows that fluorination corresponds to a barrier 13.5 kcal·mol-1 higher than chlorination. Addnl., our results present a mol. description of SalL acting as a chlorinase instead of a methyl-halide transferase.
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  19. 19
    Hauer, B. Embracing Nature’s Catalysts: A Viewpoint on the Future of Biocatalysis. ACS Catal. 2020, 10 (15), 8418– 8427,  DOI: 10.1021/acscatal.0c01708
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    19
    Embracing Nature's Catalysts: A Viewpoint on the Future of Biocatalysis
    Hauer, Bernhard
    ACS Catalysis (2020), 10 (15), 8418-8427CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    A review with commentary. Enzymes are highly efficient biocatalysts researched for industrial-scale catalysis because of their several distinct advantages that range from their operation in milder reaction conditions, to their exceptional selectivity, and to their lower environmental and physiol. toxicity. Further work remains however to demonstrate that biocatalysis is economically competitive in industries other than the pharmaceutical, food, and beverage industries. In this Viewpoint, I analyze certain aspects of transferring biocatalysis into processes to manuf. chem. compds. I focus on pointing out the obstacles in the application of biocatalysts in industries and further discuss promising research directions and solns. to successfully expand the application of enzymes.
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  20. 20
    Martinelli, L.; Nikel, P. I. Breaking the State-of-the-Art in the Chemical Industry with New-to-Nature Products via Synthetic Microbiology. Microb. Biotechnol. 2019, 12 (2), 187– 190,  DOI: 10.1111/1751-7915.13372
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    20
    Breaking the state-of-the-art in the chemical industry with new-to-Nature products via synthetic microbiology
    Martinelli Laura; Nikel Pablo I
    Microbial biotechnology (2019), 12 (2), 187-190 ISSN:.
    There is no expanded citation for this reference.
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  21. 21
    Nieto-Domínguez, M.; Nikel, P. I. Intersecting Xenobiology and Neo-Metabolism to Bring Novel Chemistries to Life. ChemBioChem. 2020, 21 (18), 2551– 2571,  DOI: 10.1002/cbic.202000091
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    21
    Intersecting Xenobiology and Neometabolism To Bring Novel Chemistries to Life
    Nieto-Dominguez, Manuel; Nikel, Pablo I.
    ChemBioChem (2020), 21 (18), 2551-2571CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. The diversity of life relies on a handful of chem. elements (carbon, oxygen, hydrogen, nitrogen, sulfur and phosphorus) as part of essential building blocks; some other atoms are needed to a lesser extent, but most of the remaining elements are excluded from biol. This circumstance limits the scope of biochem. reactions in extant metab. - yet it offers a phenomenal playground for synthetic biol. Xenobiol. aims to bring novel bricks to life that could be exploited for (xeno)metabolite synthesis. In particular, the assembly of novel pathways engineered to handle nonbiol. elements (neometabolism) will broaden chem. space beyond the reach of natural evolution. In this review, xeno-elements that could be blended into nature's biosynthetic portfolio are discussed together with their physicochem. properties and tools and strategies to incorporate them into biochem. We argue that current bioprodn. methods can be revolutionized by bridging xenobiol. and neometabolism for the synthesis of new-to-nature mols., such as organohalides.
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  22. 22
    Walker, M. C.; Thuronyi, B. W.; Charkoudian, L. K.; Lowry, B.; Khosla, C.; Chang, M. C. Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways. Science 2013, 341 (6150), 1089– 1094,  DOI: 10.1126/science.1242345
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    22
    Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways
    Walker, Mark C.; Thuronyi, Benjamin W.; Charkoudian, Louise K.; Lowry, Brian; Khosla, Chaitan; Chang, Michelle C. Y.
    Science (Washington, DC, United States) (2013), 341 (6150), 1089-1094CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    Organofluorines represent a rapidly expanding proportion of mols. that are used in pharmaceuticals, diagnostics, agrochems., and materials. Despite the prevalence of fluorine in synthetic compds., the known biol. scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural-product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems, and we show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be inserted site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the prodn. of complex fluorinated natural products using synthetic biol.
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  23. 23
    O’Hagan, D.; Deng, H. Enzymatic Fluorination and Biotechnological Developments of the Fluorinase. Chem. Rev. 2015, 115 (2), 634– 649,  DOI: 10.1021/cr500209t
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    23
    Enzymatic Fluorination and Biotechnological Developments of the Fluorinase
    O'Hagan, David; Deng, Hai
    Chemical Reviews (Washington, DC, United States) (2015), 115 (2), 634-649CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review focusing on the biosynthesis fluorinated metabolites in nature, the enzymes involeved in fluorination and possible biotech. applications of these enzymes.
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  24. 24
    Sun, H.; Yeo, W. L.; Lim, Y. H.; Chew, X.; Smith, D. J.; Xue, B.; Chan, K. P.; Robinson, R. C.; Robins, E. G.; Zhao, H.; Ang, E. L. Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-Native Substrate. Angew. Chem., Int. Ed. 2016, 55 (46), 14277– 14280,  DOI: 10.1002/anie.201606722
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    24
    Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate
    Sun, Huihua; Yeo, Wan Lin; Lim, Yee Hwee; Chew, Xinying; Smith, Derek John; Xue, Bo; Chan, Kok Ping; Robinson, Robert C.; Robins, Edward G.; Zhao, Huimin; Ang, Ee Lui
    Angewandte Chemie, International Edition (2016), 55 (46), 14277-14280CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5'-[18F]FDA, with overall radiochem. conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved kcat value in the conversion of 5'-ClDA into S-adenosyl-L-methionine (SAM) but a reduced kcat value in the conversion of SAM into 5'-FDA.
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    Sun, H.; Zhao, H.; Ang, E. L. A Coupled Chlorinase–Fluorinase System with a High Efficiency of trans-Halogenation and a Shared Substrate Tolerance. Chem. Commun. 2018, 54 (68), 9458– 9461,  DOI: 10.1039/C8CC04436H
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    25
    A coupled chlorinase-fluorinase system with a high efficiency of trans-halogenation and a shared substrate tolerance
    Sun, H.; Zhao, H.; Ang, E. L.
    Chemical Communications (Cambridge, United Kingdom) (2018), 54 (68), 9458-9461CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    Enzymic trans-halogenation enables radiolabeling under mild and aq. conditions, but rapid reactions are desired. We developed a coupled chlorinase-fluorinase system for rapid trans-halogenation. Notably, the chlorinase shares a substrate tolerance with the fluorinase, enabling these two enzymes to cooperatively produce 5'-fluorodeoxy-2-ethynyladenosine (5'-FDEA) in up to 91.6% yield in 1 h.
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  26. 26
    Thomsen, M.; Vogensen, S. B.; Buchardt, J.; Burkart, M. D.; Clausen, R. P. Chemoenzymatic Synthesis and in situ Application of S-Adenosyl-L-Methionine Analogs. Org. Biomol. Chem. 2013, 11 (43), 7606– 7610,  DOI: 10.1039/c3ob41702f
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    26
    Chemoenzymatic synthesis and in situ application of S-adenosyl-L-methionine analogs
    Thomsen, Marie; Vogensen, Stine B.; Buchardt, Jens; Burkart, Michael D.; Clausen, Rasmus P.
    Organic & Biomolecular Chemistry (2013), 11 (43), 7606-7610CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Analogs of S-adenosyl-L-methionine (SAM) are increasingly applied to the methyltransferase (MT) catalyzed modification of biomols. including proteins, nucleic acids, and small mols. However, SAM and its analogs suffer from an inherent instability, and their chem. synthesis is challenged by low yields and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymic synthesis of a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases, SalL and FDAS. Mol. modeling studies are used to guide the rational design of mutants, and the enzymic conversion of L-Met and other analogs into SAM analogs is demonstrated. We also apply this in situ enzymic synthesis to the modification of a small peptide substrate by protein arginine methyltransferase 1 (PRMT1). This technique offers an attractive alternative to chem. synthesis and can be applied in situ to overcome stability and activity issues.
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  27. 27
    Calero, P.; Volke, D. C.; Lowe, P. T.; Gotfredsen, C. H.; O’Hagan, D.; Nikel, P. I. A Fluoride-Responsive Genetic Circuit Enables in vivo Biofluorination in Engineered Pseudomonas putida. Nat. Commun. 2020, 11 (1), 5045,  DOI: 10.1038/s41467-020-18813-x
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    27
    A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida
    Calero, Patricia; Volke, Daniel C.; Lowe, Phillip T.; Gotfredsen, Charlotte H.; O'Hagan, David; Nikel, Pablo I.
    Nature Communications (2020), 11 (1), 5045CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Fluorine is a key element in the synthesis of mols. broadly used in medicine, agriculture and materials. Addn. of fluorine to org. structures represents a unique strategy for tuning mol. properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochem. of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida. By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochem. reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chem. landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks.
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  28. 28
    Scherlach, K.; Hertweck, C. Mining and Unearthing Hidden Biosynthetic Potential. Nat. Commun. 2021, 12 (1), 3864,  DOI: 10.1038/s41467-021-24133-5
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    28
    Mining and unearthing hidden biosynthetic potential
    Scherlach, Kirstin; Hertweck, Christian
    Nature Communications (2021), 12 (1), 3864CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    A review. Genetically encoded small mols. (secondary metabolites) play eminent roles in ecol. interactions, as pathogenicity factors and as drug leads. Yet, these chem. mediators often evade detection, and the discovery of novel entities is hampered by low prodn. and high rediscovery rates. These limitations may be addressed by genome mining for biosynthetic gene clusters, thereby unveiling cryptic metabolic potential. The development of sophisticated data mining methods and genetic and anal. tools has enabled the discovery of an impressive array of previously overlooked natural products. This review shows the newest developments in the field, highlighting compd. discovery from unconventional sources and microbiomes.
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  29. 29
    Hon, J.; Borko, S.; Stourac, J.; Prokop, Z.; Zendulka, J.; Bednar, D.; Martinek, T.; Damborský, J. EnzymeMiner: Automated Mining of Soluble Enzymes with Diverse Structures, Catalytic Properties and Stabilities. Nucleic Acids Res. 2020, 48 (W1), W104– W109,  DOI: 10.1093/nar/gkaa372
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    29
    EnzymeMiner: automated mining of soluble enzymes with diverse structures, catalytic properties and stabilities
    Hon, Jiri; Borko, Simeon; Stourac, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Bednar, David; Martinek, Tomas; Damborsky, Jiri
    Nucleic Acids Research (2020), 48 (W1), W104-W109CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
    Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochem. characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochem. characterization based on rational and robust anal. of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner-a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab expts. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a sol. form in Escherichia coli. The soly. prediction employs the inhouse SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step anal., sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page.
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  30. 30
    Vanacek, P.; Sebestova, E.; Babkova, P.; Bidmanova, S.; Daniel, L.; Dvořák, P.; Stepankova, V.; Chaloupkova, R.; Brezovsky, J.; Prokop, Z.; Damborský, J. Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization. ACS Catal. 2018, 8 (3), 2402– 2412,  DOI: 10.1021/acscatal.7b03523
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    Exploration of enzyme diversity by integrating bioinformatics with expression analysis and biochemical characterization
    Vanacek, Pavel; Sebestova, Eva; Babkova, Petra; Bidmanova, Sarka; Daniel, Lukas; Dvorak, Pavel; Stepankova, Veronika; Chaloupkova, Radka; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri
    ACS Catalysis (2018), 8 (3), 2402-2412CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression anal. and activity screening of selected proteins, and (iii) complete biochem./biophys. characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression anal. and the robotic screening of enzymic activity provided 8 sol. proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also Archaea. Overall, the integrated system provided biocatalysts with broad catalytic diversity showing unique substrate specificity profiles, covering a wide range of optimal operational temp. from 20 to 70 °C and an unusually broad pH range from 5.7 to 10. We obtained the most catalytically proficient native haloalkane dehalogenase enzyme to date (kcat/K0.5 = 96.8 mM-1s-1), the most thermostable enzyme with melting temp. 71 °C, three different cold-adapted enzymes showing dehalogenase activity at near-to-zero temps., and a biocatalyst degrading the warfare chem. sulfur mustard. The established strategy can be adapted to other enzyme families for exploration of their biocatalytic diversity in a large sequence space continuously growing due to the use of next-generation sequencing technologies.
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    Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. Mega X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35 (6), 1547– 1549,  DOI: 10.1093/molbev/msy096
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    MEGA X: molecular evolutionary genetics analysis across computing platforms
    Kumar, Sudhir; Stecher, Glen; Li, Michael; Knyaz, Christina; Tamura, Koichiro
    Molecular Biology and Evolution (2018), 35 (6), 1547-1549CODEN: MBEVEO; ISSN:1537-1719. (Oxford University Press)
    The Mol. Evolutionary Genetics Anal. (Mega) software implements many anal. methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has addnl. been upgraded to use multiple computing cores for many mol. evolutionary analyses.
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    Huang, F.; Haydock, S. F.; Spiteller, D.; Mironenko, T.; Li, T. L.; O’Hagan, D.; Leadlay, P. F.; Spencer, J. B. The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A. Chem. Biol. 2006, 13 (5), 475– 484,  DOI: 10.1016/j.chembiol.2006.02.014
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    32
    The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A
    Huang, Fanglu; Haydock, Stephen F.; Spiteller, Dieter; Mironenko, Tatiana; Li, Tsung-Lin; O'Hagan, David; Leadlay, Peter F.; Spencer, Jonathan B.
    Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (5), 475-484CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    A genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the prodn. of fluorine-contg. metabolites. In addn. to the previously described fluorinase FlA which catalyzes the formation of 5'-fluoro-5'-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was detd. to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5'-fluoro-5'-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to.
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    McMurry, J. L.; Chang, M. C. Y. Fluorothreonyl-tRNA Deacylase Prevents Mistranslation in the Organofluorine Producer Streptomyces cattleya. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (45), 11920– 11925,  DOI: 10.1073/pnas.1711482114
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    33
    Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya
    McMurry, Jonathan L.; Chang, Michelle C. Y.
    Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (45), 11920-11925CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small mols., ranging from therapeutics to materials. In contrast, only a few fluorinated compds. made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in Streptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochem., genetic, physiol., and proteomic studies, we show that FthB is a trans-acting tRNA editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While trans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metab. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.
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    Ma, L.; Bartholomé, A.; Tong, M. H.; Qin, Z.; Yu, Y.; Shepherd, T.; Kyeremeh, K.; Deng, H.; O’Hagan, D. Identification of a Fluorometabolite from Streptomyces sp. MA37: (2R3S4S)-5-Fluoro-2,3,4-Trihydroxypentanoic Acid. Chem. Sci. 2015, 6, 1414,  DOI: 10.1039/C4SC03540B
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    34
    Identification of a fluorometabolite from Streptomyces sp. MA37: (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid
    Ma, Long; Bartholome, Axel; Tong, Ming Him; Qin, Zhiwei; Yu, Yi; Shepherd, Thomas; Kyeremeh, Kwaku; Deng, Hai; O'Hagan, David
    Chemical Science (2015), 6 (2), 1414-1419CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    (2R3S4S)-5-Fluoro-2,3,4-trihydroxypentanoic acid (5-FHPA) has been discovered as a new fluorometabolite in the soil bacterium Streptomyces sp. MA37. Exogenous addn. of 5-fluoro-5-deoxy-D-ribose (5-FDR) into the cell free ext. of MA37 demonstrated that 5-FDR was an intermediate to a range of unidentified fluorometabolites, distinct from fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Bioinformatics anal. allowed identification of a gene cluster (fdr), encoding a pathway to the biosynthesis of 5-FHPA. Over-expression and in vitro assay of FdrC indicated that FdrC is a NAD+ dependent dehydrogenase responsible for oxidn. of 5-FDR into 5-fluoro-5-deoxy-lactone, followed by hydrolysis to 5-FHPA. The identity of 5-FHPA in the fermn. broth was confirmed by synthesis of a ref. compd. and then co-correlation by 19F-NMR and GC-MS anal. The occurrence of 5-FHPA proves the existence of a new fluorometabolite pathway.
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  35. 35
    Eustáquio, A. S.; McGlinchey, R. P.; Liu, Y.; Hazzard, C.; Beer, L. L.; Florova, G.; Alhamadsheh, M. M.; Lechner, A.; Kale, A. J.; Kobayashi, Y.; Reynolds, K. A.; Moore, B. S. Biosynthesis of the Salinosporamide A Polyketide Synthase Substrate Chloroethylmalonyl-Coenzyme A from S-Adenosyl-L-Methionine. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (30), 12295– 12300,  DOI: 10.1073/pnas.0901237106
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    Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine
    Eustaquio Alessandra S; McGlinchey Ryan P; Liu Yuan; Hazzard Christopher; Beer Laura L; Florova Galina; Alhamadsheh Mamoun M; Lechner Anna; Kale Andrew J; Kobayashi Yoshihisa; Reynolds Kevin A; Moore Bradley S
    Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (30), 12295-300 ISSN:.
    Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.
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    Zhao, C.; Li, P.; Deng, Z.; Ou, H. Y.; McGlinchey, R. P.; O’Hagan, D. Insights into Fluorometabolite Biosynthesis in Streptomyces cattleya DSM46488 through Genome Sequence and Knockout Mutants. Bioorg. Chem. 2012, 44, 1– 7,  DOI: 10.1016/j.bioorg.2012.06.002
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    Insights into fluorometabolite biosynthesis in Streptomyces cattleya DSM46488 through genome sequence and knockout mutants
    Zhao, Chunhua; Li, Peng; Deng, Zixin; Ou, Hong-Yu; McGlinchey, Ryan P.; O'Hagan, David
    Bioorganic Chemistry (2012), 44 (), 1-7CODEN: BOCMBM; ISSN:0045-2068. (Elsevier B.V.)
    Streptomyces cattleya DSM 46488 is unusual in its ability to biosynthesize fluorine contg. natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S-adenosyl-L-methionine:fluoride adenosyltransferase (fluorinase, SCATT_41540), 5'-fluoro-5'-deoxyadenosine phosphorylase (SCATT_41550), fluoroacetyl-CoA thioesterase (SCATT_41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT_20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT_p11780) confirm that they are essential for fluorometabolite prodn. Notably gene disruption of the transaldolase (SCATT_p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesize 4-fluorothreonine, revealing a branch-point role for the PLP-transaldolase. Sequence data are deposited in GenBank/EMBL/DDBJ with accession nos. CP003219 and CP003229.
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    Boël, G.; Letso, R.; Neely, H.; Price, W. N.; Wong, K. H.; Su, M.; Luff, J.; Valecha, M.; Everett, J. K.; Acton, T. B.; Xiao, R.; Montelione, G. T.; Aalberts, D. P.; Hunt, J. F. Codon Influence on Protein Expression in E. coli Correlates with mRNA Levels. Nature 2016, 529 (7586), 358– 363,  DOI: 10.1038/nature16509
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    Codon influence on protein expression in E. coli correlates with mRNA levels
    Boel, Gregory; Letso, Reka; Neely, Helen; Price, W. Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D.; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.
    Nature (London, United Kingdom) (2016), 529 (7586), 358-363CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 expts. using bacteriophage T7 polymerase to synthesize mRNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiol. protein concns. and also mRNA concns. and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ∼16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degrdn. that is a central feature of the physiol. and also possibly the regulation of translation in E. coli.
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    Kittilä, T.; Calero, P.; Fredslund, F.; Lowe, P. T.; Tezé, D.; Nieto-Domínguez, M.; O’Hagan, D.; Nikel, P. I.; Welner, D. H. Oligomerization Engineering of the Fluorinase Enzyme Leads to an Active Trimer That Supports Synthesis of Fluorometabolites in vitro. Microb. Biotechnol. 2022, 15 (5), 1622– 1632,  DOI: 10.1111/1751-7915.14009
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    Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro
    Kittila, Tiia; Calero, Patricia; Fredslund, Folmer; Lowe, Phillip T.; Teze, David; Nieto-Dominguez, Manuel; O'Hagan, David; Nikel, Pablo I.; Welner, Ditte H.
    Microbial Biotechnology (2022), 15 (5), 1622-1632CODEN: MBIIB2; ISSN:1751-7915. (Wiley-Blackwell)
    Summary : The fluorinase enzyme represents the only biol. mechanism capable of forming stable C-F bonds characterized in nature thus far, offering a biotechnol. route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis const. (KM) for S-adenosyl-L-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-L-methionine affinity points to a long-range effect of hexamerization on substrate binding - likely decreasing substrate dissocn. and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.
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    Wirth, N. T.; Nikel, P. I. Combinatorial Pathway Balancing Provides Biosynthetic Access to 2-Fluoro-cis,cis-Muconate in Engineered Pseudomonas putida. Chem. Catal. 2021, 1 (6), 1234– 1259,  DOI: 10.1016/j.checat.2021.09.002
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    Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida
    Wirth Nicolas T; Nikel Pablo I
    Chem catalysis (2021), 1 (6), 1234-1259 ISSN:.
    The wealth of bio-based building blocks produced by engineered microorganisms seldom include halogen atoms. Muconate is a platform chemical with a number of industrial applications that could be broadened by introducing fluorine atoms to tune its physicochemical properties. The soil bacterium Pseudomonas putida naturally assimilates benzoate via the ortho-cleavage pathway with cis,cis-muconate as intermediate. Here, we harnessed the native enzymatic machinery (encoded within the ben and cat gene clusters) to provide catalytic access to 2-fluoro-cis,cis-muconate (2-FMA) from fluorinated benzoates. The reactions in this pathway are highly imbalanced, leading to accumulation of toxic intermediates and limited substrate conversion. By disentangling regulatory patterns of ben and cat in response to fluorinated effectors, metabolic activities were adjusted to favor 2-FMA biosynthesis. After implementing this combinatorial approach, engineered P. putida converted 3-fluorobenzoate to 2-FMA at the maximum theoretical yield. Hence, this study illustrates how synthetic biology can expand the diversity of nature's biochemical catalysis.
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    Nikel, P. I.; de Lorenzo, V. Pseudomonas putida as a Functional chassis for Industrial Biocatalysis: From Native Biochemistry to Trans-Metabolism. Metab. Eng. 2018, 50, 142– 155,  DOI: 10.1016/j.ymben.2018.05.005
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    Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism
    Nikel, Pablo I.; de Lorenzo, Victor
    Metabolic Engineering (2018), 50 (), 142-155CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
    The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochem. stresses. Over the years, these properties have been capitalized biotechnol. owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biol. have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biol. systems. Much of the biotechnol. interest on P. putida stems from the distinct architecture of its central carbon metab. The native biochem. is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochem. network of P. putida (cis-metab.) has sufficed to obtain target compds. of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metab.) for making complex, new-to-Nature mols. A no. of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufg. of fine and bulk chems. in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochem. beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chem. elements into the extant biochem., as well as genomic stability and scaling-up issues.
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    Volke, D. C.; Calero, P.; Nikel, P. I. Pseudomonas putida. Trends Microbiol. 2020, 28 (6), 512– 513,  DOI: 10.1016/j.tim.2020.02.015
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    Pseudomonas putida
    Volke, Daniel C.; Calero, Patricia; Nikel, Pablo I.
    Trends in Microbiology (2020), 28 (6), 512-513CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
    There is no expanded citation for this reference.
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    Sánchez-Pascuala, A.; Fernández-Cabezón, L.; de Lorenzo, V.; Nikel, P. I. Functional Implementation of a Linear Glycolysis for Sugar Catabolism in Pseudomonas putida. Metab. Eng. 2019, 54, 200– 211,  DOI: 10.1016/j.ymben.2019.04.005
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    Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida
    Sanchez-Pascuala, Alberto; Fernandez-Cabezon, Lorena; de Lorenzo, Victor; Nikel, Pablo I.
    Metabolic Engineering (2019), 54 (), 200-211CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
    The core metab. for glucose assimilation of the soil bacterium and platform strain Pseudomonas putida KT2440 has been reshaped from the native, cyclically-operating Entner-Doudoroff (ED) pathway to a linear Embden-Meyerhof-Parnas (EMP) glycolysis. The genetic strategy deployed to obtain a suitable host for the synthetic EMP route involved not only eliminating enzymic activities of the ED pathway, but also erasing peripheral reactions for glucose oxidn. that divert carbon skeletons into the formation of org. acids in the periplasm. Heterologous glycolytic enzymes, recruited from Escherichia coli, were genetically knocked-in in the mutant strain to fill the metabolic gaps for the complete metab. of glucose to pyruvate through a synthetic EMP route. A suite of genetic, physiol., and biochem. tests in the thereby-refactored P. putida strain-which grew on glucose as the sole carbon and energy source-demonstrated the functional replacement of the native sugar metab. by a synthetic catabolism. 13C-labeling expts. indicated that the bulk of pyruvate in the resulting strain was generated through the metabolic device grafted in P. putida. Strains carrying the synthetic glycolysis were further engineered for carotenoid synthesis from glucose, indicating that the implanted EMP route enabled higher carotenoid content on biomass and yield on sugar as compared with strains running the native hexose catabolism. Taken together, our results highlight how conserved metabolic features in a platform bacterium can be rationally reshaped for enhancing physiol. traits of interest.
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    Bitzenhofer, N. L.; Kruse, L.; Thies, S.; Wynands, B.; Lechtenberg, T.; Rönitz, J.; Kozaeva, E.; Wirth, N. T.; Eberlein, C.; Jaeger, K. E.; Nikel, P. I.; Heipieper, H. J.; Wierckx, N.; Loeschcke, A. Towards Robust Pseudomonas Cell Factories to Harbour Novel Biosynthetic Pathways. Essays Biochem. 2021, 65 (2), 319– 336,  DOI: 10.1042/EBC20200173
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    Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways
    Bitzenhofer, Nora Lisa; Kruse, Luzie; Thies, Stephan; Wynands, Benedikt; Lechtenberg, Thorsten; Roenitz, Jakob; Kozaeva, Ekaterina; Wirth, Nicolas Thilo; Eberlein, Christian; Jaeger, Karl-Erich; Nikel, Pablo Ivan; Heipieper, Hermann J.; Wierckx, Nick; Loeschcke, Anita
    Essays in Biochemistry (2021), 65 (2), 319-336CODEN: ESBIAV; ISSN:1744-1358. (Portland Press Ltd.)
    Biotechnol. prodn. in bacteria enables access to numerous valuable chem. compds. Nowadays, advanced mol. genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compds. within reach. However, the assocd. substrates and biosynthetic products often cause severe chem. stress to the bacterial hosts. Species of the Pseudomonas clade thus represent esp. valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chems. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compds. Finally, toxic chems. such as reactive aldehydes can be eliminated by oxidn. and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective prodn. of natural compds. and new chems. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnol. context. We further highlight strategies for the identification of yet unknown tolerance-assocd. genes and their utilization for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective prodn. of natural compds. and novel biochems.
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  2. Eimear Hegarty, Johannes Büchler, Rebecca M. Buller. Halogenases for the synthesis of small molecules. Current Opinion in Green and Sustainable Chemistry 2023, 41 , 100784. https://doi.org/10.1016/j.cogsc.2023.100784
  3. Robert Haas, Pablo I. Nikel. Challenges and opportunities in bringing nonbiological atoms to life with synthetic metabolism. Trends in Biotechnology 2023, 41 (1) , 27-45. https://doi.org/10.1016/j.tibtech.2022.06.004
  4. Sanyuan Shi, Jingrui Tian, Yunzi Luo. Recent advances in fluorinated products biosynthesis. Bioresource Technology Reports 2022, 20 , 101288. https://doi.org/10.1016/j.biteb.2022.101288
  5. Patricia Calero, Nicolás Gurdo, Pablo I. Nikel. Role of the CrcB transporter of Pseudomonas putida in the multi‐level stress response elicited by mineral fluoride. Environmental Microbiology 2022, 24 (11) , 5082-5104. https://doi.org/10.1111/1462-2920.16110
  6. Lawrence P. Wackett. Toward a molecular understanding of fluoride stress in a model Pseudomonas strain. Environmental Microbiology 2022, 24 (11) , 4981-4983. https://doi.org/10.1111/1462-2920.16114
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  8. Pablo I. Nikel, Ditte H. Welner, Antonin Cros, Daniel C. Volke. In vivo fluorine biocatalysis: Six enzymes in search of a cell factory. Chem Catalysis 2022, 2 (10) , 2403-2405. https://doi.org/10.1016/j.checat.2022.10.007
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  • Abstract

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    Scheme 1

    Scheme 1. Fluorometabolite Biosynthesis Pathways and Reactions Catalyzed by Fluorinase/Chlorinasea
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    aReactions catalyzed by fluorinase/chlorinase are indicated in gray: (I) forward fluorination reaction, (II) forward chlorination reaction, and (III) reverse chlorination reaction. The common step in fluorometabolite biosynthetic pathways is shaded in orange. The canonical fluoroacetate and 4-fluoro-l-threonine biosynthetic pathway are show in purple. The 5′-fluoro-5′-deoxy-d-ribose biosynthetic route is indicated in light blue. Compound abbreviations (blue): 5′-ClDA, 5′-chloro-5′-deoxyadenosine; SAM, S-adenosyl-l-methionine; 5′-FDA, 5′-fluoro-5′-deoxyadenosine; 5-FDRP, 5′-fluoro-5′-deoxy-d-ribose 1-phosphate; 5-FDRulP, 5-fluoro-5-deoxy-d-ribulose 1-phosphate; FAld, fluoroacetaldehyde; FAc, fluoroacetate; 4-FT, 4-fluoro-l-threonine; 5-FDR, 5′-fluoro-5′-deoxy-d-ribose; 5-FHPA, 5-fluoro-2,3,4-trihydroxypentanoic acid. Enzyme abbreviations (black bold): FlA, fluorinase; FlB, 5′-fluoro-5′-deoxyadenosine phosphorylase; FlIso, 5-fluoro-5-deoxy-d-ribose 1-phosphate isomerase; FlFT, 4-fluoro-l-threonine transaldolase; FdrA, 5-fluoro-5-deoxy-d-ribose 1-phosphate phosphoesterase; and FdrC, 5-fluoro-5-deoxy-d-ribose dehydrogenase.

    Figure 1

    Figure 1. Putative fluorinases identified by genome mining. (a) Residues specified as essential for the EnzymeMiner search, based on the crystal structure of FlAMA37 (PDB ID 5B6I). The SAM substrate is shown as a ball-and-stick representation. (b) Phylogenetic tree of retrieved fluorinase sequences obtained using the MEGAX software, (31) inferred using the Neighbor-Joining method with a bootstrap of 10 000 iterations. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Sequences sourced from Actinomycetes are highlighted as blue squares. Enzymes previously characterized in the literature are indicated in blue bold font. (c-e) 3D structures for FlAMA37 (c), wild-type SalLStro (d, PDB ID 6RYZ) and FlAPtaU1 (e, modeled with the SWISS-MODEL Alignment Mode tool using the FlAScat crystal structure PDB ID 2 V7 V as template). The loop hypothesized to differentiate fluorinases from chlorinases is circled in a dashed gray line. Two chains from the homotrimer for each structure are shown as cartoon and surface representations, respectively.

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    Figure 2

    Figure 2. Fluorination gene clusters in actinomycetes. For clarity, the clusters are drawn centered on flA (identified as A) in the sense orientation. Numbers under dashed lines indicate the distance between open reading frames (ORFs) found in the same sequence entry; ORFs in separate entries are not connected by a line. Italicized letters indicate orthologues to the corresponding fl genes from S. cattleya. J′ indicates duplicate flJ copies (encoding DUF190 domain-containing protein). FT* is a truncated pseudogene homologous to flFT. Orthologues to fdr genes from Streptomyces sp. MA37 are indicated as white blocks with blue outlines. ORFs outlined in black represent genes with other/unknown functions. MFS, major facilitator superfamily; HTH, helix-turn-helix.

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    Figure 3

    Figure 3. Biochemical characterization and residue conservation of selected fluorinases. (a) Steady-state fluorination assays using increasing SAM concentrations. Reactions were carried out at 37 °C in 50 mM HEPES buffer, pH = 7.8, with 75 mM KF. Dotted lines show fits to the Michaelis–Menten equation (R2 > 0.95 in all cases). (b) End-point (1 h) transhalogenation assays with increasing 5′-ClDA concentrations. Reactions were carried out at 37 °C in 50 mM HEPES buffer, pH = 7.8, with 75 mM KF and 1 mM l-Met. Error bars represent standard deviations from triplicate independent assays. Symbols and color codes are kept in both panels. Simplified schematics for the corresponding reactions are shown above each panel. (c–f) Variable residues in the substrate binding pocket of FlAMA37 (c), FlASAJ15 (d), FlAPtaU1 (e), and SalLStro (f). Residues that differ from those of FlAMA37 are labeled in bold font, whereas conserved residues are labeled in italics. FlASxin residues are identical with those of FlAMA37. The SAM substrate is shown in ball-and-stick representation.

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    Figure 4

    Figure 4. Engineering in vivo biofluorination in P. putida. (a) Schematic representation of the fluoride-responsive genetic circuit based on the T7 phage RNA polymerase (T7RNAP) (17) and workflow for the biofluorination assay. Expression of the different fluorinase genes was induced when the cultures reached an OD600 = 0.4–0.6 by adding NaF at 15 mM. Next, following an incubation at 30 °C for 20 h, aliquots were taken for metabolite extraction and quantification by LC-MS. Further details are provided in the Supporting Information. (b) Quantification of the intracellular 5′-FDA content in engineered P. putida expressing the different fluorinase genes. In this case, the intracellular 5′-FDA concentration is normalized by the cell dry weight (CDW). Black dots show individual values from six independent biological replicates, and the error bars represent standard deviations. Asterisks indicate significant differences with p-values <0.1 (*) or <0.05 (**) for a two-sample, one-sided Welch’s t-test.

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  • References

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    This article references 43 other publications.

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      Walker, Mark C.; Chang, Michelle C. Y.
      Chemical Society Reviews (2014), 43 (18), 6527-6536CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Both natural products and synthetic organofluorines play important roles in the discovery and design of pharmaceuticals. The combination of these two classes of mols. has the potential to be useful in the ongoing search for new bioactive compds. but our ability to produce site-selectively fluorinated natural products remains limited by challenges in compatibility between their high structural complexity and current methods for fluorination. Living systems provide an alternative route to chem. fluorination and could enable the prodn. of organofluorine natural products through synthetic biol. approaches. While the identification of biogenic organofluorines has been limited, the study of the native organisms and enzymes that utilize these compds. can help to guide efforts to engineer the incorporation of this unusual element into complex pharmacol. active natural products. This review covers recent advances in understanding both natural and engineered prodn. of organofluorine natural products.
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    3. 3
      Harsanyi, A.; Sandford, G. Organofluorine Chemistry: Applications, Sources and Sustainability. Green Chem. 2015, 17 (4), 2081– 2086,  DOI: 10.1039/C4GC02166E
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      Organofluorine chemistry: applications, sources and sustainability
      Harsanyi, Antal; Sandford, Graham
      Green Chemistry (2015), 17 (4), 2081-2086CODEN: GRCHFJ; ISSN:1463-9262. (Royal Society of Chemistry)
      A review. Fluorine is an essential element for life in the developed world that impacts hugely on the general public because many pharmaceuticals, agrochems., anesthetics, materials and air conditioning materials owe their important properties to the presence of fluorine atoms within their structures. All fluorine atoms used in org. chem. are ultimately sourced from a mined raw material, fluorspar (CaF2), but, given current usage and global reserve ests., there is only sufficient fluorspar available for a further 100 years. New large scale raw material sources of fluorine are available but must be sufficiently developed for the benefits of fluorinated systems to continue in the long term.
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    4. 4
      Inoue, M.; Sumii, Y.; Shibata, N. Contribution of Organofluorine Compounds to Pharmaceuticals. ACS Omega 2020, 5 (19), 10633– 10640,  DOI: 10.1021/acsomega.0c00830
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      Contribution of Organofluorine Compounds to Pharmaceuticals
      Inoue, Munenori; Sumii, Yuji; Shibata, Norio
      ACS Omega (2020), 5 (19), 10633-10640CODEN: ACSODF; ISSN:2470-1343. (American Chemical Society)
      A review. Inspired by the success of fluorinated corticosteroids in the 1950s and fluoroquinolones in the 1980s, fluorine-contg. pharmaceuticals, which are also known as fluoro-pharmaceuticals, have been attracting attention for more than half of a century. Presently, about 20% of the com. pharmaceuticals are fluoro-pharmaceuticals. In this mini-review, we analyze the prevalence of fluoro-pharmaceuticals in the market and categorize them into several groups based on the chemotype of the fluoro-functional groups, their therapeutic purpose, and the presence of heterocycles and/or chirality to highlight the structural motifs, patterns, and promising trends in fluorine-based drug design. Our database contains 340 fluoro-pharmaceuticals, from the first fluoro-pharmaceutical, Florinef, to the latest fluoro-pharmaceuticals registered in 2019 and drugs that have been withdrawn. The names and chem. structures of all the 340 fluorinated drugs discussed are provided in the Supporting Information.
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    5. 5
      Ogawa, Y.; Tokunaga, E.; Kobayashi, O.; Hirai, K.; Shibata, N. Current Contributions of Organofluorine Compounds to the Agrochemical Industry. iScience 2020, 23 (9), 101467,  DOI: 10.1016/j.isci.2020.101467
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      Current Contributions of Organofluorine Compounds to the Agrochemical Industry
      Ogawa, Yuta; Tokunaga, Etsuko; Kobayashi, Osamu; Hirai, Kenji; Shibata, Norio
      iScience (2020), 23 (9), 101467CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
      A review. Currently, more than 1,200 agrochems. are listed and many of these are regularly used by farmers to generate the food supply to support the expanding global population. However, resistance to pesticides is an ever more frequently occurring phenomenon, and thus, a continuous supply of novel agrochems. with high efficiency, selectivity, and low toxicity is required. Moreover, the demand for a more sustainable society, by reducing the risk chems. pose to human health and by minimizing their environmental footprint, renders the development of novel agrochems. an ever more challenging undertaking. In the last two decades, fluoro-chems. have been assocd. with significant advances in the agrochem. development process. We herein analyze the contribution that organofluorine compds. make to the agrochem. industry. Our database covers 424 fluoro-agrochems. and is subdivided into several categories including chemotypes, mode of action, heterocycles, and chirality. This in-depth anal. reveals the unique relationship between fluorine and agrochems.
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    6. 6
      O’Hagan, D. Understanding Organofluorine Chemistry. An Introduction to the C–F Bond. Chem. Soc. Rev. 2008, 37, 308– 319,  DOI: 10.1039/B711844A
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      6
      Understanding organofluorine chemistry. An introduction to the C-F bond
      O'Hagan, David
      Chemical Society Reviews (2008), 37 (2), 308-319CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      A review. Fluorine is the most electroneg. element in the periodic table. When bound to carbon it forms the strongest bonds in org. chem. and this makes fluorine substitution attractive for the development of pharmaceuticals and a wide range of specialty materials. Although highly polarized, the C-F bond gains stability from the resultant electrostatic attraction between the polarized Cδ+ and Fδ- atoms. This polarity suppresses lone pair donation from fluorine and in general fluorine is a weak coordinator. However, the C-F bond has interesting properties which can be understood either in terms of electrostatic/dipole interactions or by considering stereoelectronic interactions with neighboring bonds or lone pairs. In this tutorial review these fundamental aspects of the C-F bond are explored to rationalize the geometry, conformation and reactivity of individual organofluorine compds.
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    7. 7
      Carvalho, M. F.; Oliveira, R. S. Natural Production of Fluorinated Compounds and Biotechnological Prospects of the Fluorinase Enzyme. Crit. Rev. Biotechnol. 2017, 37 (7), 880– 897,  DOI: 10.1080/07388551.2016.1267109
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      7
      Natural production of fluorinated compounds and biotechnological prospects of the fluorinase enzyme
      Carvalho, Maria F.; Oliveira, Rui S.
      Critical Reviews in Biotechnology (2017), 37 (7), 880-897CODEN: CRBTE5; ISSN:0738-8551. (Taylor & Francis Ltd.)
      Fluorinated compds. are finding increasing uses in several applications. They are employed in almost all areas of modern society. These compds. are all produced by chem. synthesis and their abundance highly contrasts with fluorinated mols. of natural origin. To date, only some plants and a handful of actinomycetes species are known to produce a small no. of fluorinated compds. that include fluoroacetate (FA), some ω-fluorinated fatty acids, nucleocidin, 4-fluorothreonine (4-FT), and the more recently identified (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid. This largely differs from other naturally produced halogenated compds., which totals more than 5000. The mechanisms underlying biol. fluorination have been uncovered after discovering the first actinomycete species, Streptomyces cattleya, that is capable of producing FA and 4-FT, and a fluorinase has been identified as the enzyme responsible for the formation of the C-F bond. The discovery of this enzyme has opened new perspectives for the biotechnol. prodn. of fluorinated compds. and many advancements have been achieved in its application mainly as a biocatalyst for the synthesis of [18F]-labeled radiotracers for medical imaging. Natural fluorinated compds. may also be derived from abiogenic sources, such as volcanoes and rocks, though their concns. and prodn. mechanisms are not well known. This review provides an outlook of what is currently known about fluorinated compds. with natural origin. The paucity of these compds. and the biol. mechanisms responsible for their prodn. are addressed. Due to its relevance, special emphasis is given to the discovery, characterization and biotechnol. potential of the unique fluorinase enzyme.
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    8. 8
      Deng, H.; O’Hagan, D.; Schaffrath, C. Fluorometabolite Biosynthesis and the Fluorinase from Streptomyces cattleya. Nat. Prod. Rep. 2004, 21 (6), 773– 784,  DOI: 10.1039/b415087m
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      8
      Fluorometabolite biosynthesis and the fluorinase from Streptomyces cattleya
      Deng, Hai; O'Hagan, David; Schaffrath, Christoph
      Natural Product Reports (2004), 21 (6), 773-784CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. This review outlines the recent developments in uncovering the enzymes and intermediates involved in fluorometabolite biosynthesis in the bacterium Streptomyces cattleya. A particular emphasis is placed on the purifn. and characterization of the fluorinase, the C-F bond forming enzyme which initiates the biosynthesis. Nature has hardly developed a biochem. around fluorine, yet fluorinated orgs. are important com. entities, therefore a biotransformation from inorg. to org. fluorine is novel and of contemporary interest.
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    9. 9
      O’Hagan, D.; Schaffrath, C.; Cobb, S. L.; Hamilton, J. T.; Murphy, C. D. Biochemistry: Biosynthesis of an Organofluorine Molecule. Nature 2002, 416 (6878), 279,  DOI: 10.1038/416279a
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      9
      Biochemistry: Biosynthesis of an organofluorine molecule
      O'Hagan, David; Schaffrath, Christoph; Cobb, Steven L.; Hamilton, John T. G.; Murphy, Cormac D.
      Nature (London, United Kingdom) (2002), 416 (6878), 279CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      An enzymic reaction that occurs in the bacterium Streptomyces cattleya and which catalyzes the conversion of fluoride ion and S-adenosylmethionine (SAM) to 5'-fluoro-5'-deoxyadenosine is described. This is the first fluorinase enzyme to be identified, a discovery that opens up a new biotechnol. opportunity for the prepn. of organofluorine compds. The crude cell-free protein prepn. from S. cattleya cells was able to mediate the biotransformation of SAM and fluoride ion all the way to fluoroacetate, suggesting that this organism also contains the necessary enzyme activities to convert 5'-FDA to fluoroacetate. The fluorination reaction appears to involve a nucleophilic attack by fluoride ion at the C-5' carbon of SAM, generating 5'-FDA and concomitantly displacing L-methionine.
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    10. 10
      Schaffrath, C.; Deng, H.; O’Hagan, D. Isolation and Characterisation of 5′-Fluorodeoxyadenosine Synthase, a Fluorination Enzyme from Streptomyces cattleya. FEBS Lett. 2003, 547 (1–3), 111– 114,  DOI: 10.1016/S0014-5793(03)00688-4
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      10
      Isolation and characterization of 5'-fluorodeoxyadenosine synthase, a fluorination enzyme from Streptomyces cattleya
      Schaffrath, Christoph; Deng, Hai; O'Hagan, David
      FEBS Letters (2003), 547 (1-3), 111-114CODEN: FEBLAL; ISSN:0014-5793. (Elsevier Science B.V.)
      5'-Fluorodeoxyadenosine synthase/fluorinase (I), a C-F bond-forming enzyme, was purified from S. cattleya and characterized. I mediates a reaction between F- and S-adenosyl-L-methionine (SAM) to generate 5'-fluoro-5'-deoxyadenosine. The mol. wt. of the monomeric protein was shown to be 32.2 kDa by electrospray mass spectrometry. The kinetic parameters for SAM (Km = 0.42 mM, Vmax = 1.28 U/mg) and F- (Km = 8.56 mM, Vmax = 1.59 U/mg) were evaluated. Cl- was also studied up to 10 mM concn. but was not found to be a substrate or inhibitor of I. Both S-adenosyl-L-homocysteine (SAH) and sinefungin were explored as inhibitors of the enzyme. SAH emerged as a potent competitive inhibitor (Ki = 29 μM), whereas sinefungin was only weakly inhibitory. The N-terminal sequence (24 residues) of I was detd.
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    11. 11
      Zhu, X.; Robinson, D. A.; McEwan, A. R.; O’Hagan, D.; Naismith, J. H. Mechanism of Enzymatic Fluorination in Streptomyces cattleya. J. Am. Chem. Soc. 2007, 129 (47), 14597– 14604,  DOI: 10.1021/ja0731569
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      11
      Mechanism of Enzymatic Fluorination in Streptomyces cattleya
      Zhu, Xiaofeng; Robinson, David A.; McEwan, Andrew R.; O'Hagan, David; Naismith, James H.
      Journal of the American Chemical Society (2007), 129 (47), 14597-14604CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Recently a fluorination enzyme was identified and isolated from Streptomyces cattleya, as the first committed step on the metabolic pathway to the fluorinated metabolites, fluoroacetate and 4-fluorothreonine. This enzyme, 5'-fluoro-5'-deoxy adenosine synthetase (FDAS), has been shown to catalyze C-F bond formation by nucleophilic attack of fluoride ion to S-adenosyl-L-methionine (SAM) with the concomitant displacement of L-methionine to generate 5'-fluoro-5'-deoxy adenosine (5'-FDA). Although the structures of FDAS bound to both SAM and products have been solved, the mol. mechanism remained to be elucidated. We now report site-directed mutagenesis studies, structural analyses, and isothermal calorimetry (ITC) expts. The data establish the key residues required for catalysis and the order of substrate binding. Fluoride ion is not readily distinguished from water by protein X-ray crystallog.; however, using chloride ion (also a substrate) with a mutant of low activity has enabled the halide ion to be located in nonproductive co-complexes with SAH and SAM. The kinetic data suggest the pos. charged sulfur of SAM is a key requirement in stabilizing the transition state. We propose a mol. mechanism for FDAS in which fluoride weakly assocs. with the enzyme exchanging two water mols. for protein ligation. The binding of SAM expels remaining water assocd. with fluoride ion and traps the ion in a pocket positioned to react with SAM, generating L-methionine and 5'-FDA. L-Methionine then dissocs. from the enzyme followed by 5'-FDA.
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    12. 12
      Deng, H.; Ma, L.; Bandaranayaka, N.; Qin, Z.; Mann, G.; Kyeremeh, K.; Yu, Y.; Shepherd, T.; Naismith, J. H.; O’Hagan, D. Identification of Fluorinases from Streptomyces sp Ma37, Nocardia brasiliensis, and Actinoplanes sp N902–109 by Genome Mining. ChemBioChem. 2014, 15 (3), 364– 368,  DOI: 10.1002/cbic.201300732
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      12
      Identification of Fluorinases from Streptomyces sp MA37, Nocardia brasiliensis, and Actinoplanes sp N902-109 by Genome Mining
      Deng, Hai; Ma, Long; Bandaranayaka, Nouchali; Qin, Zhiwei; Mann, Greg; Kyeremeh, Kwaku; Yu, Yi; Shepherd, Thomas; Naismith, James H.; O'Hagan, David
      ChemBioChem (2014), 15 (3), 364-368CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The fluorinase is an enzyme that catalyzes the combination of S-adenosyl-L-methionine (SAM) and a fluoride ion to generate 5'-fluorodeoxy adenosine (FDA) and L-methionine through a nucleophilic substitution reaction with a fluoride ion as the nucleophile. It is the only native fluorination enzyme that has been characterized. The fluorinase was isolated in 2002 from Streptomyces cattleya, and, to date, this has been the only source of the fluorinase enzyme. Herein, the authors report three new fluorinase isolates that have been identified by genome mining. The novel fluorinases from Streptomyces sp. MA37, Nocardia brasiliensis, and an Actinoplanes sp. have high homol. (80-87% identity) to the original S. cattleya enzyme. They all possess a characteristic 21-residue loop. The three newly identified genes were overexpressed in E. coli and are fluorination enzymes. An x-ray crystallog. study of the Streptomyces sp. MA37 enzyme demonstrated that it is almost identical in structure to the original fluorinase. Culturing of the Streptomyces sp. MA37 strain demonstrated that it not only also elaborates the fluorometabolites, fluoroacetate and 4-fluorothreonine, similar to S. cattleya, but this strain also produces a range of unidentified fluorometabolites. These are the first new fluorinases to be reported since the first isolate, over a decade ago, and their identification extends the range of fluorination genes available for fluorination biotechnol.
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    13. 13
      Ma, L.; Li, Y.; Meng, L.; Deng, H.; Li, Y.; Zhang, Q.; Diao, A. Biological Fluorination from the Sea: Discovery of a SAM-Dependent Nucleophilic Fluorinating Enzyme from the Marine-Derived Bacterium Streptomyces xinghaiensis NRRL B24674. RSC Adv. 2016, 6, 27047– 27051,  DOI: 10.1039/C6RA00100A
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      13
      Biological fluorination from the sea: discovery of a SAM-dependent nucleophilic fluorinating enzyme from the marine-derived bacterium Streptomyces xinghaiensis NRRL B24674
      Ma, Long; Li, Yufeng; Meng, Lingpei; Deng, Hai; Li, Yuyin; Zhang, Qiang; Diao, Aipo
      RSC Advances (2016), 6 (32), 27047-27051CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      The authors have discovered and characterised a fluorinating enzyme from marine-derived Streptomyces xinghaiensis. It is able to install a fluorine atom into S-adenosyl-L-methionine to form 5'-fluorodeoxy adenosine. Apparently, this is the first fluorinase unveiled from a marine source.
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    14. 14
      Sooklal, S. A.; de Koning, C.; Brady, D.; Rumbold, K. Identification and Characterisation of a Fluorinase from Actinopolyspora mzabensis. Protein Expr. Purif. 2020, 166, 105508,  DOI: 10.1016/j.pep.2019.105508
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      14
      Identification and characterisation of a fluorinase from Actinopolyspora mzabensis
      Sooklal, Selisha Ann; De Koning, Charles; Brady, Dean; Rumbold, Karl
      Protein Expression and Purification (2020), 166 (), 105508CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier)
      The incorporation of fluorine has been shown to improve the biophys. and bioactive properties of several org. compds. However, sustainable strategies of fluorination are needed. Fluorinases have the unique ability to catalyze a C-F bond, hence, have vast potential to be applied as biocatalysts in the prepn. of fine chems. But fluorinases are extremely rare in nature with only five representatives isolated thus far. Moreover, the heterologous expression of fluorinases is challenged by low yields of sol. protein. This study describes the identification of a fluorinase from Actinopolyspora mzabensis. Overexpression of the Am-fluorinase in E. coli BL21 (DE3) resulted in the formation of inclusion bodies (IBs). The enzyme was recovered from IBs, solubilised in 8 M urea, and successfully refolded into a biol. active form. Following hydrophobic interaction chromatog., >80 mg of the active fluorinase was obtained at a purity suitable for biocatalytic applications. An addnl. gel filtration step gave ≥95% pure Am-fluorinase. Using LC-MS/MS, the optimal pH for activity was found at 7.2 while the optimal temp. was 65 °C. At these conditions, the enzyme exhibited an activity of 0.44 ± 0.03 μM min-1 mg-1. Furthermore, the Am-fluorinase showed exceptional stability at 25 °C. Preliminary results suggest that the newly identified Am-fluorinase is relatively thermostable.
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    15. 15
      Eustáquio, A. S.; Pojer, F.; Noel, J. P.; Moore, B. S. Discovery and Characterization of a Marine Bacterial SAM-Dependent Chlorinase. Nat. Chem. Biol. 2008, 4 (1), 69– 74,  DOI: 10.1038/nchembio.2007.56
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      15
      Discovery and characterization of a marine bacterial SAM-dependent chlorinase
      Eustaquio, Alessandra S.; Pojer, Florence; Noel, Joseph P.; Moore, Bradley S.
      Nature Chemical Biology (2008), 4 (1), 69-74CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      Halogen atom incorporation into a scaffold of bioactive compds. often amplifies biol. activity, as is the case for the anticancer agent salinosporamide A, a chlorinated natural product from the marine bacterium Salinispora tropica. Significant effort in understanding enzymic chlorination shows that oxidative routes predominate to form reactive electrophilic or radical chlorine species. Here we report the genetic, biochem. and structural characterization of the chlorinase SalL, which halogenates S-adenosyl-L-methionine with chloride to generate 5'-chloro-5'-deoxyadenosine and L-methionine in a rarely obsd. nucleophilic substitution strategy analogous to that of Streptomyces cattleya fluorinase. Further metabolic tailoring produces a halogenated polyketide synthase substrate specific for salinosporamide A biosynthesis. SalL also accepts bromide and iodide as substrates, but not fluoride. High-resoln. crystal structures of SalL and active site mutants complexed with substrates and products support the SN2 nucleophilic substitution mechanism and further illuminate halide specificity in this newly discovered halogenase family.
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    16. 16
      Deng, H.; Cobb, S. L.; McEwan, A. R.; McGlinchey, R. P.; Naismith, J. H.; O’Hagan, D.; Robinson, D. A.; Spencer, J. B. The Fluorinase from Streptomyces cattleya is Also a Chlorinase. Angew. Chem., Int. Ed. Engl. 2006, 45 (5), 759– 762,  DOI: 10.1002/anie.200503582
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      16
      The fluorinase from Streptomyces cattleya is also a chlorinase
      Deng Hai; Cobb Steven L; McEwan Andrew R; McGlinchey Ryan P; Naismith James H; O'Hagan David; Robinson David A; Spencer Jonathan B
      Angewandte Chemie (International ed. in English) (2006), 45 (5), 759-62 ISSN:1433-7851.
      There is no expanded citation for this reference.
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    17. 17
      Deng, H.; O’Hagan, D. The Fluorinase, the Chlorinase and the Duf-62 Enzymes. Curr. Opin. Chem. Biol. 2008, 12 (5), 582– 592,  DOI: 10.1016/j.cbpa.2008.06.036
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      17
      The fluorinase, the chlorinase and the duf-62 enzymes
      Deng, Hai; O'Hagan, David
      Current Opinion in Chemical Biology (2008), 12 (5), 582-592CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      A review. The fluorinase from Streptomyces cattleya and chlorinase from Salinispora tropica have a commonality in that they mediate nucleophilic reactions of their resp. halide ions to the C-5' carbon of S-adenosyl-L-methionine (SAM). These enzyme reactions fall into the relatively small group of SN2 substitution reactions found in enzymol. These enzymes have some homol. to a larger class of proteins expressed by the duf-62 gene, of which around 200 representatives have been sequenced and deposited in databases. The duf-62 genes express a protein which mediates a hydrolytic cleavage of SAM to generate adenosine and L-methionine. Superficially this enzyme operates very similarly to the halogenases in that water/hydroxide replaces the halide ion. However structural examn. of the duf-62 gene product reveals a very different organization of the active site suggesting a novel mechanism for water activation.
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    18. 18
      Pereira, P. R. M.; Araújo, J. O.; Silva, J. R. A.; Alves, C. N.; Lameira, J.; Lima, A. H. Exploring Chloride Selectivity and Halogenase Regioselectivity of the Sall Enzyme through Quantum Mechanical/Molecular Mechanical Modeling. J. Chem. Inf. Model. 2020, 60 (2), 738– 746,  DOI: 10.1021/acs.jcim.9b01079
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      18
      Exploring Chloride Selectivity and Halogenase Regioselectivity of the SalL Enzyme through Quantum Mechanical/Molecular Mechanical Modeling
      Pereira, Paulo R. M.; Araujo, Jessica de O.; Silva, Jose Rogerio A.; Alves, Claudio N.; Lameira, Jeronimo; Lima, Anderson H.
      Journal of Chemical Information and Modeling (2020), 60 (2), 738-746CODEN: JCISD8; ISSN:1549-9596. (American Chemical Society)
      The catalytic mechanism of SalL chlorinase has been investigated by combining quantum mech./mol. mech. (QM/MM) techniques and umbrella sampling simulations to compute free energy profiles. Our results shed light on the interesting fact that the substitution of chloride with fluorine in SalL chlorinase leads to a loss of halogenase activity. The potential of mean force based on DFTB3/MM anal. shows that fluorination corresponds to a barrier 13.5 kcal·mol-1 higher than chlorination. Addnl., our results present a mol. description of SalL acting as a chlorinase instead of a methyl-halide transferase.
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    19. 19
      Hauer, B. Embracing Nature’s Catalysts: A Viewpoint on the Future of Biocatalysis. ACS Catal. 2020, 10 (15), 8418– 8427,  DOI: 10.1021/acscatal.0c01708
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      19
      Embracing Nature's Catalysts: A Viewpoint on the Future of Biocatalysis
      Hauer, Bernhard
      ACS Catalysis (2020), 10 (15), 8418-8427CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      A review with commentary. Enzymes are highly efficient biocatalysts researched for industrial-scale catalysis because of their several distinct advantages that range from their operation in milder reaction conditions, to their exceptional selectivity, and to their lower environmental and physiol. toxicity. Further work remains however to demonstrate that biocatalysis is economically competitive in industries other than the pharmaceutical, food, and beverage industries. In this Viewpoint, I analyze certain aspects of transferring biocatalysis into processes to manuf. chem. compds. I focus on pointing out the obstacles in the application of biocatalysts in industries and further discuss promising research directions and solns. to successfully expand the application of enzymes.
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    20. 20
      Martinelli, L.; Nikel, P. I. Breaking the State-of-the-Art in the Chemical Industry with New-to-Nature Products via Synthetic Microbiology. Microb. Biotechnol. 2019, 12 (2), 187– 190,  DOI: 10.1111/1751-7915.13372
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      20
      Breaking the state-of-the-art in the chemical industry with new-to-Nature products via synthetic microbiology
      Martinelli Laura; Nikel Pablo I
      Microbial biotechnology (2019), 12 (2), 187-190 ISSN:.
      There is no expanded citation for this reference.
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    21. 21
      Nieto-Domínguez, M.; Nikel, P. I. Intersecting Xenobiology and Neo-Metabolism to Bring Novel Chemistries to Life. ChemBioChem. 2020, 21 (18), 2551– 2571,  DOI: 10.1002/cbic.202000091
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      21
      Intersecting Xenobiology and Neometabolism To Bring Novel Chemistries to Life
      Nieto-Dominguez, Manuel; Nikel, Pablo I.
      ChemBioChem (2020), 21 (18), 2551-2571CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. The diversity of life relies on a handful of chem. elements (carbon, oxygen, hydrogen, nitrogen, sulfur and phosphorus) as part of essential building blocks; some other atoms are needed to a lesser extent, but most of the remaining elements are excluded from biol. This circumstance limits the scope of biochem. reactions in extant metab. - yet it offers a phenomenal playground for synthetic biol. Xenobiol. aims to bring novel bricks to life that could be exploited for (xeno)metabolite synthesis. In particular, the assembly of novel pathways engineered to handle nonbiol. elements (neometabolism) will broaden chem. space beyond the reach of natural evolution. In this review, xeno-elements that could be blended into nature's biosynthetic portfolio are discussed together with their physicochem. properties and tools and strategies to incorporate them into biochem. We argue that current bioprodn. methods can be revolutionized by bridging xenobiol. and neometabolism for the synthesis of new-to-nature mols., such as organohalides.
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    22. 22
      Walker, M. C.; Thuronyi, B. W.; Charkoudian, L. K.; Lowry, B.; Khosla, C.; Chang, M. C. Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways. Science 2013, 341 (6150), 1089– 1094,  DOI: 10.1126/science.1242345
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      22
      Expanding the Fluorine Chemistry of Living Systems Using Engineered Polyketide Synthase Pathways
      Walker, Mark C.; Thuronyi, Benjamin W.; Charkoudian, Louise K.; Lowry, Brian; Khosla, Chaitan; Chang, Michelle C. Y.
      Science (Washington, DC, United States) (2013), 341 (6150), 1089-1094CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      Organofluorines represent a rapidly expanding proportion of mols. that are used in pharmaceuticals, diagnostics, agrochems., and materials. Despite the prevalence of fluorine in synthetic compds., the known biol. scope is limited to a single pathway that produces fluoroacetate. Here, we demonstrate that this pathway can be exploited as a source of fluorinated building blocks for introduction of fluorine into natural-product scaffolds. Specifically, we have constructed pathways involving two polyketide synthase systems, and we show that fluoroacetate can be used to incorporate fluorine into the polyketide backbone in vitro. We further show that fluorine can be inserted site-selectively and introduced into polyketide products in vivo. These results highlight the prospects for the prodn. of complex fluorinated natural products using synthetic biol.
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    23. 23
      O’Hagan, D.; Deng, H. Enzymatic Fluorination and Biotechnological Developments of the Fluorinase. Chem. Rev. 2015, 115 (2), 634– 649,  DOI: 10.1021/cr500209t
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      23
      Enzymatic Fluorination and Biotechnological Developments of the Fluorinase
      O'Hagan, David; Deng, Hai
      Chemical Reviews (Washington, DC, United States) (2015), 115 (2), 634-649CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review focusing on the biosynthesis fluorinated metabolites in nature, the enzymes involeved in fluorination and possible biotech. applications of these enzymes.
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    24. 24
      Sun, H.; Yeo, W. L.; Lim, Y. H.; Chew, X.; Smith, D. J.; Xue, B.; Chan, K. P.; Robinson, R. C.; Robins, E. G.; Zhao, H.; Ang, E. L. Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-Native Substrate. Angew. Chem., Int. Ed. 2016, 55 (46), 14277– 14280,  DOI: 10.1002/anie.201606722
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      24
      Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate
      Sun, Huihua; Yeo, Wan Lin; Lim, Yee Hwee; Chew, Xinying; Smith, Derek John; Xue, Bo; Chan, Kok Ping; Robinson, Robert C.; Robins, Edward G.; Zhao, Huimin; Ang, Ee Lui
      Angewandte Chemie, International Edition (2016), 55 (46), 14277-14280CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5'-[18F]FDA, with overall radiochem. conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved kcat value in the conversion of 5'-ClDA into S-adenosyl-L-methionine (SAM) but a reduced kcat value in the conversion of SAM into 5'-FDA.
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    25. 25
      Sun, H.; Zhao, H.; Ang, E. L. A Coupled Chlorinase–Fluorinase System with a High Efficiency of trans-Halogenation and a Shared Substrate Tolerance. Chem. Commun. 2018, 54 (68), 9458– 9461,  DOI: 10.1039/C8CC04436H
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      25
      A coupled chlorinase-fluorinase system with a high efficiency of trans-halogenation and a shared substrate tolerance
      Sun, H.; Zhao, H.; Ang, E. L.
      Chemical Communications (Cambridge, United Kingdom) (2018), 54 (68), 9458-9461CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      Enzymic trans-halogenation enables radiolabeling under mild and aq. conditions, but rapid reactions are desired. We developed a coupled chlorinase-fluorinase system for rapid trans-halogenation. Notably, the chlorinase shares a substrate tolerance with the fluorinase, enabling these two enzymes to cooperatively produce 5'-fluorodeoxy-2-ethynyladenosine (5'-FDEA) in up to 91.6% yield in 1 h.
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    26. 26
      Thomsen, M.; Vogensen, S. B.; Buchardt, J.; Burkart, M. D.; Clausen, R. P. Chemoenzymatic Synthesis and in situ Application of S-Adenosyl-L-Methionine Analogs. Org. Biomol. Chem. 2013, 11 (43), 7606– 7610,  DOI: 10.1039/c3ob41702f
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      26
      Chemoenzymatic synthesis and in situ application of S-adenosyl-L-methionine analogs
      Thomsen, Marie; Vogensen, Stine B.; Buchardt, Jens; Burkart, Michael D.; Clausen, Rasmus P.
      Organic & Biomolecular Chemistry (2013), 11 (43), 7606-7610CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Analogs of S-adenosyl-L-methionine (SAM) are increasingly applied to the methyltransferase (MT) catalyzed modification of biomols. including proteins, nucleic acids, and small mols. However, SAM and its analogs suffer from an inherent instability, and their chem. synthesis is challenged by low yields and difficulties in stereoisomer isolation and inhibition. Here we report the chemoenzymic synthesis of a series of SAM analogs using wild-type (wt) and point mutants of two recently identified halogenases, SalL and FDAS. Mol. modeling studies are used to guide the rational design of mutants, and the enzymic conversion of L-Met and other analogs into SAM analogs is demonstrated. We also apply this in situ enzymic synthesis to the modification of a small peptide substrate by protein arginine methyltransferase 1 (PRMT1). This technique offers an attractive alternative to chem. synthesis and can be applied in situ to overcome stability and activity issues.
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    27. 27
      Calero, P.; Volke, D. C.; Lowe, P. T.; Gotfredsen, C. H.; O’Hagan, D.; Nikel, P. I. A Fluoride-Responsive Genetic Circuit Enables in vivo Biofluorination in Engineered Pseudomonas putida. Nat. Commun. 2020, 11 (1), 5045,  DOI: 10.1038/s41467-020-18813-x
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      27
      A fluoride-responsive genetic circuit enables in vivo biofluorination in engineered Pseudomonas putida
      Calero, Patricia; Volke, Daniel C.; Lowe, Phillip T.; Gotfredsen, Charlotte H.; O'Hagan, David; Nikel, Pablo I.
      Nature Communications (2020), 11 (1), 5045CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Fluorine is a key element in the synthesis of mols. broadly used in medicine, agriculture and materials. Addn. of fluorine to org. structures represents a unique strategy for tuning mol. properties, yet this atom is rarely found in Nature and approaches to integrate fluorometabolites into the biochem. of living cells are scarce. In this work, synthetic gene circuits for organofluorine biosynthesis are implemented in the platform bacterium Pseudomonas putida. By harnessing fluoride-responsive riboswitches and the orthogonal T7 RNA polymerase, biochem. reactions needed for in vivo biofluorination are wired to the presence of fluoride (i.e. circumventing the need of feeding expensive additives). Biosynthesis of fluoronucleotides and fluorosugars in engineered P. putida is demonstrated with mineral fluoride both as only fluorine source (i.e. substrate of the pathway) and as inducer of the synthetic circuit. This approach expands the chem. landscape of cell factories by providing alternative biosynthetic strategies towards fluorinated building-blocks.
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    28. 28
      Scherlach, K.; Hertweck, C. Mining and Unearthing Hidden Biosynthetic Potential. Nat. Commun. 2021, 12 (1), 3864,  DOI: 10.1038/s41467-021-24133-5
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      28
      Mining and unearthing hidden biosynthetic potential
      Scherlach, Kirstin; Hertweck, Christian
      Nature Communications (2021), 12 (1), 3864CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      A review. Genetically encoded small mols. (secondary metabolites) play eminent roles in ecol. interactions, as pathogenicity factors and as drug leads. Yet, these chem. mediators often evade detection, and the discovery of novel entities is hampered by low prodn. and high rediscovery rates. These limitations may be addressed by genome mining for biosynthetic gene clusters, thereby unveiling cryptic metabolic potential. The development of sophisticated data mining methods and genetic and anal. tools has enabled the discovery of an impressive array of previously overlooked natural products. This review shows the newest developments in the field, highlighting compd. discovery from unconventional sources and microbiomes.
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    29. 29
      Hon, J.; Borko, S.; Stourac, J.; Prokop, Z.; Zendulka, J.; Bednar, D.; Martinek, T.; Damborský, J. EnzymeMiner: Automated Mining of Soluble Enzymes with Diverse Structures, Catalytic Properties and Stabilities. Nucleic Acids Res. 2020, 48 (W1), W104– W109,  DOI: 10.1093/nar/gkaa372
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      EnzymeMiner: automated mining of soluble enzymes with diverse structures, catalytic properties and stabilities
      Hon, Jiri; Borko, Simeon; Stourac, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Bednar, David; Martinek, Tomas; Damborsky, Jiri
      Nucleic Acids Research (2020), 48 (W1), W104-W109CODEN: NARHAD; ISSN:1362-4962. (Oxford University Press)
      Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Despite genomic databases growing exponentially, classical biochem. characterization techniques are time-demanding, cost-ineffective and low-throughput. Therefore, computational methods are being developed to explore the unmapped sequence space efficiently. Selection of putative enzymes for biochem. characterization based on rational and robust anal. of all available sequences remains an unsolved problem. To address this challenge, we have developed EnzymeMiner-a web server for automated screening and annotation of diverse family members that enables selection of hits for wet-lab expts. EnzymeMiner prioritizes sequences that are more likely to preserve the catalytic activity and are heterologously expressible in a sol. form in Escherichia coli. The soly. prediction employs the inhouse SoluProt predictor developed using machine learning. EnzymeMiner reduces the time devoted to data gathering, multi-step anal., sequence prioritization and selection from days to hours. The successful use case for the haloalkane dehalogenase family is described in a comprehensive tutorial available on the EnzymeMiner web page.
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    30. 30
      Vanacek, P.; Sebestova, E.; Babkova, P.; Bidmanova, S.; Daniel, L.; Dvořák, P.; Stepankova, V.; Chaloupkova, R.; Brezovsky, J.; Prokop, Z.; Damborský, J. Exploration of Enzyme Diversity by Integrating Bioinformatics with Expression Analysis and Biochemical Characterization. ACS Catal. 2018, 8 (3), 2402– 2412,  DOI: 10.1021/acscatal.7b03523
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      Exploration of enzyme diversity by integrating bioinformatics with expression analysis and biochemical characterization
      Vanacek, Pavel; Sebestova, Eva; Babkova, Petra; Bidmanova, Sarka; Daniel, Lukas; Dvorak, Pavel; Stepankova, Veronika; Chaloupkova, Radka; Brezovsky, Jan; Prokop, Zbynek; Damborsky, Jiri
      ACS Catalysis (2018), 8 (3), 2402-2412CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      Millions of protein sequences are being discovered at an incredible pace, representing an inexhaustible source of biocatalysts. Here, we describe an integrated system for automated in silico screening and systematic characterization of diverse family members. The workflow consists of (i) identification and computational characterization of relevant genes by sequence/structural bioinformatics, (ii) expression anal. and activity screening of selected proteins, and (iii) complete biochem./biophys. characterization and was validated against the haloalkane dehalogenase family. The sequence-based search identified 658 potential dehalogenases. The subsequent structural bioinformatics prioritized and selected 20 candidates for exploration of protein functional diversity. Out of these 20, the expression anal. and the robotic screening of enzymic activity provided 8 sol. proteins with dehalogenase activity. The enzymes discovered originated from genetically unrelated Bacteria, Eukaryota, and also Archaea. Overall, the integrated system provided biocatalysts with broad catalytic diversity showing unique substrate specificity profiles, covering a wide range of optimal operational temp. from 20 to 70 °C and an unusually broad pH range from 5.7 to 10. We obtained the most catalytically proficient native haloalkane dehalogenase enzyme to date (kcat/K0.5 = 96.8 mM-1s-1), the most thermostable enzyme with melting temp. 71 °C, three different cold-adapted enzymes showing dehalogenase activity at near-to-zero temps., and a biocatalyst degrading the warfare chem. sulfur mustard. The established strategy can be adapted to other enzyme families for exploration of their biocatalytic diversity in a large sequence space continuously growing due to the use of next-generation sequencing technologies.
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      Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. Mega X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35 (6), 1547– 1549,  DOI: 10.1093/molbev/msy096
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      MEGA X: molecular evolutionary genetics analysis across computing platforms
      Kumar, Sudhir; Stecher, Glen; Li, Michael; Knyaz, Christina; Tamura, Koichiro
      Molecular Biology and Evolution (2018), 35 (6), 1547-1549CODEN: MBEVEO; ISSN:1537-1719. (Oxford University Press)
      The Mol. Evolutionary Genetics Anal. (Mega) software implements many anal. methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has addnl. been upgraded to use multiple computing cores for many mol. evolutionary analyses.
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    32. 32
      Huang, F.; Haydock, S. F.; Spiteller, D.; Mironenko, T.; Li, T. L.; O’Hagan, D.; Leadlay, P. F.; Spencer, J. B. The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A. Chem. Biol. 2006, 13 (5), 475– 484,  DOI: 10.1016/j.chembiol.2006.02.014
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      The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A
      Huang, Fanglu; Haydock, Stephen F.; Spiteller, Dieter; Mironenko, Tatiana; Li, Tsung-Lin; O'Hagan, David; Leadlay, Peter F.; Spencer, Jonathan B.
      Chemistry & Biology (Cambridge, MA, United States) (2006), 13 (5), 475-484CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      A genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the prodn. of fluorine-contg. metabolites. In addn. to the previously described fluorinase FlA which catalyzes the formation of 5'-fluoro-5'-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was detd. to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5'-fluoro-5'-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to.
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    33. 33
      McMurry, J. L.; Chang, M. C. Y. Fluorothreonyl-tRNA Deacylase Prevents Mistranslation in the Organofluorine Producer Streptomyces cattleya. Proc. Natl. Acad. Sci. U.S.A. 2017, 114 (45), 11920– 11925,  DOI: 10.1073/pnas.1711482114
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      Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya
      McMurry, Jonathan L.; Chang, Michelle C. Y.
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (45), 11920-11925CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Fluorine is an element with unusual properties that has found significant utility in the design of synthetic small mols., ranging from therapeutics to materials. In contrast, only a few fluorinated compds. made by living organisms have been found to date, most of which derive from the fluoroacetate/fluorothreonine biosynthetic pathway first discovered in Streptomyces cattleya. While fluoroacetate has long been known to act as an inhibitor of the tricarboxylic acid cycle, the fate of the amino acid fluorothreonine is still not well understood. Here, we show that fluorothreonine can be misincorporated into protein in place of the proteinogenic amino acid threonine. We have identified two conserved proteins from the organofluorine biosynthetic locus, FthB and FthC, that are involved in managing fluorothreonine toxicity. Using a combination of biochem., genetic, physiol., and proteomic studies, we show that FthB is a trans-acting tRNA editing protein, which hydrolyzes fluorothreonyl-tRNA 670-fold more efficiently than threonyl-RNA, and assign a role to FthC in fluorothreonine transport. While trans-acting tRNA editing proteins have been found to counteract the misacylation of tRNA with commonly occurring near-cognate amino acids, their role has yet to be described in the context of secondary metab. In this regard, the recruitment of tRNA editing proteins to biosynthetic clusters may have enabled the evolution of pathways to produce specialized amino acids, thereby increasing the diversity of natural product structure while also attenuating the risk of mistranslation that would ensue.
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    34. 34
      Ma, L.; Bartholomé, A.; Tong, M. H.; Qin, Z.; Yu, Y.; Shepherd, T.; Kyeremeh, K.; Deng, H.; O’Hagan, D. Identification of a Fluorometabolite from Streptomyces sp. MA37: (2R3S4S)-5-Fluoro-2,3,4-Trihydroxypentanoic Acid. Chem. Sci. 2015, 6, 1414,  DOI: 10.1039/C4SC03540B
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      Identification of a fluorometabolite from Streptomyces sp. MA37: (2R3S4S)-5-fluoro-2,3,4-trihydroxypentanoic acid
      Ma, Long; Bartholome, Axel; Tong, Ming Him; Qin, Zhiwei; Yu, Yi; Shepherd, Thomas; Kyeremeh, Kwaku; Deng, Hai; O'Hagan, David
      Chemical Science (2015), 6 (2), 1414-1419CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      (2R3S4S)-5-Fluoro-2,3,4-trihydroxypentanoic acid (5-FHPA) has been discovered as a new fluorometabolite in the soil bacterium Streptomyces sp. MA37. Exogenous addn. of 5-fluoro-5-deoxy-D-ribose (5-FDR) into the cell free ext. of MA37 demonstrated that 5-FDR was an intermediate to a range of unidentified fluorometabolites, distinct from fluoroacetate (FAc) and 4-fluorothreonine (4-FT). Bioinformatics anal. allowed identification of a gene cluster (fdr), encoding a pathway to the biosynthesis of 5-FHPA. Over-expression and in vitro assay of FdrC indicated that FdrC is a NAD+ dependent dehydrogenase responsible for oxidn. of 5-FDR into 5-fluoro-5-deoxy-lactone, followed by hydrolysis to 5-FHPA. The identity of 5-FHPA in the fermn. broth was confirmed by synthesis of a ref. compd. and then co-correlation by 19F-NMR and GC-MS anal. The occurrence of 5-FHPA proves the existence of a new fluorometabolite pathway.
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    35. 35
      Eustáquio, A. S.; McGlinchey, R. P.; Liu, Y.; Hazzard, C.; Beer, L. L.; Florova, G.; Alhamadsheh, M. M.; Lechner, A.; Kale, A. J.; Kobayashi, Y.; Reynolds, K. A.; Moore, B. S. Biosynthesis of the Salinosporamide A Polyketide Synthase Substrate Chloroethylmalonyl-Coenzyme A from S-Adenosyl-L-Methionine. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (30), 12295– 12300,  DOI: 10.1073/pnas.0901237106
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      35
      Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine
      Eustaquio Alessandra S; McGlinchey Ryan P; Liu Yuan; Hazzard Christopher; Beer Laura L; Florova Galina; Alhamadsheh Mamoun M; Lechner Anna; Kale Andrew J; Kobayashi Yoshihisa; Reynolds Kevin A; Moore Bradley S
      Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (30), 12295-300 ISSN:.
      Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.
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    36. 36
      Zhao, C.; Li, P.; Deng, Z.; Ou, H. Y.; McGlinchey, R. P.; O’Hagan, D. Insights into Fluorometabolite Biosynthesis in Streptomyces cattleya DSM46488 through Genome Sequence and Knockout Mutants. Bioorg. Chem. 2012, 44, 1– 7,  DOI: 10.1016/j.bioorg.2012.06.002
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      36
      Insights into fluorometabolite biosynthesis in Streptomyces cattleya DSM46488 through genome sequence and knockout mutants
      Zhao, Chunhua; Li, Peng; Deng, Zixin; Ou, Hong-Yu; McGlinchey, Ryan P.; O'Hagan, David
      Bioorganic Chemistry (2012), 44 (), 1-7CODEN: BOCMBM; ISSN:0045-2068. (Elsevier B.V.)
      Streptomyces cattleya DSM 46488 is unusual in its ability to biosynthesize fluorine contg. natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S-adenosyl-L-methionine:fluoride adenosyltransferase (fluorinase, SCATT_41540), 5'-fluoro-5'-deoxyadenosine phosphorylase (SCATT_41550), fluoroacetyl-CoA thioesterase (SCATT_41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT_20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT_p11780) confirm that they are essential for fluorometabolite prodn. Notably gene disruption of the transaldolase (SCATT_p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesize 4-fluorothreonine, revealing a branch-point role for the PLP-transaldolase. Sequence data are deposited in GenBank/EMBL/DDBJ with accession nos. CP003219 and CP003229.
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    37. 37
      Boël, G.; Letso, R.; Neely, H.; Price, W. N.; Wong, K. H.; Su, M.; Luff, J.; Valecha, M.; Everett, J. K.; Acton, T. B.; Xiao, R.; Montelione, G. T.; Aalberts, D. P.; Hunt, J. F. Codon Influence on Protein Expression in E. coli Correlates with mRNA Levels. Nature 2016, 529 (7586), 358– 363,  DOI: 10.1038/nature16509
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      Codon influence on protein expression in E. coli correlates with mRNA levels
      Boel, Gregory; Letso, Reka; Neely, Helen; Price, W. Nicholson; Wong, Kam-Ho; Su, Min; Luff, Jon D.; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.
      Nature (London, United Kingdom) (2016), 529 (7586), 358-363CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 expts. using bacteriophage T7 polymerase to synthesize mRNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiol. protein concns. and also mRNA concns. and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ∼16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degrdn. that is a central feature of the physiol. and also possibly the regulation of translation in E. coli.
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    38. 38
      Kittilä, T.; Calero, P.; Fredslund, F.; Lowe, P. T.; Tezé, D.; Nieto-Domínguez, M.; O’Hagan, D.; Nikel, P. I.; Welner, D. H. Oligomerization Engineering of the Fluorinase Enzyme Leads to an Active Trimer That Supports Synthesis of Fluorometabolites in vitro. Microb. Biotechnol. 2022, 15 (5), 1622– 1632,  DOI: 10.1111/1751-7915.14009
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      38
      Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro
      Kittila, Tiia; Calero, Patricia; Fredslund, Folmer; Lowe, Phillip T.; Teze, David; Nieto-Dominguez, Manuel; O'Hagan, David; Nikel, Pablo I.; Welner, Ditte H.
      Microbial Biotechnology (2022), 15 (5), 1622-1632CODEN: MBIIB2; ISSN:1751-7915. (Wiley-Blackwell)
      Summary : The fluorinase enzyme represents the only biol. mechanism capable of forming stable C-F bonds characterized in nature thus far, offering a biotechnol. route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis const. (KM) for S-adenosyl-L-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-L-methionine affinity points to a long-range effect of hexamerization on substrate binding - likely decreasing substrate dissocn. and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.
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    39. 39
      Wirth, N. T.; Nikel, P. I. Combinatorial Pathway Balancing Provides Biosynthetic Access to 2-Fluoro-cis,cis-Muconate in Engineered Pseudomonas putida. Chem. Catal. 2021, 1 (6), 1234– 1259,  DOI: 10.1016/j.checat.2021.09.002
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      39
      Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida
      Wirth Nicolas T; Nikel Pablo I
      Chem catalysis (2021), 1 (6), 1234-1259 ISSN:.
      The wealth of bio-based building blocks produced by engineered microorganisms seldom include halogen atoms. Muconate is a platform chemical with a number of industrial applications that could be broadened by introducing fluorine atoms to tune its physicochemical properties. The soil bacterium Pseudomonas putida naturally assimilates benzoate via the ortho-cleavage pathway with cis,cis-muconate as intermediate. Here, we harnessed the native enzymatic machinery (encoded within the ben and cat gene clusters) to provide catalytic access to 2-fluoro-cis,cis-muconate (2-FMA) from fluorinated benzoates. The reactions in this pathway are highly imbalanced, leading to accumulation of toxic intermediates and limited substrate conversion. By disentangling regulatory patterns of ben and cat in response to fluorinated effectors, metabolic activities were adjusted to favor 2-FMA biosynthesis. After implementing this combinatorial approach, engineered P. putida converted 3-fluorobenzoate to 2-FMA at the maximum theoretical yield. Hence, this study illustrates how synthetic biology can expand the diversity of nature's biochemical catalysis.
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    40. 40
      Nikel, P. I.; de Lorenzo, V. Pseudomonas putida as a Functional chassis for Industrial Biocatalysis: From Native Biochemistry to Trans-Metabolism. Metab. Eng. 2018, 50, 142– 155,  DOI: 10.1016/j.ymben.2018.05.005
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      40
      Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism
      Nikel, Pablo I.; de Lorenzo, Victor
      Metabolic Engineering (2018), 50 (), 142-155CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
      The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochem. stresses. Over the years, these properties have been capitalized biotechnol. owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biol. have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biol. systems. Much of the biotechnol. interest on P. putida stems from the distinct architecture of its central carbon metab. The native biochem. is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochem. network of P. putida (cis-metab.) has sufficed to obtain target compds. of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metab.) for making complex, new-to-Nature mols. A no. of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufg. of fine and bulk chems. in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochem. beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chem. elements into the extant biochem., as well as genomic stability and scaling-up issues.
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      Volke, D. C.; Calero, P.; Nikel, P. I. Pseudomonas putida. Trends Microbiol. 2020, 28 (6), 512– 513,  DOI: 10.1016/j.tim.2020.02.015
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      41
      Pseudomonas putida
      Volke, Daniel C.; Calero, Patricia; Nikel, Pablo I.
      Trends in Microbiology (2020), 28 (6), 512-513CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
      There is no expanded citation for this reference.
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    42. 42
      Sánchez-Pascuala, A.; Fernández-Cabezón, L.; de Lorenzo, V.; Nikel, P. I. Functional Implementation of a Linear Glycolysis for Sugar Catabolism in Pseudomonas putida. Metab. Eng. 2019, 54, 200– 211,  DOI: 10.1016/j.ymben.2019.04.005
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      42
      Functional implementation of a linear glycolysis for sugar catabolism in Pseudomonas putida
      Sanchez-Pascuala, Alberto; Fernandez-Cabezon, Lorena; de Lorenzo, Victor; Nikel, Pablo I.
      Metabolic Engineering (2019), 54 (), 200-211CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)
      The core metab. for glucose assimilation of the soil bacterium and platform strain Pseudomonas putida KT2440 has been reshaped from the native, cyclically-operating Entner-Doudoroff (ED) pathway to a linear Embden-Meyerhof-Parnas (EMP) glycolysis. The genetic strategy deployed to obtain a suitable host for the synthetic EMP route involved not only eliminating enzymic activities of the ED pathway, but also erasing peripheral reactions for glucose oxidn. that divert carbon skeletons into the formation of org. acids in the periplasm. Heterologous glycolytic enzymes, recruited from Escherichia coli, were genetically knocked-in in the mutant strain to fill the metabolic gaps for the complete metab. of glucose to pyruvate through a synthetic EMP route. A suite of genetic, physiol., and biochem. tests in the thereby-refactored P. putida strain-which grew on glucose as the sole carbon and energy source-demonstrated the functional replacement of the native sugar metab. by a synthetic catabolism. 13C-labeling expts. indicated that the bulk of pyruvate in the resulting strain was generated through the metabolic device grafted in P. putida. Strains carrying the synthetic glycolysis were further engineered for carotenoid synthesis from glucose, indicating that the implanted EMP route enabled higher carotenoid content on biomass and yield on sugar as compared with strains running the native hexose catabolism. Taken together, our results highlight how conserved metabolic features in a platform bacterium can be rationally reshaped for enhancing physiol. traits of interest.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXosVCisrc%253D&md5=0b08372afa4ebea65ca8b9804db5ba33
    43. 43
      Bitzenhofer, N. L.; Kruse, L.; Thies, S.; Wynands, B.; Lechtenberg, T.; Rönitz, J.; Kozaeva, E.; Wirth, N. T.; Eberlein, C.; Jaeger, K. E.; Nikel, P. I.; Heipieper, H. J.; Wierckx, N.; Loeschcke, A. Towards Robust Pseudomonas Cell Factories to Harbour Novel Biosynthetic Pathways. Essays Biochem. 2021, 65 (2), 319– 336,  DOI: 10.1042/EBC20200173
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      43
      Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways
      Bitzenhofer, Nora Lisa; Kruse, Luzie; Thies, Stephan; Wynands, Benedikt; Lechtenberg, Thorsten; Roenitz, Jakob; Kozaeva, Ekaterina; Wirth, Nicolas Thilo; Eberlein, Christian; Jaeger, Karl-Erich; Nikel, Pablo Ivan; Heipieper, Hermann J.; Wierckx, Nick; Loeschcke, Anita
      Essays in Biochemistry (2021), 65 (2), 319-336CODEN: ESBIAV; ISSN:1744-1358. (Portland Press Ltd.)
      Biotechnol. prodn. in bacteria enables access to numerous valuable chem. compds. Nowadays, advanced mol. genetic toolsets, enzyme engineering as well as the combinatorial use of biocatalysts, pathways, and circuits even bring new-to-nature compds. within reach. However, the assocd. substrates and biosynthetic products often cause severe chem. stress to the bacterial hosts. Species of the Pseudomonas clade thus represent esp. valuable chassis as they are endowed with multiple stress response mechanisms, which allow them to cope with a variety of harmful chems. A built-in cell envelope stress response enables fast adaptations that sustain membrane integrity under adverse conditions. Further, effective export machineries can prevent intracellular accumulation of diverse harmful compds. Finally, toxic chems. such as reactive aldehydes can be eliminated by oxidn. and stress-induced damage can be recovered. Exploiting and engineering these features will be essential to support an effective prodn. of natural compds. and new chems. In this article, we therefore discuss major resistance strategies of Pseudomonads along with approaches pursued for their targeted exploitation and engineering in a biotechnol. context. We further highlight strategies for the identification of yet unknown tolerance-assocd. genes and their utilization for engineering next-generation chassis and finally discuss effective measures for pathway fine-tuning to establish stable cell factories for the effective prodn. of natural compds. and novel biochems.
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