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Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola
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† Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa 50011 United States
‡ Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093 United States
*E-mail: [email protected]
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Journal of Natural Products

Cite this: J. Nat. Prod. 2014, 77, 9, 2144–2147
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https://doi.org/10.1021/np500422d
Published September 9, 2014

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Abstract

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While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms.

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Subjects

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The production of terpenoids is most commonly associated with plants, which have clearly expanded their ability to produce this class of natural products. For example, terpene synthases form moderate sized gene families, with more than 10 such enzymatic genes found in each of the known vascular plant genome sequences. (1) By contrast, there appear to be just over 100 terpene synthases among the more than 1000 sequenced bacterial genomes, suggesting the relative scarcity of terpene synthases and, hence, terpenoid production, among commonly studied bacterial genera. (2)
This relative paucity of bacterial terpenoid production is also illustrated by the labdane-related diterpenoids, a large superfamily of ∼7000 known natural products whose biosynthesis is characterized by the initiating reaction. Specifically, acid–base catalyzed bicyclization of the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP, 1), which is mediated by class II diterpene cyclases that generally form the eponymous labdadienyl/copalyl diphosphate (CPP). This is typically followed by an additional cyclization and/or rearrangement reaction initiated by ionization of the allylic diphosphate catalyzed by a class I diterpene synthase. (3) All vascular plants have at least one class II diterpene cyclase in order to produce the requisite gibberellin phytohormones, and many plant species have multiple such enzymes. (4) However, less than 100 class II diterpene cyclases are present in the known bacterial genomes (5) and less than 10 have been characterized. (6-12)
Nevertheless, there are a handful of bacteria that produce labdane-related diterpenoids of significant interest. The relevant class II diterpene cyclases have been identified for a number of these bacterial natural products, including such enzymes involved in the production of gibberellin phytohormones by plant symbiotic rhizobia and phytopathogens, (9, 11, 12) the potential antibacterial and antidiabetic compounds platencin and platensimycin by Streptomyces platensis, (10) and an immuno-modulatory factor by Mycobacterium tuberculosis. (8) Intriguingly, it has been reported that an epiphytic marine bacterium produces a labdane-related diterpenoid that acts at subpicomolar levels to promote the aggregation of marine green macroalgae (e.g., sea lettuce). (13)
With the advent of next generation sequencing, there has been a tremendous increase in the availability of microbial genome sequences, (14) which has generally revealed that there are many more putative natural product biosynthetic gene clusters than known metabolites. (15) A variety of approaches have been taken toward identifying the compounds resulting from such orphan operons. (16) Here is reported the use of recombinant expression to characterize a bioinformatics-predicted labdane-related diterpenoid biosynthetic operon from the marine bacterium Salinispora arenicola CNS-205.
The genome sequence of the CNS-205 strain of the widely distributed marine actinobacterium S. arenicola was previously reported, with bioinformatic analysis indicating the presence of 30 putative natural product biosynthetic gene clusters. (17) Included among these was terp1, annotated as producing an unidentified diterpene. This small operon contains genes encoding a putative class II diterpene cyclase (Sare_1288) and class I (di)terpene synthase (Sare_1287) as well as a cytochrome P450 (CYP) mono-oxygenase (Sare_1286) that has been assigned as CYP1051A1 (Figure 1). Thus, the terp1 operon is predicted to encode for the production of a labdane-related diterpenoid, although this appears to be unique to the originally sequenced CNS-205 strain, (17) as similar sequences do not seem to be present in the other 36 strains whose genome sequences have recently been reported. (18)

Figure 1

Figure 1. Schematic of the terp1 diterpenoid biosynthetic operon from S. arenicola CNS-205.

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The putative class II diterpene cyclase is most closely related to two previously characterized ent-CPP synthases (CPSs) from Streptomyces species of terrestrial actinobacteria (42–45% amino acid sequence identity) (7, 10) and is referred to henceforth as SaCPS. While class II diterpene cyclases almost invariably contain a DxDD motif that cooperatively acts as the catalytic acid, (19) SaCPS contains a Thr in place of the last Asp (Supporting Information (SI), Figure S1). Such a variant DxDT motif has been previously noted in the active class II diterpene cyclase from M. tuberculosis, (8) suggesting that SaCPS might be functional.
Similarly, the putative class I diterpene synthase is most closely related to a previously characterized ent-pimaradiene synthase from Streptomyces sp. KO-3988 (∼30% amino acid sequence identity) (20) and is referred to henceforth as SaDTS. Although class I terpene synthases almost invariably contain a DDxxD motif involved in binding the requisite divalent magnesium ion cofactors, (21) SaDTS does not contain a corresponding sequence. Amino acid sequence alignments indicate that the corresponding sequence in SaDTS is EDWQVD83–88 instead (SI, Figure S2). While the ent-kaurene synthase from S. platensis also does not have the canonical DDxxD motif at this position, sequence conforming to the DDxxD motif can be found nearby, (10) which is not true in SaDTS. On the other hand, SaDTS does contain the NDxxSxxxE motif also involved in binding the requisite divalent magnesium ion co-factors, (21) leaving open the possibility that SaDTS may be active as well.
Characterization of the bioinformatics-predicted labdane-related diterpenoid product of this operon was undertaken with a previously developed modular metabolic engineering system. (22) This enabled recombinant expression of SaCPS with a GGPP synthase (GGPS) in Escherichia coli, which led to the production of CPP, observed as the dephosphorylated copalol by GC-MS analysis of hexane extracts of the induced culture (Figure 2A). To determine the absolute configuration of this CPS, SaCPS was co-expressed with GGPS and selective class I diterpene synthases, much as previously described for investigation of other class II diterpene cyclases. Briefly, SaCPS and the GGPS were co-expressed with either the ent-kaurene synthase from Arabidopsis thaliana (AtKS) that selectively reacts with ent-CPP, (9) or a mutant of the abietadiene synthase from Abies grandis (AgAS) that no longer exhibits class II activity and only reacts with normal CPP (AgAS:D404A). (23) No ent-kaurene was observed upon co-expression of SaCPS (and GGPS) with AtKS, while the same products made by wild-type AgAS were readily observed upon co-expression with AgAS:D440A (SI, Figure S3). Thus, SaCPS makes normal (5S,9S,10S) CPP (2).

Figure 2

Figure 2. GC-MS extracted ion (m/z = 257) chromatograms and associated mass spectra for (A) production of CPP (2), detected as dephosphorylated copalol (2′; retention time, RT = 17.16 min), from expression of SaCPS in a strain of E. coli engineered to make GGPP (1) by SaCPS. (B) Isopimara-8,15-diene (3; RT = 15.27 min) from expression of SaDTS in a strain engineered to make 2. (C) Isopimara-8,15-dien-19-ol (4; RT = 17.04 min) from co-expression of CYP1051A1 with an Fdx and Fdr in a strain engineered to make 3.

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Co-expression of SaDTS with GGPS and SaCPS led to production of an unknown diterpene, observed by GC-MS analysis of hexane extracts of the induced culture. To determine the structure of this compound, SaDTS was co-expressed with GGPS and a plant CPS in E. coli (Figure 2B), which produces larger quantities of 2 (i.e., than SaCPS), and the culture volume was increased to 2 L. This enabled isolation of ∼2 mg for NMR analysis (SI, Figures S4–S6 and Table S1), which indicated that this was a (iso)pimara-8,15-diene, with resolution of the configuration at C-13 derived from comparison to previously reported NMR chemical shift data, (24, 25) which led to assignment of the SaDTS product as isopimara-8,15-diene (3).
To functionally characterize CYP1051A1, it was necessary to account for the fact that CYPs require the input of electrons, generally obtained from NADPH, and in the case of bacterial CYPs typically provided by a ferredoxin (Fdx) that has been reduced by a ferredoxin reductase (FdR). (26) Accordingly, CYP1051A1 was co-expressed with an Fdx and FdR from S. arenicola (Sare_4141 and Sare_0646, respectively) in a strain of E. coli also engineered to produce 3. The resulting recombinant strain produced a hydroxylated derivative of 3, observed by GC-MS analysis of an organic solvent extract (Figure 2C). Attempts to scale up production of the CYP1051A1 product were not fruitful, with a net yield of only ∼100 μg from 5 L of culture. Fortuitously, it was discovered that CYP99A3 from rice (27) catalyzed the same reaction (SI, Figure S7) and was more amendable to scale-up. Thus, CYP99A3 was used to produce enough compound (∼1 mg), which was mixed with the ∼0.1 mg isolated from CYP1051A1 for NMR analysis (SI, Figures S4, S8, and S9 and Table S2). Only a single peak was found in the alcohol region of the 13C spectra (SI, Figure S9), consistent with equivalence of the CYP99A3 and CYP1051A1 products, which the collected data indicated was isopimara-8,15-dien-19-ol (4). The configuration at C-4 was suggested by the observation of an NOE signal between the secondary alcohol hydrogens on C-19 and the methyl hydrogens of C-20, whose configuration was already known (vide supra) and verified by comparison to previously reported NMR chemical shift data. (28) Thus, the S. arenicolaterp1 operon presumably can lead to the production of isopimara-8,15-dien-19-ol (Scheme 1).

Scheme 1

Scheme 1. Labdane-Related Diterpenoid Biosynthetic Pathway Encoded by S. arenicola CNS-205 terp1 Operon
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It has been previously reported that at least two species of Streptomyces grown in certain media produce the diterpenoid viguiepinol (3α-hydroxy-ent-pimara-9(11),15-diene) and derived oxaloterpins, which are very similar to 4. (29, 30) To determine whether terp1 pathway metabolites were produced by S. arenicola CNS-205, this actinomycete was fermented in four different media (SI), and chemical extracts were evaluated by GC-MS with selected ion monitoring for 3 and 4. None of the evaluated fermentation conditions afforded production of 3 or 4 at levels detectable by GC-MS. Chemical profiles were also compared among wild type S. arenicola CNS-205 and PCR-targeted terp1 gene replacement mutants to facilitate the detection of terp1 operon-derived metabolites that are modified by additional enzymes in vivo. No differences in GC-MS or LC-MS metabolite profiles were noted for extracts from wild type S. arenicola CNS-205, a sare_1286::AprR mutant lacking the CYP1051A1 cytochrome P450 enzyme and a sare_1287::AprR mutant lacking the DTS class I (di)terpene synthase enzyme. These data suggest that the terp1 gene cluster is either inactive in S. arenicola CNS-205 under evaluated conditions or that diterpenoid titers are below the limit of detection.
In conclusion, functional characterization of the orphan terp1 biosynthetic operon from S. arenicola is reported here. While the terp1 operon is only found in strain CNS-205, these results further illuminate the natural products capacity of these widely distributed marine actinobacteria. Although the resulting diterpenoid 4 cannot be found in pure cultures of S. arenicola CNS-205 grown in a variety of media, it is possible that 4 serves an ecological function and is only produced by S. arenicola CNS-205 under specific environmental conditions (e.g., in the presence of other organisms). Alternatively, it has been previously noted that all other characterized bacterial diterpenoid biosynthetic operons contain a GGPS as bacteria do not generally produce 1 otherwise, (7, 10, 12, 31-36) and the lack of a GGPS in the S. arenicola CNS-205 terp1 operon may underlie the observed lack of production of 4. In any case, these results demonstrate the applicability of the utilized modular metabolic engineering system to elucidation of (di)terpenoid biosynthetic operons uncovered by genome mining of actino- and potentially other bacteria as well.

Experimental Section

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General Experimental Procedures

NMR spectroscopic data were recorded on a Bruker Avance 500 spectrometer equipped with cryogenic probe for 1H and 13C measurements (Bruker). GC-MS analyses were carried out using a Varian 3900 GC with Saturn 2100 ion trap mass spectrometer equipped with HP-5 ms capillary column (30 m × 0.25 mm × 0.25 μm) in electron ionization (70 eV) mode (Agilent/Varian). Column chromatography was performed with 80 × 200 mesh silica gel (Fisher Scientific). HPLC was carried out with a ZORBAX Eclipse XDB-C8 column (4.6 mm × 150 mm, 5 μm) on an Agilent 1100 series system equipped with fraction collector and diode array detector. Solvents for chromatographic separations were purchased from Fisher Scientific.

Methods Summary

The genes from S. arenicola described herein were cloned from the fosmids used in its genome sequencing project. (17) These were cloned into the Gateway vector system (Invitrogen) to enable their use in the metabolic engineering system, including co-expression with previously characterized enzymes (i.e., to investigate configurations or increase yield). Enzymatic products were analyzed by GC-MS of organic extracts from the relevant recombinant cultures. Where necessary (i.e., for 3 and 4), these cultures were scaled up to enable production and purification of larger quantities for structural characterization by NMR.

Isopimara-8,15-diene (3)

Previously described as a colorless solid; (37, 38)1H and 13C NMR, as well as MS, data largely match literature values, (24, 25, 39) with the few significant differences supported here by HMBC correlations (SI, Table S1).

Isopimara-8,15-dien-19-ol (4)

Previously described as a colorless solid; (38)1H and 13C NMR data largely match literature values, (28) again with the few significant differences supported here by HMBC correlations (SI, Table S2).

Supporting Information

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Detailed description of experimental methods and characterization of the various compounds, along with supplemental figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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  • Corresponding Author
    • Reuben J. Peters - Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa 50011 United States;  Email: [email protected]
  • Authors
    • Meimei Xu - Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa 50011 United States
    • Matthew L. Hillwig - Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa 50011 United States
    • Amy L. Lane - Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093 United States
    • Mollie S. Tiernan - Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa 50011 United States
    • Bradley S. Moore - Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093 United States
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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This work was supported by funds from the NIH, an IRACDA postdoctoral fellowship to A.L.L. (GM068524) and research grants to B.S.M. (GM085770) and R.J.P. (GM076324).

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Cite this: J. Nat. Prod. 2014, 77, 9, 2144–2147
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  • Abstract

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    Figure 1

    Figure 1. Schematic of the terp1 diterpenoid biosynthetic operon from S. arenicola CNS-205.

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    Figure 2

    Figure 2. GC-MS extracted ion (m/z = 257) chromatograms and associated mass spectra for (A) production of CPP (2), detected as dephosphorylated copalol (2′; retention time, RT = 17.16 min), from expression of SaCPS in a strain of E. coli engineered to make GGPP (1) by SaCPS. (B) Isopimara-8,15-diene (3; RT = 15.27 min) from expression of SaDTS in a strain engineered to make 2. (C) Isopimara-8,15-dien-19-ol (4; RT = 17.04 min) from co-expression of CYP1051A1 with an Fdx and Fdr in a strain engineered to make 3.

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    Scheme 1

    Scheme 1. Labdane-Related Diterpenoid Biosynthetic Pathway Encoded by S. arenicola CNS-205 terp1 Operon
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  • References

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      Natural Product Reports (2010), 27 (11), 1521-1530CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. Unlike the majority of terpenoids, a significant fraction of the polycyclic diterpenoids (∼7000 already known) are now understood to originate from dual, rather than single, biosynthetic cyclization and/or rearrangement reactions, which proceed via a bicyclic diphosphate intermediate. The trivial name for the hydrocarbon skeleton of the most commonly found version of this biosynthetic intermediate forms the basis for a unifying "labdane-related" designation for this large super-family of natural products. Notably, many of these are found in plants, where the requisite biosynthetic machinery for gibberellin phytohormones, particularly the relevant diterpene cyclases, provides a biosynthetic reservoir that appears to have been repeatedly drawn upon to evolve new labdane-related diterpenoids. The potent biol. activity of the "ancestral" gibberellins, which has led to the independent evolution of distinct gibberellin biosynthetic pathways in plants, fungi, and bacteria, is further discussed as an archetypical example of the selective pressure driving evolution of the large super-family of labdane-related diterpenoid natural products, with the obsd. diversification suggesting that their underlying hydrocarbon skeletal structures might serve as privileged scaffolds from which biol. activity is readily derived.
    4. 4
      Zi, J.; Mafu, S.; Peters, R. J. Annu. Rev. Plant Biol. 2014, 65, 259– 286
      4
      To gibberellins and beyond! Surveying the evolution of (Di)terpenoid metabolism
      Zi, Jiachen; Mafu, Sibongile; Peters, Reuben J.
      Annual Review of Plant Biology (2014), 65 (), 259-286CODEN: ARPBDW; ISSN:1543-5008. (Annual Reviews)
      A review. The diterpenoids are classically defined by their compn.-four isoprenyl units (20 carbons)-and are generally derived from [E,E,E]-geranylgeranyl diphosphate (GGPP). Such metab. seems to be ancient and has been extensively diversified, with ∼12,000 diterpenoid natural products known. Particularly notable are the gibberellin phytohormones, whose requisite biosynthesis has provided a genetic reservoir that gave rise to not only a large superfamily of ∼7000 diterpenoids but also, to some degree, all plant terpenoid natural products. This review focuses on the diterpenoids, particularly the defining biosynthetic characteristics of the major superfamilies defined by the cyclization and/or rearrangement of GGPP catalyzed by diterpene synthases/cyclases, although it also includes some discussion of the important subsequent elaboration in the few cases where sufficient mol. genetic information is available. It addnl. addresses the array of biol. activity providing the selective pressures that drive the obsd. gene family expansion and diversification, along with biosynthetic gene clustering.
    5. 5
      Mann, F. M.; Peters, R. J. Med. Chem. Commun. 2012, 3, 899– 904
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    6. 6
      Dairi, T.; Hamano, Y.; Kuzuyama, Y.; Itoh, N.; Furihata, K.; Seto, H. J. Bacteriol. 2001, 183, 6085– 6092
      6
      Eubacterial diterpene cyclase genes essential for production of the isoprenoid antibiotic terpentecin
      Dairi, Tohru; Hamano, Yoshimitsu; Kuzuyama, Tomohisa; Itoh, Nobuya; Furihata, Kazuo; Seto, Haruo
      Journal of Bacteriology (2001), 183 (20), 6085-6094CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      A gene cluster contg. the mevalonate pathway genes (open reading frame 2 [ORF2] to ORF7) for the formation of isopentenyl diphosphate and a geranylgeranyl diphosphate (GGDP) synthase gene (ORF1) had previously been cloned from Streptomyces griseolosporeus strain MF730-N6, a diterpenoid antibiotic, terpentecin (TP) producer. Sequence anal. in the upstream region of the cluster revealed seven new ORFs, ORF8 to ORF14, which were suggested to encode TP biosynthetic genes. Two mutants were constructed, in which ORF11 and ORF12, which encode a protein showing similarities to eukaryotic diterpene cyclases (DCs) and a eubacterial pentalenene synthase, resp., were inactivated by gene disruptions. The mutants produced no TP, confirming that these cyclase genes are essential for the prodn. of TP. The two cyclase genes were also expressed in Streptomyces lividans together with the GGDP synthase gene under the control of the ermE* constitutive promoter. The transformant produced a novel cyclic diterpenoid, ent-clerod-3,13(16),14-triene (terpentetriene), which has the same basic skeleton as TP. The two enzymes, each of which was overproduced in Escherichia coli and purified to homogeneity, converted GGDP into terpentetriene. To the best of our knowledge, this is the first report of a eubacterial DC.
    7. 7
      Kawasaki, T.; Kuzuyama, T.; Kuwamori, Y.; Matsuura, N.; Itoh, N.; Furihata, K.; Seto, H.; Dairi, T. J. Antibiot. 2004, 57, 739– 747
      There is no corresponding record for this reference.
    8. 8
      Nakano, C.; Okamura, T.; Sato, T.; Dairi, T.; Hoshino, T. Chem. Commun. 2005, 2005, 1016– 1018
      There is no corresponding record for this reference.
    9. 9
      Morrone, D.; Chambers, J.; Lowry, L.; Kim, G.; Anterola, A.; Bender, K.; Peters, R. J. FEBS Lett. 2009, 583, 475– 480
      There is no corresponding record for this reference.
    10. 10
      Smanski, M. J.; Yu, Z.; Casper, J.; Lin, S.; Peterson, R. M.; Chen, Y.; Wendt-Pienkowski, E.; Rajski, S. R.; Shen, B. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 13498– 503
      There is no corresponding record for this reference.
    11. 11
      Lu, X.; Hershey, D. M.; Wang, L.; Bogdanove, A. J.; Peters, R. J. New Phytol.

      submitted for publication

      .
      There is no corresponding record for this reference.
    12. 12
      Hershey, D. M.; Lu, X.; Zi, J.; Peters, R. J. J. Bacteriol. 2014, 196, 100– 106
      12
      Functional conservation of the capacity for ent-kaurene biosynthesis and an associated operon in certain rhizobia
      Hershey, David M.; Lu, Xuan; Zi, Jiachen; Peters, Reuben J.
      Journal of Bacteriology (2014), 196 (1), 100-106, 8 pp.CODEN: JOBAAY; ISSN:1098-5530. (American Society for Microbiology)
      Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a crit. link to central metab. In addn., the rest of the operon consists of enzymic genes that presumably lead to a more elaborated diterpenoid, although the prodn. of gibberellins was not obsd. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid prodn. may modulate the interaction of these particular symbionts with their host plants.
    13. 13
      Matsuo, Y.; Imagawa, H.; Nishizawa, M.; Shizuri, Y. Science 2005, 307, 1598
      There is no corresponding record for this reference.
    14. 14
      Nett, M.; Ikeda, H.; Moore, B. S. Nat. Prod. Rep. 2009, 26, 1362– 1384
      14
      Genomic basis for natural product biosynthetic diversity in the actinomycetes
      Nett, Markus; Ikeda, Haruo; Moore, Bradley S.
      Natural Product Reports (2009), 26 (11), 1362-1384CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. Covering: up to mid-2009. The phylum Actinobacteria hosts diverse high G + C, Gram-pos. bacteria that have evolved a complex chem. language of natural product chem. to help navigate their fascinatingly varied lifestyles. To date, 71 Actinobacteria genomes have been completed and annotated, with the vast majority representing the Actinomycetales, which are the source of numerous antibiotics and other drugs from genera such as Streptomyces, Saccharopolyspora and Salinispora. These genomic analyses have illuminated the secondary metabolic proficiency of these microbes - underappreciated for years based on conventional isolation programs - and have helped set the foundation for a new natural product discovery paradigm based on genome mining. Trends in the secondary metabolomes of natural product-rich actinomycetes are highlighted in this review article, which contains 199 refs.
    15. 15
      Challis, G. L. J. Med. Chem. 2008, 51, 2618– 28
      There is no corresponding record for this reference.
    16. 16
      Corre, C.; Challis, G. L. Nat. Prod. Rep. 2009, 26, 977– 986
      16
      New natural product biosynthetic chemistry discovered by genome mining
      Corre, Christophe; Challis, Gregory L.
      Natural Product Reports (2009), 26 (8), 977-986CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. Covering: 2000 to 2008. Analyses of plant and microbial genome sequences have revealed many genes and gene clusters encoding proteins similar to those known to be involved in the biosynthesis of structurally-complex natural products. Many of these genes and gene clusters do not direct the prodn. of known metabolites of the organism in which they are found. Others represent novel gene clusters that direct the biosynthesis of known natural products. This article highlights several examples of new biosynthetic chem. discovered in the course of investigating such gene clusters.
    17. 17
      Penn, K.; Jenkins, C.; Nett, M.; Udwary, D. W.; Gontang, E. A.; McGlinchey, R. P.; Foster, B.; Lapidus, A.; Podell, S.; Allen, E. E.; Moore, B. S.; Jensen, P. R. ISME J. 2009, 3, 1193– 1203
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      Ziemert, N.; Lechner, A.; Wietz, M.; Millan-Aguinaga, N.; Chavarria, K. L.; Jensen, P. R. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, E1130– E1139
      There is no corresponding record for this reference.
    19. 19
      Prisic, S.; Xu, J.; Coates, R. M.; Peters, R. J. ChemBioChem 2007, 8, 869– 874
      There is no corresponding record for this reference.
    20. 20
      Kawasaki, T.; Hayashi, Y.; Kuzuyama, T.; Furihata, K.; Itoh, N.; Seto, H.; Dairi, T. J. Bacteriol. 2006, 188, 1236– 1244
      20
      Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster
      Kawasaki, Takashi; Hayashi, Yutaka; Kuzuyama, Tomohisa; Furihata, Kazuo; Itoh, Nobuya; Seto, Haruo; Dairi, Tohru
      Journal of Bacteriology (2006), 188 (4), 1236-1244CODEN: JOBAAY; ISSN:0021-9193. (American Society for Microbiology)
      Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compd. that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compd., we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compd. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic anal., these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).
    21. 21
      Aaron, J. A.; Christianson, D. W. Pure Appl. Chem. 2010, 82, 1585– 1597
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    22. 22
      Cyr, A.; Wilderman, P. R.; Determan, M.; Peters, R. J. J. Am. Chem. Soc. 2007, 129, 6684– 6685
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    23. 23
      Peters, R. J.; Ravn, M. M.; Coates, R. M.; Croteau, R. B. J. Am. Chem. Soc. 2001, 123, 8974– 8978
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      Wenkert, E.; Buckwalter, B. L. J. Am. Chem. Soc. 1972, 94, 4367– 4369
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      Chu, M.; Coates, R. M. J. Org. Chem. 1992, 57, 4590– 4597
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      Munro, A. W.; Girvan, H. M.; McLean, K. J. Nat. Prod. Rep. 2007, 24, 585– 609
      26
      Variations on a (t)heme - novel mechanisms, redox partners and catalytic functions in the cytochrome P450 superfamily
      Munro, Andrew W.; Girvan, Hazel M.; McLean, Kirsty J.
      Natural Product Reports (2007), 24 (3), 585-609CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)
      A review. The catalytic potential of cytochrome P 450 superfamily isoforms in org. biotransformations is discussed with emphasis on the breadth of P 450 redox systems now recognized and the catalytic versatility of these biotechnol. important enzymes.
    27. 27
      Wang, Q.; Hillwig, M. L.; Peters, R. J. Plant J. 2011, 65, 87– 95
      27
      CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice
      Wang, Qiang; Hillwig, Matthew L.; Peters, Reuben J.
      Plant Journal (2011), 65 (1), 87-95CODEN: PLJUED; ISSN:0960-7412. (Wiley-Blackwell)
      Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochems. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone prodn., located on rice chromosome 4, which contains two cytochrome P 450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochem. characterization of CYP99A2 and CYP99A3, which was ultimately achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidns. of the C19 Me group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiol. relevance for the obsd. activity of CYP99A3. In addn., we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene-derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis.
    28. 28
      Ceccherelli, P.; Curini, M.; Marcotullio, M. C. J. Chem. Soc., Perkin Trans. 1 1985, 2173– 2175
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    29. 29
      Motohashi, K.; Ueno, R.; Sue, M.; Furihata, K.; Matsumoto, T.; Dairi, T.; Omura, S.; Seto, H. J. Nat. Prod. 2007, 70, 1712– 1717
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    30. 30
      Xie, P.; Ma, M.; Rateb, M. E.; Shaaban, K. A.; Yu, Z.; Huang, S. X.; Zhao, L. X.; Zhu, X.; Yan, Y.; Peterson, R. M.; Lohman, J. R.; Yang, D.; Yin, M.; Rudolf, J. D.; Jiang, Y.; Duan, Y.; Shen, B. J. Nat. Prod. 2014, 77, 377– 387
      30
      Biosynthetic potential-based strain prioritization for natural product discovery: A showcase for diterpenoid-producing actinomycetes
      Xie, Pengfei; Ma, Ming; Rateb, Mostafa E.; Shaaban, Khaled A.; Yu, Zhiguo; Huang, Sheng-Xiong; Zhao, Li-Xing; Zhu, Xiangcheng; Yan, Yijun; Peterson, Ryan M.; Lohman, Jeremy R.; Yang, Dong; Yin, Min; Rudolf, Jeffrey D.; Jiang, Yi; Duan, Yanwen; Shen, Ben
      Journal of Natural Products (2014), 77 (2), 377-387CODEN: JNPRDF; ISSN:0163-3864. (American Chemical Society-American Society of Pharmacognosy)
      Natural products remain the best sources of drugs and drug leads and serve as outstanding small-mol. probes to dissect fundamental biol. processes. A great challenge for the natural product community is to discover novel natural products efficiently and cost effectively. Here the authors report the development of a practical method to survey biosynthetic potential in microorganisms, thereby identifying the most promising strains and prioritizing them for natural product discovery. Central to this approach is the innovative prepn., by a two-tiered PCR method, of a pool of pathway-specific probes, thereby allowing the survey of all variants of the biosynthetic machineries for the targeted class of natural products. The utility of the method was demonstrated by surveying 100 strains, randomly selected from the authors' actinomycete collection, for their biosynthetic potential of four classes of natural products, arom. polyketides, reduced polyketides, nonribosomal peptides, and diterpenoids, identifying 16 talented strains. One of the talented strains, Streptomyces griseus CB00830, was finally chosen to showcase the discovery of the targeted classes of natural products, resulting in the isolation of three diterpenoids, six nonribosomal peptides and related metabolites, and three polyketides. Variations of this method should be applicable to the discovery of other classes of natural products.
    31. 31
      Hamano, Y.; Dairi, T.; Yamamoto, M.; Kawasaki, T.; Kaneda, K.; Kuzuyama, T.; Itoh, N.; Seto, H. Biosci., Biotechnol., Biochem. 2001, 65, 1627– 1635
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      Durr, C.; Schnell, H.-J.; Luzhetskyy, A.; Murillo, R.; Weber, M.; Welzel, K.; Vente, A.; Bechthold, A. Chem. Biol. 2006, 13, 365– 377
      32
      Biosynthesis of the terpene phenalinolactone in Streptomyces sp. Tu6071: analysis of the gene cluster and generation of derivatives
      Durr Clemens; Schnell Hans-Jorg; Luzhetskyy Andriy; Murillo Renato; Weber Monika; Welzel Katrin; Vente Andreas; Bechthold Andreas
      Chemistry & biology (2006), 13 (4), 365-77 ISSN:1074-5521.
      Phenalinolactones are terpene glycosides with antibacterial activity. A striking structural feature is a highly oxidized gamma-butyrolactone of elusive biosynthetic origin. To investigate the genetic basis of the phenalinolactones biosynthesis, we cloned and sequenced the corresponding gene cluster from the producer strain Streptomyces sp. Tu6071. Spanning a 42 kbp region, 35 candidate genes could be assigned to putatively encode biosynthetic, regulatory, and resistance-conferring functions. Targeted gene inactivations were carried out to specifically manipulate the phenalinolactones pathway. The inactivation of a sugar methyltransferase gene and a cytochrome P450 monoxygenase gene led to the production of modified phenalinolactone derivatives. The inactivation of a Fe(II)/alpha-ketoglutarate-dependent dioxygenase gene disrupted the biosynthetic pathway within gamma-butyrolactone formation. The structure elucidation of the accumulating intermediate indicated that pyruvate is the biosynthetic precursor of the gamma butyrolactone moiety.
    33. 33
      Hayashi, Y.; Matsuura, N.; Toshima, H.; Itoh, N.; Ishikawa, J.; Mikami, Y.; Dairi, T. J. Antibiot. (Tokyo) 2008, 61, 164– 174
      33
      Cloning of the gene cluster responsible for the biosynthesis of brasilicardin A, a unique diterpenoid
      Hayashi Yutaka; Matsuura Nobuyasu; Toshima Hiroaki; Itoh Nobuya; Ishikawa Jun; Mikami Yuzuru; Dairi Tohru
      The Journal of antibiotics (2008), 61 (3), 164-74 ISSN:0021-8820.
      Brasilicardin A (BCA), produced by Nocardia brasiliensis IFM 0406 (currently referred to as N. terpenica), has a unique structure consisting of a diterpene skeleton with L-rhamnose, N-acetylglucosamine, amino acid, and 3-hydroxybenzoate moieties, and exhibits potent biological activities. To understand the biosynthetic machinery of this unique compound, we have cloned the corresponding gene cluster. Firstly, we cloned a gene by PCR that encodes geranylgeranyl diphosphate synthase (GGPPS), which produces a direct precursor of diterpene compounds. We obtained four candidate genes and one of the genes was confirmed to encode a GGPPS. By sequence analysis of regions flanking the GGPPS gene, we identified eleven genes (bra1-11), all oriented in the same direction. We did not, however, detect any genes related to L-rhamnose and N-acetylglucosamine biosyntheses in the flanking regions. A gene disruption experiment did indeed show that this gene cluster was responsible for BCA biosynthesis.
    34. 34
      Kim, S. Y.; Zhao, P.; Igarashi, M.; Sawa, R.; Tomita, T.; Nishiyama, M.; Kuzuyama, T. Chem. Biol. 2009, 16, 736– 743
      34
      Cloning and Heterologous Expression of the Cyclooctatin Biosynthetic Gene Cluster Afford a Diterpene Cyclase and Two P450 Hydroxylases
      Kim, Seung-Young; Zhao, Ping; Igarashi, Masayuki; Sawa, Ryuichi; Tomita, Takeo; Nishiyama, Makoto; Kuzuyama, Tomohisa
      Chemistry & Biology (Cambridge, MA, United States) (2009), 16 (7), 736-743CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Cyclooctatin, a diterpene characterized by a 5-8-5 fused ring system, is a potent inhibitor of lysophospholipase. Here we report the cloning and characterization of a complete cyclooctatin biosynthetic gene cluster from Streptomyces melanosporofaciens MI614-43F2 and heterologous prodn. of cyclooctatin in S. albus. Sequence anal. coupled with subcloning and gene deletion revealed that the minimal cyclooctatin biosynthetic gene cluster consists of four genes, cotB1 to cotB4, encoding geranylgeranyl diphosphate (GGDP) synthase, terpene cyclase (CotB2), and two cytochromes P 450, resp. Incubation of the recombinant CotB2 with GGDP resulted in the formation of cyclooctat-9-en-7-ol, an unprecedented tricyclic diterpene alc. The present study establishes the complete biosynthetic pathway of cyclooctatin and provides insights into both the stereospecific diterpene cyclization mechanism of the GGDP cyclase and the mol. bases for the stereospecific and regiospecific hydroxylation.
    35. 35
      Mann, F. M.; Xu, M.; Davenport, E. K.; Peters, R. J. Front. Microbiol. 2012, 3, DOI:  DOI: 10.3389/fmicb.2012.00368 .
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      Meguro, A.; Tomita, T.; Nishiyama, M.; Kuzuyama, T. ChemBioChem 2013, 14, 316– 321
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