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Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi

  • William Rostain
    William Rostain
    Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
  • Tom Zaplana
    Tom Zaplana
    Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    More by Tom Zaplana
  • Magali Boutard
    Magali Boutard
    Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
  • Chloé Baum
    Chloé Baum
    Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938, United States
    More by Chloé Baum
  • Sibylle Tabuteau
    Sibylle Tabuteau
    Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
  • Mary Sanitha
    Mary Sanitha
    Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur-603 203, TN, India
    More by Mary Sanitha
  • Mohandass Ramya
    Mohandass Ramya
    Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur-603 203, TN, India
  • Adam Guss
    Adam Guss
    Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6038, United States
    More by Adam Guss
  • Laurence Ettwiller
    Laurence Ettwiller
    New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938, United States
  • , and 
  • Andrew C. Tolonen*
    Andrew C. Tolonen
    Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    *Email: [email protected]
Cite this: ACS Synth. Biol. 2022, 11, 12, 4077–4088
Publication Date (Web):November 25, 2022
https://doi.org/10.1021/acssynbio.2c00385

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.

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Introduction

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The ability to experimentally control expression of target genes is needed for reliable modification of biological systems. While numerous tools to modulate gene expression are available for a few well-studied models such as Escherichia coli, they are lacking in many other bacterial taxa with applications in biotechnology. For example, the Clostridia are Gram-positive, anaerobic, spore-forming bacteria with important roles in industry and health. Clostridia include species that transform plant biomass or carbon dioxide into value-added chemicals, intestinal commensals that metabolize dietary fiber to produce health-promoting metabolites, and important pathogens. (1−4) Clostridium phytofermentans (also called Lachnoclostridium phytofermentans) is a member of the family Lachnospiraceae that is distinguished by its ability to ferment lignocellulosic biomass with ethanol, hydrogen, and acetate as major products. (5,6)
Advances in genetic manipulation are needed to exploit the therapeutic and industrial potential of additional Clostridia species. In particular, experimental approaches for in vivo modulation of gene expression have a few prerequisites. First, a method is needed to deliver foreign DNA into the cell. Among DNA delivery methods, electroporation is advantageous for simultaneous delivery of multiple DNA constructs or linear DNA fragments. Electroporation methods have been demonstrated in a few other model Clostridia, including biomass-fermenting species, (7,8) but typically use an anaerobic glovebox that requires significant infrastructure investment. Second, modulation of gene expression using genetic regulatory circuits often requires more than one plasmid, so a set of compatible resistance markers and plasmid replicons are needed. A set of modular shuttle plasmids, called pMTL plasmids, has been constructed to test replicons and selectable markers in Clostridia. (9) Third, defined regulatory elements are needed to finely control expression of target genes. A set of constitutive promoters of different strengths can be applied to specify transcription levels, (10) and flanking a promoter with operator sites has been applied to modulate transcription in Clostridium acetobutylicum using the tet repressor (TetR) and anhydrotetracycline (aTc). (11)
CRISPR interference (CRISPRi) is a method to repress transcription that does not require changing regulatory elements of the target gene. (12) By customizing the guide RNA (gRNA) sequence, the DNase-dead CRISPR effector, e.g., Cas9 (dCas9) or Cas12a (dCas12a), can be programmed to bind any DNA sequence containing a protospacer adjacent motif (PAM) element without cleaving the DNA. Transcriptional repression results because the dCas protein interferes with RNA polymerase by binding either at the promoter to inhibit transcript initiation or downstream to prevent transcript elongation. While Cas9-mediated CRISPRi has been more widely developed in Clostridia, (13−15) Cas12a (also called Cpf1) presents certain advantages. (16) Cas12a does not require a tracrRNA and can process a tandem array of guides using its intrinsic RNase activity. (17,18) Multiplexing targets is thus simpler with Cas12a than with Cas9, which requires RNase III for crRNA cleavage and an independently transcribed gRNA cassette for each guide. Cas12a also appears to be less toxic than Cas9 in many bacteria. (19)
The goal of this study is to develop a suite of approaches for efficient genetic transformation and experimental modulation of gene expression in Clostridia using C. phytofermentans as a model. We demonstrate a simple benchtop method for electrotransformation of C. phytofermentans that does not require an anaerobic glovebox, and we apply it to systematically test antibiotic resistance cassettes and plasmid origins of replication. We use a luminescent reporter to characterize a collection of synthetic promoters of varying strengths and a TetR-based system for inducible gene expression. We unite these tools into a CRISPRi system for gene repression based on a TetR-regulated dCas12a and show that it can be used for efficient in vivo CRISPRi-mediated repression of reporter and metabolic genes. We discuss how these approaches advance C. phytofermentans as a model system and can be applied to engineer other Clostridia.

Results

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Restriction Modification Systems in C. phytofermentans

Electroporation of some Clostridia species requires methylation of vector DNA to circumvent endogenous restriction modification systems (RMSs), (20,21) leading us to investigate the presence of RMSs in C. phytofermentans. REBASE (22) predicts that the C. phytofermentans genome encodes two type II RMSs, Cphy0266–8 and Cphy2923–5, and one type IV restriction enzyme, Cphy1615. Cphy0267 was predicted to methylate the sequence 5′-CTGAAG-3′, whereas no specificities could be predicted for Cphy2924. Three forms of DNA methylation are common in bacterial genomes: 5-methylcytosine (m5C), N4-methylcytosine (m4C), and N6-methyladenine (m6A). (23) We profiled the C. phytofermentans genome for these three methylation patterns using two complementary sequencing methods: RIMS-seq identifies m5C as C-to-T mutations on Read 1, (23) and SMRT sequencing identifies m4C and m6A based on nucleotide incorporation rates measured as pulse width and interpulse duration. (24)
RIMS-seq of the C. phytofermentans genome revealed m5C modification at 5′-GATC-3′ based on elevated C-to-T mutations (p = 1.23 × 10–4061) (Figure 1A). DNA modification analysis of SMRT sequencing supported the presence of m6A methylation in the C. phytofermentans genome based on an increased proportion of adenine bases with elevated modification quality values (QVs) relative to other bases (Figure 1B). SMRT motif analysis (25) localized the m6A modification to 5′-CTKCAG-3′ (97.2% modified, mean score 100.6) and 5′-CTGAAG-3′ (96.4% modified, mean score 95.3) (Figure 1C). Together, sequence analysis and methylome profiling of the C. phytofermentans genome suggest that Cphy0267 performs m6A methylation at 5′-CTGAAG-3′ and Cphy2924 performs m5C methylation at 5′-GATC-3′.

Figure 1

Figure 1. Base modification detection in the C. phytofermentans genome by (A) RIMS-seq sequencing and (B, C) SMRT sequencing. (A) RIMS-seq data analysis showing frequency of C-to-T mutations on Read 1 (blue bars) and Read 2 (yellow bars), indicating m5C methylation at the 5′-GATC-3′ motif (p = 1.23 × 10–4061). (B) Kinetic detection histogram of the number of bases at each modification QV. (C) Modified base motif analysis identified two motifs with predicted m6A modification: CTKCAG and CTGAAG, where K is G/T. Modified bases are underlined for both sites. Abbreviations: SMRT, single molecule real-time; RIMS, rapid identification of methylase specificity; QV, quality value; m6A, 6-methyladenosine; m5C, 5-methylcytosine.

On the basis of these methylation activities, we examined the restriction activity of C. phytofermentans lysate on plasmid DNA isolated from ER2796, a DNA-methylation-deficient E. coli strain. (26) In vitro treatment of unmethylated plasmid DNA with C. phytofermentans lysate did not result in DNA cleavage (Figure 2A), suggesting that the putative type II and type IV restriction systems are inactive. Further, the electroporation efficiencies of C. phytofermentans using the methods described below were similar whether pQexp (Table 1) was isolated from C. phytofermentans, E. coli DH5α, or E. coli ER2796, supporting that premethylation of plasmid DNA with C. phytofermentans methylases does not improve electroporation (data not shown). We thus concluded that although the C. phytofermentans genome is methylated, the associated restriction enzymes are likely inactive and do not impede electrotransformation.

Figure 2

Figure 2. Factors affecting efficacy of C. phytofermentans electroporation. (A) Treatment of linearized plasmid DNA with C. phytofermentans culture lysate. Three plasmids (pQexp, pMTL83353, and pBR322) isolated from methylation-deficient E. coli ER2796 were linearized with NheI, incubated with (+) or without (−) C. phytofermentans culture lysate, and resolved by gel electrophoresis. (B) Electroporation efficiencies (colonies per μg of DNA) of 3 μg of plasmid DNA delivered using different electropulse voltages and capacitances. (C) Colonies after electroporation with 1–6 μg of plasmid DNA. (D) Electroporation efficiencies (colonies per μg of DNA) after storage of competent cells at −80 °C for 0–12 weeks. (B–D) Electrotransformations were performed with pQexp and transformants selected with 40 μg mL–1 erythromycin. Data points show each electrotransformation; bars show mean ± SD. Treatment comparisons were analyzed by Tukey’s test after confirmation that the data distribution requirements were met using the Shapiro–Wilk test; p values < 0.05 are shown. SD, standard deviation.

Table 1. Plasmids and Strains Used in This Study
namedescriptionsource
Plasmids
pQexperm, pAMβ1 Gram(+) origin, pUC Gram(−) origin, oriTref (27)
pBR322tet, bla, colE1 Gram(−) originNew England Biolabs
pMTL82254ermB, pBP1 Gram(+) origin, ColEl Gram(−) origin + tra, catP reporterChain Biotech
pMTL83353aad9, pCB102 Gram(+) origin, ColE1 Gram(−) origin + tra, Pfdx + MCSChain Biotech
pMTL84422tetA, pCD6 Gram(+) origin, p15a Gram(−) origin + tra, Pthl + MCSChain Biotech
pMTL85141catP, pIM13 Gram(+) origin, ColEI, MCSChain Biotech
pQmod1EermB, pIM13 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod2EermB, pBP1 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod3EermB, pCB102 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod4EermB, pCD6 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod2CcatP, pBP1 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod2Saad9, pBP1 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod2TtetA, pBP1 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod3CcatP, pCB102 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod3Saad9, pCB102 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod3TtetA, pCB102 Gram(+) origin, ColE1 Gram(−) origin, MCSthis study
pQmod2E-GGpQmod2E, Plac-RFP flanked by BsaI sitesthis study (Addgene 191345)
pQmod2S-GGpQmod2S, Plac-RFP flanked by BsaI sitesthis study (Addgene 191346)
pQmod2C-GGpQmod2C, Plac-RFP flanked by BsaI sitesthis study (Addgene 191347)
pQmod3E-GGpQmod3E, Plac-RFP flanked by BsaI sitesthis study (Addgene 191348)
pQmod3S-GGpQmod3S, Plac-RFP flanked by BsaI sitesthis study (Addgene 191349)
pQmod3C-GGpQmod3C, Plac-RFP flanked by BsaI sitesthis study (Addgene 191350)
pQmod4E-GGpQmod4E, Plac-RFP flanked by BsaI sitesthis study (Addgene 191351)
pATmin-GGerm, pAMβ1 Gram(+) origin, pUC Gram(−) origin, Plac-RFP flanked by BsaI sitesthis study (Addgene 191352)
pSB1C3-J04450cat, pUC Gram(−) origin, Plac-mRFP1 cassetteRegistry of Standard Biological Parts (J04450)
pNL1.1[Nluc]source of NanoLuc genePromega
pQnl_Pcons17erm, pAMβ1 Gram(+) origin, pUC Gram(−) origin, Pcons17-Nanolucthis study (Addgene 191353)
pQnl_Pcphy1–24pQnl_cons17 with Pcons17 replaced with promoters Pcphy1 to Pcphy24this study
pQnl_Pcphy23erm, pAMβ1 Gram(+) origin, pUC Gram(−) origin, Pcphy23-Nanolucthis study (Addgene 191354)
pQnl_tetermB, pBP1 Gram(+) origin, ColE1 Gram(−) origin, PGusA2-TetO2/1-Nanoluc, miniPthl-tetRthis study (Addgene 191355)
pY109source of Cas12a (LbCpf1)Addgene 84740
pQdC12aPGusA2-TetO2/1-dCas12a, miniPthl-tetR, gRNA cassette, pQmod3C-GG backbonethis study (Addgene 191356)
Strains
E. coli NEB 5-alphacompetent E. coli cells used in transformationsNew England Biolabs
E. coli ER2796E. coli strain deficient in DNA methylationref (26)
C. phytofermentans ISDgreference strainATCC 700394

Electroporation of C. phytofermentans

Previously, the transfer of plasmid DNA into C. phytofermentans was demonstrated by conjugation with E. coli. (27−31) Conjugation showed that pQexp, which bears the pAMβ1 replicon and erythromycin resistance gene, stably replicates C. phytofermentans. (27) To develop methods for electrotransformation of C. phytofermentans, we evaluated the effects of electropulse, DNA concentration, and cell wall-weakening osmolytes on electroporation of pQexp using an exponential-decay wave pulse. All electroporations were performed at the bench without an anaerobic glovebox, making these methods generally accessible to microbiology laboratories.
We varied the pulse voltage and capacitance to find the highest transformation efficiency at 1.2 kV and 25 μF (Figure 2B), which typically yielded 80–100 colonies per μg of DNA and a time constant of ∼3 ms. Similar numbers of colonies were observed at DNA concentrations from 1 to 6 μg, supporting that DNA uptake does not limit the transformation efficiency (Figure 2C). Glycine improves electrotransformation in other Clostridia by incorporating into the cell wall in place of d-alanine, weakening the cell wall by reducing peptidoglycan linkages. (32,33) Supplementing GS2 medium with >100 mM glycine significantly reduced the growth rate, but 50, 100, or 150 mM glycine supplementation did not affect the transformation efficiency (data not shown). We also found that electrocompetent C. phytofermentans cells can be stored frozen at −80 °C without loss of transformation efficiency, even when the cells were frozen for 12 weeks (Figure 2D).

Plasmid Replicons and Resistance Markers

The ability to independently select for more than one plasmid in the same cell requires a set of compatible plasmid replicons and antibiotic resistance genes. To identify plasmid replicons that function in C. phytofermentans, we used the pMTL plasmid backbone (Figure 3A) (9) to construct plasmids pQmod1–4E, each bearing the erythromycin resistance gene and a different Gram-positive replicon: pIM13, pBP1, pCB102, and pCD6, respectively (Table 1). Plasmids with the pBP1 and pCB102 origins were electroporated with efficiencies similar to that of the pAMβ1 origin used previously (Figure 3B), and transformants with plasmids bearing any of these three origins can be maintained in liquid medium with erythromycin at growth rates similar to wild-type (WT) without selection. The transformation efficiencies were significantly lower with the plasmids bearing a pCD6 origin (p = 0.039) or pIM13 origin (p = 0.037) relative to the pAMβ1 origin (Figure 3B). Cells retain pCD6 plasmids in liquid medium with erythromycin at a growth rate 3 times lower than WT without selection. The pIM13 transformant colonies were unable to grow under selection in liquid medium, suggesting that the pIM13 origin is too unstable to be maintained in C. phytofermentans even with antibiotic selection.

Figure 3

Figure 3. Plasmid replicons and antibiotic resistance genes that function in C. phytofermentans. (A) Structure of modular pMTL plasmids showing the MCS, Gram-positive replicon, antibiotic resistance marker, and Gram-negative ColE1 replicon. Restriction enzyme sites between modular units are shown along with the SacI and NheI sites in the MCS used to insert the Golden Gate RFP cassette in pQmod-GG plasmids. Gram-positive origins and resistance markers tested in C. phytofermentans are listed. (B) Efficiencies of C. phytofermentans electrotransformation using erythromycin-resistant plasmids with different Gram-positive replicons. Pairwise treatment comparisons were analyzed by Tukey’s test after confirmation that the data distribution requirements were met by the Shapiro–Wilk test; significant p values relative to pAMβ1 (pQexp) transformations are shown. (C) Antibiotic concentrations to select for C. phytofermentans transformants on solid medium and in liquid culture. (D) PCR of C. phytofermentans culture showing stable simultaneous maintenance of three plasmids with different Gram-positive replicons and resistance markers: pQexp (pAMβ1, erm), pQmod2S (pBP1, aad9), and pQmod3C (pCB102, catP). PCR was performed with primers for genomic DNA (primers 1575F/R) and plasmid origins pAMβ1 (primers PR46/47), pBP1 (primers PR28/29), and pCB102 (primers PR30/31). Abbreviations: MCS, multiple cloning site; RFP, red fluorescent protein; ND, no data.

To identify selectable markers for C. phytofermentans, we constructed pQmod2 plasmids bearing the pBP1 replicon and resistance genes for spectinomycin (aad9), thiamphenicol (catP), or tetracycline (tetA) (Table 1). We found that aad9-containing plasmids can be maintained using 600 μg mL–1 spectinomycin and catP plasmids can be maintained using 40 μg mL–1 thiamphenicol (Figure 3C). Although C. phytofermentans is sensitive to 15 μg mL–1 tetracycline in liquid and solid media, we were unable to isolate transformants with plasmids bearing the tetA gene, (34) supporting that this resistance gene is not functional in C. phytofermentans.
Having identified three plasmid replicons (pAMβ1, pBP1, and pCB102) and three resistance cassettes (ermB, aad9, and catP) that function in C. phytofermentans, we tested whether they can be simultaneously maintained in the same cell. Sequential transformation of C. phytofermentans with pQexp (pAMβ1, erm), pQmod2S (pBP1, aad9), and pQmod3C (pCB102, catP) yielded transformants that after five serial transfers in liquid medium with triple antibiotic selection were confirmed by PCR to retain all three plasmids (Figure 3D). To simplify cloning and testing of genetic elements, we built a series of pQmod-GG plasmids containing an IPTG-inducible Plac promoter driving expression of the red fluorescent protein (RFP) gene for red/white selection in E. coli (Figure S1). The Plac-RFP cassette is flanked with BsaI sites to facilitate gene replacement by Golden Gate assembly. (35) pQmod-GG plasmids with pBP1 and pCB102 replicons were constructed with all three resistance markers, and plasmids with the pCD6 and pAMβ1 replicons were made with erythromycin resistance (Table 1).

Constitutive and Inducible Promoters

A set of constitutive promoters of varying strengths can be applied to customize the expression of target genes and dissect promoter sequence–function relationships. We designed the synthetic Pcons17 promoter, a benchmark for high expression in C. phytofermentans. Pcons17 incorporates the consensus promoter elements (UP element, −35 box, −10 box) defined by genome-wide mapping of transcription start sites in C. phytofermentans (36) and computationally optimized sequences between the promoter elements (37,38) and the 5′ UTR containing the ribosome binding site. (39) Pcons17 was cloned upstream of the Nanoluc luciferase reporter (40) in the pQexp backbone, yielding pQnl_Pcons17 (Table 1). Cell-density-normalized luminescence demonstrated high NanoLuc expression from the Pcons17 consensus promoter in pQnl_Pcons17 (Figure 4A).

Figure 4

Figure 4. Modulation of gene expression in C. phytofermentans using constitutive and inducible promoters. (A) Promoter strengths of Pcons17, Pcphy1–24, and minus-NanoLuc control (pQexp) measured as normalized luminescence. (B) Nucleotide sequence showing degenerate positions used to build the Pcphy promoter library. (C, D) Promoter-strength-weighted sequence motifs of (C) UP element and −35 box and (D) −10 box. (E) Diagram of pQnl_tet plasmid for aTc-regulated gene expression in C. phytofermentans. (F) Induction of NanoLuc gene expression in C. phytofermentans expressing pQnl_tet measured as normalized luminescence at different aTc concentrations. (A, F) Culture luminescence was normalized to OD600; bars show means ± SD of triplicate cultures. Degenerate nucleotides: W, A/T; R, A/G; K, G/T; V, A/C/G; H, A/C/T; D, A/G/T. Abbreviations: TSS, transcription start site; RBS, ribosome binding site; TetR, tetracycline repressor; aTc, anhydrotetracycline; OD600, optical density at 600 nm; SD, standard deviation.

We created the Pcphy promoter library by systematically permuting the Pcons17 sequence using oligonucleotides with 20 degenerate positions covering all conserved nucleotides in the promoter consensus motif (Figure 4B). The Pcphy library was cloned upstream of the NanoLuc reporter to make the pQnl_Pcphy plasmid library. NanoLuc-based luminescence from different clones demonstrated that promoters in the Pcphy library span a wide range of expression from 4-fold to 2000-fold above the background luminescence of cells containing an empty plasmid (Figure 4A; see Figure S2 for promoter sequences). Sequence motifs in which the frequencies of nucleotides were weighted based on Pcphy-driven NanoLuc expression levels revealed the relative importance of nucleotide conservation across promoter elements (Figure 4C,D). These expression-weighted motifs support that expression is enhanced by an A-rich UP element upstream of the −35 box hexamer (5′-TTGACA-3′) (Figure 4C), which likely interacts with the alpha subunit of RNA polymerase. (41) In addition, there are two conserved positions upstream of the −10 hexamer box (5′-TATAAT-3′) (Figure 4D) that may increase transcription, as in some other Gram-positive bacteria. (42)
The ability to experimentally modulate gene expression using a chemical inducer simplifies expression level optimization, especially with toxic gene products that need to be repressed under certain conditions. We demonstrated a system for inducible gene expression in C. phytofermentans using the tetracycline repressor (TetR) from Tn10. TetR is a transcriptional repressor whose binding to the TetO operator sequence is inhibited by tetracycline or anhydrotetracycline (aTc). We constructed pQnl_tet (Table 1) containing tetR driven by the miniPthl promoter along with the NanoLuc gene under control of the PgusA2-tetO2/1 promoter (Figure 4E), developed for TetR-based gene repression in C. acetobutylicum. (11) Upon electroporation of C. phytofermentans with pQnl_tet, nanoLuc expression was strongly repressed in the absence of aTc (Figure 4F). NanoLuc expression was induced with 5 ng mL–1 aTc and progressively increased up to 200 ng mL–1 aTc without affecting growth (Figure 4F). Concentrations of aTc above 200 ng mL–1 further increased expression but also reduced growth, likely due to either the metabolic cost of expressing Nanoluc or aTc toxicity.

Gene Repression by CRISPRi

CRISPRi using DNase-dead CRISPR effectors of Cas9 (dCas9) or Cas12a (dCas12a) streamlines transcriptional repression of target genes by eliminating the need to modify their promoters to contain operator sites, as is required in the TetR system. In this study, we selected the Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a). (18) While CRISPRi based on LbCas12a has not yet been tested in prokaryotes, (19) we reasoned that the protein will likely be active in C. phytofermentans because it comes from a bacterium in the same family. As the rarity of Cas9 PAM (5′-NGG-3′) sites has impeded Cas9 targeting in other AT-rich Clostridia genomes, (15) we assessed the distribution of the Cas12a PAM (5′-TTTV-3′) (43) across the C. phytofermentans genome. The Cas12a PAM is less common than that of Cas9 across the C. phytofermentans genome, but it is more abundant in highly expressed promoter regions (Figure S3). Thus, Cas12a is well-suited to target C. phytofermentans promoter regions using CRISPRi.
Plasmids constitutively expressing Cas9 sometimes cannot be transformed into Clostridia due to toxicity of the Cas protein. (44) While Cas12a is reported to be less toxic to Clostridia than Cas9, (45) we mitigated potential Cas12a toxicity by constructing a Tet-repressible dCas12a-based CRISPRi plasmid, pQdC12a. To this end, pQmod3C was modified to carry the following elements: the miniPthl-tetR cassette from pQnl_tet, an aTc-inducible dLbCas12a, and a gRNA designed so that the target can be customized by Golden Gate cloning using BbsI (Figure 5A). As placement of a Rho-independent terminator immediately downstream of the guide impairs Cas activity, (46) we included the first 25 bp of the terminal repeat from the wild-type LbCas12a array downstream of the guide, which including the cloning scar separates the guide from the fdx terminator by 55 bp. To demonstrate dCas12a-mediated gene repression in C. phytofermentans, we targeted pQdC12a to two sites in the Pcphy23 promoter driving NanoLuc expression on pQnl_Pcphy23 (Figure 5A). Guide g-nl1 binds the Pcphy23 promoter at the −10 box, and guide g-nl2 binds at the −35 box (Figure S4). As both guides target the promoter region, we did not expect that binding orientation would affect repression. (47)

Figure 5

Figure 5. CRISPRi repression of reporter gene expression in C. phytofermentans. (A) Two-plasmid system demonstrating dCas12a repression of the NanoLuc reporter. pQdC12a has a tet-repressible dCas12a and gRNA cassette; pQnl_Pcphy23 has the Pcphy23 promoter driving NanoLuc expression. (B) Normalized luminescence of C. phytofermentans expressing NanoLuc (pQnl_Pcphy23) and dCas12a targeting Pcphy23 with guide g-nl1, guide g-nl2, or a no-guide control (pQdC12a). Luminescence was measured ±100 ng mL–1 aTc and normalized to OD600. Data points are individual cultures, and bars are means ± SD of triplicate cultures. The p values show treatment comparisons by two-sided Student’s t test. Abbreviations: aTc, anhydrotetracycline; T3510, transcription terminator from C. phytofermentans cphy3510 gene; T0133, transcription terminator from C. phytofermentans cphy0133 gene; Tfdx, transcriptional terminator from C. pasteurianum fdx gene; DR, direct repeat; TR, terminal repeat; OD600, optical density at 600 nm; SD, standard deviation.

We cotransformed C. phytofermentans with pQnl_Pphy23 and pQdC12a targeted with either guide g-nl1, guide g-nl2, or a no-guide control. In the presence of aTc, NanoLuc expression was strongly repressed using g-nl1 (19-fold repression, p = 1.79 × 10–3) or g-nl2 (8-fold repression, p = 1.94 × 10–3) relative to pQdC12a (Figure 5B). Mean NanoLuc expression with g-nl1 was 2.5-fold lower than with g-nl2, suggesting that preventing transcription initiation by targeting the promoter at the −10 box may be more effective than the −35 box (p = 1.90 × 10–3), but these differences could also reflect sequence dependence of gRNA activity. NanoLuc expression was repressed to a lesser extent without aTc using either g-nl1 (6-fold repression, p = 2.7 × 10–6) or g-nl2 (5-fold repression, p = 3.2 × 10–4) relative to pQdC12a (Figure 5B), likely due to leaky Tet repression of dCas12a. Together, these results support that dCas12a-mediated CRISPRi effectively represses in vivo target gene expression in C. phytofermentans.
We evaluated pQdC12a-mediated CRISPRi of chromosomal genes in C. phytofermentans by targeting dCas12a to the cphy1326 promoter using guide g-cphy1326 (Figure 6A). The cphy1326 and cphy1327 genes encode phosphate acetyltransferase and acetate kinase, respectively. These two genes encode the only predicted pathway to convert acetyl-CoA to acetate in C. phytofermentans, and their transcription and translation are elevated under conditions of high acetate production, (5,48) leading us to propose that repressing cphy1326 transcription should reduce acetate formation. Genome-wide mapping of transcription start sites (TSSs) in C. phytofermentans indicated that cphy1326 is expressed from a primary TSS 139 bp upstream of the start codon. (36) Guide g-cphy1326 was designed to bind the −10 box (5′-TATAAT-3′), which is 10 bp upstream of the cphy1326 TSS.

Figure 6

Figure 6. CRISPRi repression of fermentation gene expression in C. phytofermentans. (A) dCas12a was targeted to repress transcription of the acetate biosynthesis operon by binding the promoter −10 box upstream of the cphy1326 (pta) gene using guide g-cphy1326. (B) Transcription of fermentation genes shown as log2(fold change) in the g-cphy1326 strain relative to the no-guide control strain measured by qRT-PCR. (C–F) Yields of fermentation products (C) acetate, (D) ethanol, (E) formate, and (F) lactate in the g-cphy1326 and no-targeting control strains measured by HPLC. (B–F) Colors show data for acetate (green), ethanol (yellow), formate (gray), and lactate (blue). Bars are means ± SD of (B) four or (C–F) three cultures; data points are individual cultures. The p values show treatment comparisons by two-sided Student’s t test. Abbreviations: TSS, transcription start site; pta, phosphate acetyltransferase; ackA, acetate kinase; NS, not significant; SD, standard deviation; HPLC, high-performance liquid chromatography.

Targeting pQdC12a to the cphy1326 promoter using guide g-cphy1326 reduced cphy1326 transcription relative to the no-guide control (11.5-fold repression, p = 0.021) (Figure 6B). While cphy1326-cphy1327 constitute a putative operon, transcription of cphy1327 was not repressed, suggesting that it is also independently transcribed. Alternative routes for acetyl-CoA conversion were upregulated in the g-cphy1326 strain based on elevated transcription of genes encoding the primary ethanol dehydrogenase (cphy3925), lactate dehydrogenase (cphy1117), and pyruvate formate-lyase activating enzyme (cphy2820) (Figure 6B). Quantification of fermentation products by high-performance liquid chromatography (HPLC) showed that acetate biosynthesis was reduced in the g-cphy1326 strain (19% reduction, p = 1.5 × 10–3) (Figure 6C). Production of ethanol was not altered in the g-cphy1326 strain (Figure 6D), whereas yields were increased for formate (46% increase, p = 1.2 × 10–4) (Figure 6E) and lactate (75% increase, p = 1.6 × 10–3) (Figure 6F). Together, these results support that C. phytofermentans elevated production of alternative fermentation products in response to CRISPRi-mediated repression of acetate production.

Discussion

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In this study, we demonstrated a framework for electrotransformation and modulation of gene expression in C. phytofermentans using tools that can be applied in other Clostridia. We electrotransformed C. phytofermentans using a simple benchtop method that does not require an anaerobic glovebox. The electroporation efficiency was high enough to test plasmids (Figure 3), screen promoters (Figure 4), and construct genetic circuits (Figure 5) but lower than that with other models such as E. coli, making improvements in electrotransformation desirable. However, our data support that C. phytofermentans electrotransformation was not limited by endogenous RMSs (Figure 2A) or DNA concentration (Figure 2C) and was not improved by cell-wall-weakening agents. We grew C. phytofermentans cultures in anaerobic jars and electroporated cells on the bench, making these methods simple to apply in other laboratories. Performing electroporations under more strict anaerobic conditions in a chamber might improve the transformation efficiency.
Although Clostridia are phylogenetically diverse, our results support that their molecular biology is sufficiently conserved for the pMTL plasmid system (9) to be a valuable resource to test functional plasmid elements. We identified three plasmid replicons (pCB102, pAMβ1, and pBP1) and three resistance markers (catP, aad9, and ermB) that function in C. phytofermentans. Whereas the pIM13 origin and the tetA resistance gene are functional in other Clostridia, (34,49) they are inactive in C. phytofermentans. Further, C. phytofermentans can be sequentially transformed with all three plasmid replicons using different resistance markers (Figure 3D), supporting that these plasmids could also be simultaneously maintained in other Clostridia. Using the pQmod-GG plasmids, cargo genes can easily be cloned into backbones with these different replicons and resistance markers using Golden Gate cloning and RFP red/white screening (Table 1). Genes can be expressed at different levels on these plasmids using the Pcphy promoter library (Figure 4A) and modulated using TetR-mediated repression (Figure 4F). These tools enable in vivo expression of genetic circuits composed of multiple operons whose expression needs to be individually tuned.
We united these tools into a system for CRISPRi-mediated repression in C. phytofermentans using a Cas12a protein (dLbCas12a) from another Lachnospiraceae species, the first demonstration of CRISPR using this protein in bacteria. (19) CRISPRi is particularly useful to study genes that may have essential roles, as when redirecting carbon flux in central metabolism. As a first step toward redirecting carbon flux in C. phytofermentans, we showed that repressing genes for acetate biosynthesis reduced the acetate yields and increased the yields of other fermentation products (Figure 6). Our results show that an 11.5-fold repression in cphy1326 expression translated into only a 19% reduction in acetate formation, which could be augmented either through greater repression of cphy1326 using multiple guides or simultaneous targeting of multiple genes. Fortunately, dCas12a simplifies multiplex gene repression by CRISPRi due to its ability to process tandem guides without sacrificing efficiency relative to individual guides. (50)
The results of this study provide a foundation for construction of genetic circuits with experimentally modulated gene expression in C. phytofermentans. These approaches complement previous technologies to study C. phytofermentans genetics using targetron-based gene inactivation, (30) large-scale genome insertion and deletion, (31) and in vivo directed evolution. (51) Cas12a could be used to make genomic changes in C. phytofermentans, as has been demonstrated in some other Clostridia. (45) In the future, these strategies could be generally applied to other Clostridia, including soil species with important roles in environmental carbon cycling, intestinal commensals, and pathogens.

Methods and Materials

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Bacterial Cultivation

C. phytofermentans ISDg (ATCC 700394) was cultured anaerobically at 37 °C in GS2 medium (52) containing 5 g L–1 glucose and antibiotics at selected concentrations to maintain plasmids (Figure 3C). Cultures were incubated anaerobically in sealed jars (BD Jars, 260626) using anaerobic sachets (BD GasPaks, 260678); all experimental manipulations were performed at the bench. E. coli was cultured aerobically at 37 °C in LB medium containing 250 μg mL–1 erythromycin, 100 μg mL–1 spectinomycin, 25 μg mL–1 chloramphenicol, or 15 μg mL–1 tetracycline to select for plasmids.

Genome Methylation and DNA Restriction

We identified DNA methylation patterns in the C. phytofermentans ISDg genome using SMRT sequencing (53) and RIMS-seq. (23) SMRT DNA sequencing was performed at the U.S. Department of Energy Joint Genome Institute (DOE JGI Project ID 1100006), and sequence files are available in the NCBI SRA (accession code SRP123768). RIMS-seq was performed at Genoscope-CEA, and sequences files are available in the European Nucleotide Archive (accession code ERA17973853).
To test whether C. phytofermentans cell lysate restricts plasmid DNA, C. phytofermentans lysate was prepared by centrifuging an overnight culture and freezing the cell pellet at −80 °C for 2 h. The cell pellet was resuspended in lysis buffer (50 mM phosphate buffer, pH 8, 500 mM NaCl, 15% glycerol, 1 mM pefablock (Sigma 76307)), incubated with 1 mg mL–1 lysozyme (Novagen 71230) for 30 min at room temperature, and sonicated (Cole-Parmer Vibracell CV33) four times for 30 s at 28% with 10 s between sonications. Plasmid DNA of pQexp, pMTL83353, and pBR322 (Table 1) were isolated from DNA-methylation-deficient E. coli strain ER2796 (26) and linearized with NheI, and 1 μg of plasmid DNA was incubated with and without 250 μg mL–1 C. phytofermentans lysate overnight at 37 °C. DNA was resolved by electrophoresis to visualize whether incubation with cell lysate resulted in plasmid digestion.

C. phytofermentans Electroporation

To prepare C. phytofermentans cells for electroporation, 10 mL of late log culture was transferred into 40 mL of fresh medium and grown for 6 h to OD600 0.6–0.7. Cells were centrifuged (6245 g, 7 min, 4 °C), washed once with 4 mL of ice-cold ETM buffer (1 mM MgCl2, 256 mM sucrose, 5 mM sodium phosphate buffer, pH 7.4), and resuspended in 200 μL of ETM buffer. A 50 μL aliquot of cells was transferred to a prechilled 0.1 cm gap electroporation cuvette, mixed with plasmid DNA (3 μg of DNA unless noted otherwise), and incubated on ice for 5 min. Cells were electroporated using a Bio-Rad Genepulser II (1.2 kV, 25 μF), immediately diluted with 1 mL of GS2 medium, and incubated anaerobically at 37 °C for 15 h. Cells were plated on GS2 agar plates containing the appropriate antibiotic selection and cultured for 6 days. Transformant colonies were picked and transferred to liquid GS2 medium supplemented with the appropriate antibiotic, and the presence of plasmid DNA in the transformants was confirmed by PCR using 1 μL of log phase culture as template and primers shown in Table S1.

Plasmid Construction

The sequences of all primers used in this study are shown in Table S1. DNA samples and sequences of plasmids from this study have been deposited in Addgene. pMTL plasmids (Table 1) were modified using double digests of either AscI plus FseI to swap Gram-positive replicons or FseI plus PmeI to swap antibiotic genes (Figure 3A). To create a set of erythromycin resistance plasmids to test Gram-positive origins, the pIM13 origin of pMTL85241 was replaced with either the pBP1 origin from pMTL82254 to yield pQmod2E, the pCB102 origin from pMTL83353 to yield pQmod3E, or the pCD6 origin from pMTL84422 to yield pQmod4E (Table 1). To test antibiotic resistance genes, the ermB gene of pQmod2E was replaced with either the catP gene of pMTL85141 to yield pQmod2C, the aad9 gene of pMTL83353 to yield pQmod2S, or the tetA gene of pMTL84422 to yield pQmod2T (Table 1). A set of Golden Gate-compatible plasmids containing an RFP cassette for red/white screening flanked by BsaI sites (pQmod-GG plasmids) was constructed by PCR amplification of the J04450 biobrick RFP cassette from pSB1C3 using primers PR66/PR67 and cloning of the resulting product between the SacI and NheI sites of the multiple cloning site (Figure S1).
We designed the Pcons17 promoter using the consensus UP, −35, and −10 elements based on C. phytofermentans genome-wide transcription start site (TSS) data. (36) The sequences between the −35 and −10 elements were selected by analyzing the C. phytofermentans ISDg genome sequence (GenBank CP000885.1) with PePPER (37) to produce a consensus sequence. (38) The 5′ UTR and ribosome binding site (RBS) upstream of NanoLuc were designed to maximize translation. (39) The NanoLuc gene was PCR-amplified (primers PR1/PR2) from pNL1.1[Nluc] (Promega). The vector backbone to express the NanoLuc gene was built by PCR amplification of the erm gene (primers PR3/PR4), pAMβ1 replicon (primers PR5/PR6), and pUC replicon (primers PR7/PR8) of pQexp. (27) The fragments were assembled with the NanoLuc PCR fragment by four-part Golden Gate cloning using BsmBI, yielding pQnl_Pcons17 (Table 1). pATmin-GG was constructed by Gibson assembly from PCR of pQnl_Pcons17 with PR70/PR71 and of pQmod2C-GG with PR72/PR73.
The Pcphy plasmid library was constructed by PCR amplification of the entire pQnl_Pcons17 sequence using outward-facing primers (PR48/PR49) to introduce degeneracy to the Pcons17 promoter, followed by DpnI digestion. The PCR product was digested with BsaI, self-ligated, and transformed into E. coli. After 75 min recovery in LB medium, the transformed E. coli cells were transferred into liquid LB medium containing erythromycin and incubated at 37 °C for 16 h, and plasmid DNA was isolated to yield the pQnl_Pcphy plasmid library. Promoter diversity in the library was measured by transforming it into E. coli and sequencing 20 individual clones (primers OK2ML9F and Seq_NanoLuc_rv), which confirmed that no two promoters had identical sequences. C. phytofermentans was transformed with the pQnl_Pcphy library, and NanoLuc expression of individual clones was quantified by luciferase assay.
To assemble pQnl_tet for TetR-mediated control of gene expression, the NanoLuc gene was PCR-amplified from pQnl_Pcons17 (primers PR63/PR64), generating a product containing NanoLuc fused to the PGusA2-TetO2/1 promoter. The tetR gene (Genbank accession code EEA9527598.1) was purchased as a gBlock DNA fragment (Integrated DNA Technologies, Coralville, IA, USA) and PCR-amplified (primers PR58/PR59) to generate a product of the miniPthl promoter driving tetR expression. Subsequently, pQnl_tet was assembled by a three-fragment Golden Gate ligation reaction using BsaI to insert PgusA2-TetO2/1-NanoLuc and miniPthl-tetR into pQmod2E-GG.
pQdC12a was assembled by Golden Gate cloning with two fragments of LbCpf1/LbCas12a from pY109, (17) simultaneously incorporating the D832A mutation and ligating to cassettes containing miniPthl-tetR and gRNA expression cassettes. The dCas12a gene was fused to the aTc-inducible PgusA2-tetO2/1 promoter (11) and terminator from cphy3510 by Golden Gate cloning. The gRNA cassette contains the synthetic P4 promoter, (38) extended direct repeat, BbsI sites into which annealed oligos can be inserted to target dCas12a binding, 25 bp from the wild-type Cas12a terminal repeat, and the fdx terminator from C. pasteurianum.
pQdC12a was targeted to bind the Pcphy23 promoter in pQnl_Pcphy23 using guide sequences that bind either the −10 box (g-nl1) or the −35 box (g-nl2) (Figure S4 and Table S1). To repress cphy1326 transcription, pQdC12a was targeted using guide g-cphy1326 that binds the −10 box (5-TATAAT-3), which is 10 bp upstream of the cphy1326 TSS as previously identified by TSS mapping (36) (Table S1). To clone guide sequences, pQdC12a was digested with BbsI, gel-purified, and ligated with annealed guide sequences.

Luciferase Assay

To measure luciferase expression in C. phytofermentans containing NanoLuc plasmids, we grew cultures to late log phase, measured the cell density (OD600), and lysed the cells by addition of a 1:10 volume of PopCulture reagent (EMD Millipore) containing 25 μg mL–1 lysozyme followed by incubation at room temperature for 10 min. Cell lysate was mixed with an equal volume of NanoLuc Reagent (Promega N1110), and luminescence was measured at 454 nm using a Safas Xenius XMA. Luminescence measurements from triplicate cultures were normalized to the cell density (OD600). Following luminescence measurement of Pcphy clones, plasmids covering a range of luminescence strengths were isolated, and the promoter regions were sequenced after amplification with primers OK2ML9F and Seq_NanoLuc_rv. Luciferase measurements with pQnl_tet and CRISPRi plasmids were conducted by growing a culture through two transfers to late log phase followed by dilution into medium supplemented with antibiotics and aTc as appropriate and growth to late log phase before measurement of luciferase expression.

Quantitative PCR

We measured C. phytofermentans mRNA expression by quantitative reverse transcription PCR (qRT-PCR). Briefly, total RNA was extracted from 5 mL mid log phase (OD600 = 0.7) cultures using TRI reagent (Sigma 93289), and 10 μg of RNA was treated with 4 units of Turbo DNase (Ambion AM2238) for 30 min at 37 °C. One microgram of RNA was reverse-transcribed to single-stranded cDNA (Applied Biosystems 4368814). Real-time PCR amplification was conducted by quantitative PCR (qPCR) (KAPA KK4621) with the primers shown in Table S1 using an MJ Research DNA Engine Opticon II machine. Expression values are means of four cultures calculated by the threshold cycle method normalized to 16S rRNA levels.

High-Performance Liquid Chromatography

To quantify the fermentation products, cultures were grown for 5 days to ensure complete consumption of glucose in the medium. Culture supernatants were 0.2 μM filtered and collected. Fermentation products were measured using a Shimadzu Prominence LC20/SIL-20AC HPLC equipped with an RID-10A refractive index detector and a Biorad Aminex HPX-87H 300 mm × 7.8 mm column. The column was maintained at 60 °C with a mobile phase of 5 mM H2SO4 at a flow rate of 0.5 mL min–1. The sample injection volume was 20 μL. Purified standards of the fermentation products (Sigma) were used to define elution times and generate dilution curves, which were used to quantify the fermentation products in the culture supernatants.

Sequence Analysis and Statistics

Bioinformatic prediction of putative DNA restriction modification systems in C. phytofermentans was conducted using REBASE. (22) Methylation analysis of SMRT sequence data was performed using SMRT Tools (protocol version = 2.2.0, method = RS Modification and Motif Analysis.1). (24) Modification quality values (QVs) were computed as −10 log10(p value) for a modified base position based on DNA polymerase kinetics measured as interpulse durations. Modified sites were grouped into motifs using SMRT Tools MotifFinder (v1). (25) The RIMS-seq experiment and data analysis were performed as described previously (54) with motif context analysis calculated based on the C to T errors in Read 1 and Read 2 for all NNNCN contexts with N = ATCG. To evaluate the relative importance of nucleotides at different positions across promoter sequences, we generated sequence motifs (54) in which the relative abundances of sequence inputs were based upon the mean strength of each promoter in the Pcphy library (Figure S2). Statistical comparisons were performed by either Tukey’s test (multiple comparisons) or Student’s t test (pairwise comparisons).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.2c00385.

  • Design of Golden Gate plasmids with dropout RFP cassettes for rapid cloning and testing of genetic elements (Figure S1), sequences and expression strengths of promoters in the Pcphy promoter library (Figure S2), relative abundances of protospacer adjacent motifs in the C. phytofermentans genome and in promoter regions (Figure S3), binding locations of dCas12a guides targeted to the Pcphy23 promoter driving NanoLuc expression in pQnl_Cphy23 (Figure S4), and primers used in this study (Table S1) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • William Rostain - Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    • Tom Zaplana - Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    • Magali Boutard - Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    • Chloé Baum - Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, FranceNew England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938, United States
    • Sibylle Tabuteau - Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Évry, France
    • Mary Sanitha - Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur-603 203, TN, India
    • Mohandass Ramya - Molecular Genetics Laboratory, Department of Genetic Engineering, College of Engineering and Technology, SRM Institute of Science and Technology, SRM Nagar, Kattankulathur-603 203, TN, India
    • Adam Guss - Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6038, United States
    • Laurence Ettwiller - New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938, United States
  • Author Contributions

    W.R., M.R., L.E., and A.C.T. conceived the study. W.R., A.G., L.E., and A.C.T. designed the experiments. W.R., T.Z., M.B., C.B., S.T., and M.S. performed the experiments. W.R., C.B., A.G., L.E., and A.C.T. analyzed the results. W.R. and ACT wrote the manuscript with contributions from all authors. All of the authors read and approved the final manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Corinne Cruaud, Shahinaz Gas, and Ioana Popescu for technical expertise. This work was supported by Genoscope-CEA, the Agence Nationale de la Recherche (Grant ANR-16-CE05-0020), Évry Genopole under the “Action Thématique Incitative Genopole” Grant (funding ATIGE 2021 No. 2698), a travel grant from SRM University, and the Oak Ridge National Laboratory at the Center for Bioenergy Innovation, a U.S. Department of Energy (DOE) Bioenergy Research Center. PacBio methylome data were generated by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, supported by the Office of Science of the U.S. DOE under Contract DE-AC02-05CH11231.

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  1. Yanchao Zhang, Aleksandra M. Kubiak, Tom S. Bailey, Luuk Claessen, Philip Hittmeyer, Ludwig Dubois, Jan Theys, Philippe Lambin, . Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia. Microbiology Spectrum 2023, https://doi.org/10.1128/spectrum.02459-23
  • Abstract

    Figure 1

    Figure 1. Base modification detection in the C. phytofermentans genome by (A) RIMS-seq sequencing and (B, C) SMRT sequencing. (A) RIMS-seq data analysis showing frequency of C-to-T mutations on Read 1 (blue bars) and Read 2 (yellow bars), indicating m5C methylation at the 5′-GATC-3′ motif (p = 1.23 × 10–4061). (B) Kinetic detection histogram of the number of bases at each modification QV. (C) Modified base motif analysis identified two motifs with predicted m6A modification: CTKCAG and CTGAAG, where K is G/T. Modified bases are underlined for both sites. Abbreviations: SMRT, single molecule real-time; RIMS, rapid identification of methylase specificity; QV, quality value; m6A, 6-methyladenosine; m5C, 5-methylcytosine.

    Figure 2

    Figure 2. Factors affecting efficacy of C. phytofermentans electroporation. (A) Treatment of linearized plasmid DNA with C. phytofermentans culture lysate. Three plasmids (pQexp, pMTL83353, and pBR322) isolated from methylation-deficient E. coli ER2796 were linearized with NheI, incubated with (+) or without (−) C. phytofermentans culture lysate, and resolved by gel electrophoresis. (B) Electroporation efficiencies (colonies per μg of DNA) of 3 μg of plasmid DNA delivered using different electropulse voltages and capacitances. (C) Colonies after electroporation with 1–6 μg of plasmid DNA. (D) Electroporation efficiencies (colonies per μg of DNA) after storage of competent cells at −80 °C for 0–12 weeks. (B–D) Electrotransformations were performed with pQexp and transformants selected with 40 μg mL–1 erythromycin. Data points show each electrotransformation; bars show mean ± SD. Treatment comparisons were analyzed by Tukey’s test after confirmation that the data distribution requirements were met using the Shapiro–Wilk test; p values < 0.05 are shown. SD, standard deviation.

    Figure 3

    Figure 3. Plasmid replicons and antibiotic resistance genes that function in C. phytofermentans. (A) Structure of modular pMTL plasmids showing the MCS, Gram-positive replicon, antibiotic resistance marker, and Gram-negative ColE1 replicon. Restriction enzyme sites between modular units are shown along with the SacI and NheI sites in the MCS used to insert the Golden Gate RFP cassette in pQmod-GG plasmids. Gram-positive origins and resistance markers tested in C. phytofermentans are listed. (B) Efficiencies of C. phytofermentans electrotransformation using erythromycin-resistant plasmids with different Gram-positive replicons. Pairwise treatment comparisons were analyzed by Tukey’s test after confirmation that the data distribution requirements were met by the Shapiro–Wilk test; significant p values relative to pAMβ1 (pQexp) transformations are shown. (C) Antibiotic concentrations to select for C. phytofermentans transformants on solid medium and in liquid culture. (D) PCR of C. phytofermentans culture showing stable simultaneous maintenance of three plasmids with different Gram-positive replicons and resistance markers: pQexp (pAMβ1, erm), pQmod2S (pBP1, aad9), and pQmod3C (pCB102, catP). PCR was performed with primers for genomic DNA (primers 1575F/R) and plasmid origins pAMβ1 (primers PR46/47), pBP1 (primers PR28/29), and pCB102 (primers PR30/31). Abbreviations: MCS, multiple cloning site; RFP, red fluorescent protein; ND, no data.

    Figure 4

    Figure 4. Modulation of gene expression in C. phytofermentans using constitutive and inducible promoters. (A) Promoter strengths of Pcons17, Pcphy1–24, and minus-NanoLuc control (pQexp) measured as normalized luminescence. (B) Nucleotide sequence showing degenerate positions used to build the Pcphy promoter library. (C, D) Promoter-strength-weighted sequence motifs of (C) UP element and −35 box and (D) −10 box. (E) Diagram of pQnl_tet plasmid for aTc-regulated gene expression in C. phytofermentans. (F) Induction of NanoLuc gene expression in C. phytofermentans expressing pQnl_tet measured as normalized luminescence at different aTc concentrations. (A, F) Culture luminescence was normalized to OD600; bars show means ± SD of triplicate cultures. Degenerate nucleotides: W, A/T; R, A/G; K, G/T; V, A/C/G; H, A/C/T; D, A/G/T. Abbreviations: TSS, transcription start site; RBS, ribosome binding site; TetR, tetracycline repressor; aTc, anhydrotetracycline; OD600, optical density at 600 nm; SD, standard deviation.

    Figure 5

    Figure 5. CRISPRi repression of reporter gene expression in C. phytofermentans. (A) Two-plasmid system demonstrating dCas12a repression of the NanoLuc reporter. pQdC12a has a tet-repressible dCas12a and gRNA cassette; pQnl_Pcphy23 has the Pcphy23 promoter driving NanoLuc expression. (B) Normalized luminescence of C. phytofermentans expressing NanoLuc (pQnl_Pcphy23) and dCas12a targeting Pcphy23 with guide g-nl1, guide g-nl2, or a no-guide control (pQdC12a). Luminescence was measured ±100 ng mL–1 aTc and normalized to OD600. Data points are individual cultures, and bars are means ± SD of triplicate cultures. The p values show treatment comparisons by two-sided Student’s t test. Abbreviations: aTc, anhydrotetracycline; T3510, transcription terminator from C. phytofermentans cphy3510 gene; T0133, transcription terminator from C. phytofermentans cphy0133 gene; Tfdx, transcriptional terminator from C. pasteurianum fdx gene; DR, direct repeat; TR, terminal repeat; OD600, optical density at 600 nm; SD, standard deviation.

    Figure 6

    Figure 6. CRISPRi repression of fermentation gene expression in C. phytofermentans. (A) dCas12a was targeted to repress transcription of the acetate biosynthesis operon by binding the promoter −10 box upstream of the cphy1326 (pta) gene using guide g-cphy1326. (B) Transcription of fermentation genes shown as log2(fold change) in the g-cphy1326 strain relative to the no-guide control strain measured by qRT-PCR. (C–F) Yields of fermentation products (C) acetate, (D) ethanol, (E) formate, and (F) lactate in the g-cphy1326 and no-targeting control strains measured by HPLC. (B–F) Colors show data for acetate (green), ethanol (yellow), formate (gray), and lactate (blue). Bars are means ± SD of (B) four or (C–F) three cultures; data points are individual cultures. The p values show treatment comparisons by two-sided Student’s t test. Abbreviations: TSS, transcription start site; pta, phosphate acetyltransferase; ackA, acetate kinase; NS, not significant; SD, standard deviation; HPLC, high-performance liquid chromatography.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.2c00385.

    • Design of Golden Gate plasmids with dropout RFP cassettes for rapid cloning and testing of genetic elements (Figure S1), sequences and expression strengths of promoters in the Pcphy promoter library (Figure S2), relative abundances of protospacer adjacent motifs in the C. phytofermentans genome and in promoter regions (Figure S3), binding locations of dCas12a guides targeted to the Pcphy23 promoter driving NanoLuc expression in pQnl_Cphy23 (Figure S4), and primers used in this study (Table S1) (PDF)


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