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

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.

Keywords: CRISPRi; Cas12a/Cpf1; Clostridia; electroporation; fermentation; methylome.

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

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

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

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

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. phytofermentanscphy3510 gene; T0133, transcription terminator from C. phytofermentanscphy0133 gene; Tfdx, transcriptional terminator from C. pasteurianumfdx gene; DR, direct repeat; TR, terminal repeat; OD₆₀₀, optical density at 600 nm; SD, standard deviation.

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.