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.

Lachnospiraceae are emerging industrial biocatalysts and biotherapeutics

“…Early studies found that conjugative transposons bearing antibiotic resistance genes transfer DNA between Lachnospiraceae ( Barbosa et al, 1999 ). Experimental methods have been developed to transfer plasmid DNA into species of Lachnoclostridium , Roseburia , Eubacterium , Enterocloster , Lacrimispora , and Blautia by conjugation with Escherichia coli ( Tolonen et al, 2009 ; Cuív et al, 2015 ; Sheridan et al, 2019 ; Jin et al, 2022 ) and by electroporation into species of Lachnoclostridium and Butyrivibrio ( Beard et al, 1995 ; Rostain et al, 2022 ) ( Figure 4A ).…”

“…Native plasmids from Lachnospiraceae have been used as expression vectors ( Hefford et al, 1997 ) and the pMTL plasmid system ( Heap et al, 2009 ) can be applied to identify plasmid origins and resistance markers that function in strains of interest ( Jin et al, 2022 ; Rostain et al, 2022 ). Plasmid origins that have been shown to replicate in Lachnospiraceae include pCB102 from Clostridium butyricum , pBP1 from Clostridium botulinum , pAMβ1 from Enterococcus faecalis , pWV101 from Lactococcus lactis , pIM13 from Bacillus subtilis , and pCD6 from C. difficile , and these plasmids can be maintained using antibiotic resistance genes catP (chloramphenicol), ermB (erythromycin), or aad9 (spectinomycin) ( Sheridan et al, 2019 ; Jin et al, 2022 ; Rostain et al, 2022 ) ( Figure 4B ).…”

“…Shuttle vectors containing Gram-negative and Gram-positive origins of replication have permitted the heterologous expression of a β-(1,3-1,4)-glucanase in Eubacterium rectale and R. inulinivorans (Sheridan et al, 2019) and of a synthetic ethanol formation pathway in L. phytofermentans (Tolonen et al, 2015b). Plasmid-based expression of a NanoLuc reporter has identified a library of promoters of varying strength, and reporter gene expression can Frontiers in Bioengineering and Biotechnology frontiersin.org be regulated by anhydrotetracycline using a promoter flanked tet repressor sites (Rostain et al, 2022). Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) using dCas12a has been applied to repress the transcription of chromosomal genes for fermentation, thereby reducing production of butyrate in Blautia luti and Enterocloster boltae (Jin et al, 2022) and acetate in L. phytofermentans (Rostain et al, 2022) (Figure 4C).…”

Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia

“…Therefore, the accurate prediction of RNA folding would be beneficial for investigating off-target effects, and we encourage RNA folding to be considered in computational tools to predict efficient gRNAs ( 25 ). Recently, several dCas12a-based CRISPR interference systems have been developed for tuning gene expression in clostridia ( 13 , 47 , 49 ). In our later work, we would like to investigate the impact of the folding of pre-crRNAs on the efficiency of gene repression.…”

Tolerance in Solventogenic Clostridia for Enhanced Butanol Production: Genetic Mechanisms and Recent Strain Engineering Advances

“…This nucleasedeficient version of spCas9 binds to target sequences without cutting, effectively blocking gene expression [72]. Tested in various Clostridium species [73,74], CRISPRi offers adjustable gene downregulation, making it a valuable tool for the partial repression of essential genes [75], and the concurrent repression of multiple genes [76]. CRISPRa, on the other hand, involves a nuclease-deficient Cas protein fused with an activation domain for gene upregulation [77].…”