σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

Abstract

Microbial production of butanol and isopropanol, two high value-added chemicals, is naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its ancient discovery, the precise mechanisms controlling alcohol synthesis in this microorganism are poorly understood. In this work, an allyl alcohol tolerant strain obtained by random mutagenesis was characterized. This strain, designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the {sigma}54 sigma factor. Complementation with the wild-type sigL gene fully restored solvent production and sugar assimilation, demonstrating that {sigma}54 plays a central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic comparison with other strains further revealed that these functions are probably conserved among the C. beijerinckii strains. The importance of {sigma}54 in C. beijerinckii was further assessed by the characterization of a sigL deletion mutant of the model strain NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited phenotypic traits similar to the AA strain, and was subsequently complemented with the sigL gene from either the wild type or the AA strains. The results of this experiment confirmed the crucial role of {sigma}54 in the regulation of both solventogenesis and sugar consumption pathways in C. beijerinckii.\n\nImportanceClostridium beijerinckii shows a significant potential for producing valuable biochemicals and biofuels. One of the major hurdles impeding its widespread usage is its low endogenous production of alcohols, which could be alleviated by metabolic engineering approaches. Despite its former long-time use in the industrial acetone-butanol-ethanol process, the molecular mechanisms controlling solventogenesis in the Clostridium genus still remain elusive, preventing genetic engineering approaches for strain enhancement. In this context, our study provides novel insights into the crucial role of the {sigma}54 transcriptional factor in solvent synthesis regulation in two C. beijerinckii strains. Furthermore, we show that this sigma factor also controls sugar consumption and is therefore a key controller of carbon metabolism in this species.