σ⁵⁴ (σ^L) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

Abstract

9Microbial production of butanol and isopropanol, two high value-added chemicals, is 10 naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its 11 ancient discovery, the precise mechanisms controlling alcohol synthesis in this 12 microorganism are poorly understood. In this work, an allyl alcohol tolerant strain 13 obtained by random mutagenesis was characterized. This strain, designated as the AA 14 mutant, shows a dominant production of acids, a severely diminished butanol synthesis 15 capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-16 deficient strain was also found to have a limited consumption of two carbohydrates and 17 to be still able to form spores, highlighting its particular phenotype. Sequencing of the 18 AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which 19 encodes the σ 54 sigma factor. Complementation with the wild-type sigL gene fully 20 restored solvent production and sugar assimilation, demonstrating that σ 54 plays a 21 central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic 22 comparison with other strains further revealed that these functions are probably 23 conserved among the C. beijerinckii strains. The importance of σ 54 in C. beijerinckii was 24 further assessed by the characterization of a sigL deletion mutant of the model strain 25 NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited 26 phenotypic traits similar to the AA strain, and was subsequently complemented with the 27 sigL gene from either the wild type or the AA strains. The results of this experiment 28 confirmed the crucial role of σ 54 in the regulation of both solventogenesis and sugar 29 consumption pathways in C. beijerinckii. 30 3 Importance 31Clostridium beijerinckii shows a significant potential for producing valuable biochemicals 32 and biofuels. One of the major hurdles impeding its widespread usage is its low 33 endogenous production of alcohols, which could be alleviated by metabolic engineering 34 approaches. Despite its former long-time use in the industrial acetone-butanol-ethanol 35 process, the molecular mechanisms controlling solventogenesis in the Clostridium 36 genus still remain elusive, preventing genetic engineering approaches for strain 37 enhancement. In this context, our study provides novel insights into the crucial role of 38 the σ 54 transcriptional factor in solvent synthesis regulation in two C. beijerinckii strains. 39Furthermore, we show that this sigma factor also controls sugar consumption and is 40 therefore a key controller of carbon metabolism in this species. 41 42 80 highly conserved DNA-binding region of the encoded protein. We hypothesize that this 81 mutation results in the loss of nucleic acid binding capacity and thus impedes 82 transcription of the σ 54 regulon. According to in silico analyses, this regulon is apparently 83 conserved in other C. beijerinckii strains. To confirm this hypothesis, sigL (Cbei_0595) 84was deleted in the acet...

Genomic reconstruction of σ54 regulons in Clostridiales

“…For the acetogenic species including C. ljungdahlii, C. carboxidivorans, and C. autoethanogenum that can fix CO 2 or CO [47], the σ 54 may play an important role in regulation of both heterotrophic and autotrophic metabolism. Although the recent two studies in C. beijerinckii have obtained some similar results about the σ 54 function [49,50], our systematic analysis of the regulatory network of σ 54 yielded more complete and comprehensive regulons of σ 54 and EBPs, covering all completely sequenced Clostridiales genomes. The majority of the EBPs in Clostridiales are OCSs possessing a regulatory domain that directly recognize signal molecules and modulates the activity of the EBPs.…”

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance

“…This may be causing the acid crash phenotype. Sigma-54 regulates sugar consumption and carbon metabolism in C. beijerinckii ( Hocq et al, 2019 ). Thus, a higher glucose uptake rate and acid production rate, the reasons for the acid crash phenotype ( Maddox et al, 2000 ), may occur due to mutations in regulatory genes such as sigma-54.…”

Pathway dissection, regulation, engineering and application: lessons learned from biobutanol production by solventogenic clostridia

“…From the viewpoint of industrial applications, sporulation is an intrinsic drawback for clostridia, and a solvent-producing, non-sporulating strain may be more desirable for ABE fermentation, as avoiding sporulation without ceasing solvent formation would benefit specific cell productivity and simplify bioprocessing. However, the sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases, making the regulation mechanism of the sporulation process still incompletely understood at the molecular level [50,51]. As for solventogenic clostridia, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program [52,53].…”