Agyeman-Duah, E. | bioRxiv Science

bioRxiv2024

Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013

Bioconversion of abundant lactose-replete whey permeate to value added chemicals holds promise for valorization of this increasing food processing waste. Efficient conversion of whey-permeate-borne lactose requires adroit microbial engineering to funnel carbon to the desired chemical. Having engineered a strain of Clostridium beijerinckii NCIMB 8052 (C. beijerinckii_mgsA+mgR) that produces 87% more butanol on lactose than the control strain, in this study, we deployed RNA sequencing to profile the global transcriptome of C. beijerinckii_mgsA+mgR. The results revealed broadly contrasting gene expression patterns in C. beijerinckii_mgsA+mgR relative to the control strain. These were characterized by widespread downregulation of Fe-S proteins in C. beijerinckii_mgsA+mgR, coupled with increased expression of lactose uptake and catabolic genes, iron and phosphate uptake genes, two component signal transduction and motility genes, and genes involved in the biosynthesis of vitamin B5 and B12, aromatic amino acids, particularly tryptophan; arginine, and pyrimidines. Conversely, L-aspartate-dependent de novo biosynthesis of NAD as well as biosynthesis/metabolism of glycine, threonine, lysine, isoleucine and asparagine were downregulated. Furthermore, genes involved in cysteine and methionine biosynthesis and metabolism, including cysteine desulfurase--a central player in Fe-S cluster biosynthesis--were equally downregulated. Genes involved in biosynthesis of capsular polysaccharides and stress response were also downregulated in C. beijerinckii_mgsA+mgR. The results suggest that remodeling of cellular and metabolic networks in C. beijerinckii_mgsA+mgR to counter likely effect of methylglyoxal production following heterologous expression of methyl glyoxal synthase led to enhanced growth and butanol production in C. beijerinckii_mgsA+mgR. IMPORTANCEBiological production of commodity chemicals from abundant waste streams such as whey permeate represents a rational approach for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol, promises to arm researchers with new engineering targets that can be fine-tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii_mgsA+mgR and indeed, C. beijerinckii for improved butanol production on lactose, and ultimately in whey permeate.

microbiology↗

bioRxiv2024

Unravelling the roadblocks to 1,2-propanediol biosynthesis in select solventogenic Clostridium species

BackgroundThe compound 1,2-propanediol is an important industrial bulk chemical that has proven particularly recalcitrant to bio-production. Solvent-producing Clostridium species represent promising candidates for engineering 1,2-propaediol production. Co-production of 1,2-popanediol and butanol has the potential to improve the economics of the acetone-butanol-ethanol (ABE) fermentation. ResultsIn this study, the methylglyoxal synthase gene (mgsA) from Clostridium beijerinckii NCIMB 8052 was homologously expressed in this organism. Additionally, a separate strain of Clostridium beijerinckii NCIMB 8052 was engineered by cloning and expressing mgsA and methylglyoxal/glyoxal reductase (mgR) from Clostridium pasteurianum ATCC 6013 as a fused protein linked by polyglycine linker in the former. Both strains of C. beijerinckii NCIMB 8052 failed to produce 1,2-propaneol. Instead, traces of acetol--the precursor of 1,2-propanediol--were detected in cultures of both strains. When the recombinant strains were exposed to acetol, both strains exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. Conversely, methylglyoxal supplementation led to the production of traces of acetol but not lactaldehyde or 1,2-propanediol. When wildtype C. beijerinckii NCIMB 8052, C. pasteurianum ATCC 6013 and Clostridium tyrobutyricum ATCC 25755 were challenged with methylglyoxal, C. beijerinckii produced [~]0.1 g/L (S)-(+)-1,2-Propanediol, while C. tyrobutyricum produced traces of lactate. C. pasteurianum produced neither 1,2-propanediol nor lactate. The wild types of all three species above exhibited [~]100% acetol-to-1,2-propanediol conversion efficiency. The recombinant strain of C. beijerinckii expressing fused MgsA and MgR from C. pasteurianum ATCC 6013 showed enhanced growth and solvent production, producing as high as 88% more butanol on both glucose and lactose than the control strain and the recombinant strain of the same organism expressing the native MgsA. ConclusionsRecombinant and native strains of C. beijerinckii, C. pasteurianum and C. tyrobutyricum studied in this work exhibit extremely poor capacity to catalyze the conversion of the intermediates of the methylglyoxal bypass to 1,2-propanediol. This is indicative of lack of appropriate enzymes to catalyze the reactions from methylglyoxal to acetol or lactaldehyde. Inability to detect methylglyoxal in the recombinant strains harboring mgsA (both homologous and heterologous)-- whereas the strain expressing both mgsA and mgR from C. pasteurianum, under the same promoter (Padc) produced higher concentrations of butanol--suggests that C. beijerinckii might possess a regulatory mechanism that limits the activity of methylglyoxal-producing MgsA. The protein product of mgR from C. pasteurianum represents a promising metabolic engineering candidate towards increasing butanol production.

microbiology↗