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bioRxiv · 10.1101/2024.02.22.581430

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

Agyeman-Duah, E.·Kumar, S.·Ujor, V.
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Abstract

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.

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Agyeman-Duah, E., Kumar, S., Ujor, V.. 2024-02-22. Unravelling the roadblocks to 1,2-propanediol biosynthesis in select solventogenic Clostridium species. https://doi.org/10.1101/2024.02.22.581430

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