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bioRxiv · 10.1101/2024.02.27.582317

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

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

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.

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Kumar, S., Agyeman-Duah, E., Ujor, V. C.. 2024-02-28. Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013. https://doi.org/10.1101/2024.02.27.582317

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