(485b) Comparative Transcriptomic and Proteomics Analyses of Engineered Clostridium Tyrobutyricum for Butanol Production

Butanol is a promising feedstock for sustainable aviation fuel due to tis compatibility with existing infrastructure and potential for production from renewable resources. However, a major bottleneck in its fermentation process lies in the sporulation of Clostridium species, which suspends cellular metabolism and solvent biosynthesis. Targeting histidine kinases (HKs), key regulators in the sporulation pathway, presents a compelling strategy to enhance butanol fermentation through genome engineering.

To elucidate the underlying metabolic and regulatory mechanisms, we performed comparative transcriptomic and proteomic analyses of two engineered Clostridium tyrobutyricum strains. WT-adhE2 is a wild-type strain overexpressing aldehyde/alcohol dehydrogenase (adhE2) from C. acetobutylicum, enabling conversion of butyryl-CoA to butanol. DSpo0A-adhE2 is CRISPR-engineered strain with deletion of Spo0A, the sporulation transcription factor, along with adhE2 overexpression.

Batch fermentation studies assessed butanol production, cellular growth, glucose consumption, and metabolic intermediates profiles. Comparative transcriptomic analysis using RNA sequencing and ExpressAnalyst Pro identified 85 differentially expressed genes in the DSpo0A-adhE2 strain with 13 upregulated and 72 downregulated. It was observed that downregulation of genes included those encoding spore coat proteins (CTK_RS01415) and spore-associated proteins CotJA and CotJB (CTK_RS03515 and CTK_RS03510), while upregulated genes included ATP-binding protein (CTK_RS10890) and pyruvate formate-lyase activating protein (CTK_RS10680).

Proteomic analyses were performed on additional engineered strains (Dcat1::adhE2 and Dack_adhE2). Mass spectra (MS/MS) data were analyzed using SEQUEST against the Uniprot proteome database, and proteins were filtered, grouped, and quantified using Scaffold. It resealed 17 downregulated and 3 upregulated proteins. Key targets include CoA transferases (critical for CoA transfer in butanol biosynthesis), glycerol-3-phosphate dehydrogenase (GPDH; involved in NADH redox balance), and the DNA repair protein RecN (linked to cell robustness and survival).

Pathway enrichment and gene set enrichment analyses identified 10 significantly affected pathways, including amino acid and glutathione metabolism. Overall, this integrated transcriptomic and proteomic study provides valuable insights into the understanding of the genetic and proteomic shifts driving enhanced butanol production in C. tyrobutyricum. The identified genes and proteins offer promising targets for further metabolic engineering to enhance butanol yield, tolerance and process robustness in C. tyrobutyricum.