(499d) Metabolic Engineering of Clostridium Tyrobutyricum for Increased Intracellular NADH Pool and High-Yield N-Butanol Production

Clostridium tyrobutyricum is a native butyric acid productor. Due to its high metabolic flux toward butyryl-CoA, C. tyrobutyricum has also been engineered to overexpress a bifunctional aldehyde/alcohol dehydrogenase (ALD/ADH) for n-butanol production. However, the mutant C. tyrobutyricum OE::adhE2 (Ct-adhE2) also produced large amounts of butyrate, acetate, and ethanol, which not only reduced the butanol yield but also increased the downstream processing cost for product purification. Compared to butyrate and ethanol, butanol biosynthesis requires two more NADH. The limited intracellular NADH pools is limiting butanol production in the fermentation. In this study, we aimed to increase the reducing equivalent (NADH) for butanol biosynthesis in C. tyrobutyricum, which should increase butanol selectivity over ethanol and acids. Formate dehydrogenase (FDH) could facilitate the regeneration of NADH from NAD⁺ with format as the electron donor. Ferredoxin-NAD(P)⁺ oxidoreductase (FNR) is involved in electron transfer from the reduced ferredoxin to NAD(P)⁺. Thus, we hypothesized that overexpressing FNR (encoded by fnr and nfnAB) or FDH (encoded by fdh) could increase intracellular NAD(P)H pool and butanol biosynthesis. C. tyrobutyricum mutant strains co-expressing adhE2 with fdh, fnr, or nfnAB were constructed and evaluated for their performance in batch fermentation. In general, mutants co-expressing fdh, fnr or nfnAB with adhE2 showed increases in C4/C2, alcohol/acid, and butanol/ethanol ratios, confirming their benefits on increasing intracellular NADH pool to favor butanol biosynthesis. In batch fermentation kinetics studies carried out in serum bottles, one engineered C. tyrobutyricum strain produced butanol at 0.3 g/g glucose, more than 50% increase compared to the strain expressing only adhE2. Metabolic engineering strategies for further increasing butanol production by enhancing metabolic flux toward butyryl-CoA via overexpressing heterologous thiolase (thl) and 3-hydroxybutyryl-CoA dehydrogenase (hbd) and knockout of the CoA transferase (cat1) responsible for the biosynthesis of butyrate in C. tyrobutyricum will also be discussed in the presentation.