(166j) Metabolic Process Engineering of Clostridium Tyrobutyricum ?cat1::AdhE2 for Enhanced n-Butanol Production: Effects of Mannitol and Methyl Viologen on Flux Distribution

Clostridium tyrobutyricum, a Gram-positive, spore-forming, obligate anaerobic bacterium, is a promising microbial cell factory for butyric acid and n-butanol production because of its high metabolic flux toward butyryl-CoA, high butyric acid/butanol tolerance, and excellent ability to use both glucose and xylose present in lignocellulosic hydrolysates. Unlike Clostridium acetobutylicum and many other clostridia, C. tyrobutyricum uses CoA transferase (encoded by cat1), instead of phosphotransbutyrylase (PTA) and butyrate kinase (BK), to convert butyryl-CoA to butyrate, which transfers the CoA to acetate to form acetyl-CoA, thus allowing the reassimilation of acetate. C. tyrobutyricum has been engineered to overexpress adhE2, encoding bifunctional aldehyde/alcohol dehydrogenase, for n-butanol production from various carbon sources. More recently, CRISPR was used to insert adhE2 and replace cat1 on the genome in C. tyrobutyricum. The mutant Ct-Δcat1::adhE2 produced n-butanol at the highest titer ever reported for any known microorganisms and little butyrate, with acetate and ethanol also produced in higher amounts, resulting in a low butanol yield of ~0.2 g/g glucose due to increased acetate and ethanol production. Compared to ethanol, butanol production requires 2 more NADH. We hypothesized that increasing the intracellular NADH pool would increase butanol production. The effects of using the more reduced substrate mannitol and methyl viologen (MV) as an electron carrier on carbon flux distribution and butanol biosynthesis in CT-Δcat1::adhE2 were studied. With mannitol as the substrate, CT-Δcat1::adhE2 produced 17.4 g/L butanol at a productivity of 0.36 g/L‧h and yield of 0.27 g/g, an increase of 36% compared to glucose as the substrate. With 200 μM MV added in the medium, butanol production from glucose increased to a high yield of 0.29 g/g with significantly decreased acetate and ethanol production. Thus, high-yield n-butanol production can be realized using engineered C. tyrobutyricum with mannitol as the substrate or MV as an electron carrier in glucose fermentation.