Metabolic Engineering of Clostridium tyrobutyricum for High-Yield n-Butanol Production by Increasing Intracellular Reducing Equivalent with NADPH-Dependent 3-Hydroxybutyryl-CoA Dehydrogenase

Clostridium tyrobutyricum was engineered to overexpress adhE2 encoding the aldehyde/alcohol dehydrogenase and an exogenous NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri (Ckhbd) for n-butanol production. In general, large amounts of butyrate, acetate, and ethanol are also produced from glucose when butanol biosynthesis is hindered by limited intracellular NADH pools. In silico flux balance analysis showed that coupling NADP⁺/NADPH turnover with butanol production increased the reducing equivalent supply and butanol selectivity over ethanol and acids, thus increasing butanol production from glucose. This was verified with the coexpression of Ckhbd and adhE2 in C. tyrobutyricum wild type (WT), Ack, ΔhydA, and Δcat1 strains. Except for the Δcat1 strains, strains coexpressing Ckhbd showed significant (>5%) increase in reducing equivalents, 50-60% increase in butanol production (butanol yield: 0.24-0.28 vs. 0.15-0.18 g/g), and 2.5- to 4.5-fold increases in butanol/ethanol and alcohols/acids ratios due to increased flux from acetyl-CoA to butyryl-CoA and reducing equivalents compared to the strains expressing only adhE2. In the presence of methyl viologen, the strain Ack-adhE2-Ckhbd produced the highest butanol yield of 0.36 g/g, ∼88% of the theoretical yield from glucose, which was among the highest yields reported for known solventogenic clostridia.