Metabolic Engineering of <i>Clostridium tyrobutyricum</i> for High-Yield <i>n</i>-Butanol Production by Increasing Intracellular Reducing Equivalent with NADPH-Dependent 3-Hydroxybutyryl-CoA Dehydrogenase
Jun Feng, Qingke Wang, Xiaolong Guo, Jialei Hu, Geng Wang, Li Lü, Zhen Qin, Hongxin Fu, Jufang Wang, Shang‐Tian Yang
Abstract
Clostridium tyrobutyricum was engineered to overexpress adh E2 encoding the aldehyde/alcohol dehydrogenase and an exogenous NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri (Ck hbd ) 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 Ck hbd and adh E2 in C. tyrobutyricum wild type (WT), Ack, Δ hyd A, and Δ cat 1 strains. Except for the Δ cat 1 strains, strains coexpressing Ck hbd 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 adh E2. In the presence of methyl viologen, the strain Ack- adh E2-Ck hbd 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.