Akhtar M Kalim, Jones Patrik R
Research & Development Division, Fujirebio Inc., 51 Komiya-cho, Hachioji-shi, Tokyo 192-0031, Japan.
Metab Eng. 2009 May;11(3):139-47. doi: 10.1016/j.ymben.2009.01.002. Epub 2009 Jan 25.
A synthetic pyruvate:H(2) pathway was constructed in Escherichia coli BL21(DE3) by co-expression of six proteins: E. coli YdbK, Clostridium pasteurianum [4Fe-4S]-ferredoxin, and Clostridium acetobutylicum HydF, HydE, HydG, and HydA. The effect of cofactor addition and host strain on H(2) yield and fermentation product accumulation was studied, together with in vitro reconstitution of the entire pathway. The deletion of iscR and/or the addition of thiamine pyrophosphate to the medium enhanced the total and specific activity of recombinant YdbK and increased the yield of H(2) per glucose. It was concluded that the introduced pathway outcompeted other pyruvate-consuming reactions, and that the ability to compete for pyruvate at least in part was determined by total YdbK activity. The results demonstrate the successful construction of a high-yielding H(2) pathway in a microorganism that effectively does not synthesize any H(2). The additional co-expression of Bacillus subtilis AmyE enabled starch-dependent H(2) synthesis in minimal media.
通过共表达六种蛋白质，即大肠杆菌YdbK、巴氏梭菌[4Fe-4S]-铁氧化还原蛋白以及丙酮丁醇梭菌HydF、HydE、HydG和HydA，在大肠杆菌BL21(DE3)中构建了一条合成的丙酮酸：H₂途径。研究了辅因子添加和宿主菌株对H₂产量及发酵产物积累的影响，同时对整个途径进行了体外重构。缺失iscR和/或向培养基中添加硫胺素焦磷酸可提高重组YdbK的总活性和比活性，并增加每葡萄糖的H₂产量。得出的结论是，引入的途径胜过其他消耗丙酮酸的反应，并且至少部分竞争丙酮酸的能力由YdbK的总活性决定。结果表明，在一种实际上不合成任何H₂的微生物中成功构建了一条高产H₂途径。额外共表达枯草芽孢杆菌AmyE可在基本培养基中实现淀粉依赖性H₂合成。
