Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.
Metabolic engineering of Clostridium autoethanogenum for selective alcohol production.
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DOI：
10.1016/j.ymben.2017.01.007
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发表时间：
2017-03
影响因子：
8.4
通讯作者：
Minton NP
中科院分区：
文献类型：
--
作者：
Liew F;Henstra AM;Kӧpke M;Winzer K;Simpson SD;Minton NP
利用产乙酸梭菌等产乙酸菌进行气体发酵，为利用工业废气生产燃料乙醇提供了一条诱人的途径。乙酸乙酯通过乙醛还原为乙醛，进而通过乙醛还原为乙醇：铁氧还蛋白氧化还原酶(AOR)和乙醇脱氢酶(AOR)与乙醇脱氢酶(ADHE)形成乙醇的经典途径一样被认为是乙醛/乙醇脱氢酶(ADHE)。在这里，我们证明了AOR对乙酸菌中乙醇的形成至关重要，而ADHE的失活导致自养乙醇产量持续增加(高达180%)。利用Clostron和等位基因交换突变(首次在乙酸根中被证实)，我们产生了AOR和ADHE亚型的单突变和双突变，以确认每个基因的作用。ORAN1+2双基因敲除菌株失去了将外源醋酸酯、丙酸和丁酸转化为相应酒精的能力，进一步突出了这些酶在催化从热力学上不利的羧酸还原为酒精方面的作用。通过代谢工程使自产乙烷菌的乙醇产量提高180%。证实了AOR在乙酸菌自养乙醇生产中的作用。产生了自产乙烷假单胞菌的AoR和adhe突变体。证实了乙酸菌中稳定缺失的等位基因交换突变。灭活adhe和aor2，而不是oa1，可以提高自养乙醇的产量。
Gas fermentation using acetogenic bacteria such as Clostridium autoethanogenum offers an attractive route for production of fuel ethanol from industrial waste gases. Acetate reduction to acetaldehyde and further to ethanol via an aldehyde: ferredoxin oxidoreductase (AOR) and alcohol dehydrogenase has been postulated alongside the classic pathway of ethanol formation via a bi-functional aldehyde/alcohol dehydrogenase (AdhE). Here we demonstrate that AOR is critical to ethanol formation in acetogens and inactivation of AdhE led to consistently enhanced autotrophic ethanol production (up to 180%). Using ClosTron and allelic exchange mutagenesis, which was demonstrated for the first time in an acetogen, we generated single mutants as well as double mutants for both aor and adhE isoforms to confirm the role of each gene. The aor1+2 double knockout strain lost the ability to convert exogenous acetate, propionate and butyrate into the corresponding alcohols, further highlighting the role of these enzymes in catalyzing the thermodynamically unfavourable reduction of carboxylic acids into alcohols. 180% improvement in C. autoethanogenum ethanol production via metabolic engineering. Confirmed role of AOR in autotrophic ethanol production of acetogens. Generated both aor and adhE mutants of C. autoethanogenum.. Demonstrated allelic exchange mutagenesis for stable deletions in acetogens. Inactivation of adhE and aor2, but not aor1, improves autotrophic ethanol production.