Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase
Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase
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DOI：
10.1128/aem.00301-14
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发表时间：
2014-06-01
影响因子：
4.4
通讯作者：
Patrick, Wayne M.
中科院分区：
文献类型：
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作者：
Koepke, Michael;Gerth, Monica L.;Patrick, Wayne M.
产乙酸细菌使用CO和/或CO2加上H-2作为其唯一的碳源和能源。利用这些生物的发酵过程有望从丰富的废气原料中生产化学品和生物燃料，同时减少工业温室气体排放。已知产乙酸梭菌在气体发酵期间合成乳酸盐衍生的代谢物乳酸盐和2，3_丁二醇。在工业上，2，3-丁二醇对于化学生产是有价值的。在这里，我们确定和表征的C。用于乳酸和2，3-丁二醇生物合成的自产乙醇酶。假定的C。当在大肠杆菌中表达时，自产乙醇乳酸脱氢酶具有活性。2，3-丁二醇途径在E.通过克隆和表达乙酰乳酸合成酶、乙酰乳酸脱羧酶和2，3-丁二醇脱氢酶的候选基因，将其转化大肠杆菌。在厌氧条件下，产生的E.大肠杆菌菌株产生1.1 +/- 0.2 mM 2 R，3R-丁二醇（23 μ M h（-1）光密度单位（-1）），这与C.在含CO的废气上生长的过程中，除了2，3-丁二醇脱氢酶之外，我们还鉴定了严格依赖于NADPH的伯-仲醇脱氢酶（CaADH），其可以将乙偶姻还原为2，3-丁二醇。详细的动力学分析表明，CaADH接受一系列的2-，3-和4-碳底物，包括非生理酮丙酮和丁酮。CaADH对丙酮的高活性使我们预测，并通过实验证实，C。autoethanogenum可以作为将外源丙酮转化为异丙醇的全细胞生物催化剂。总之，我们的结果在功能上验证了来自C. autoethanogenum，鉴定CaADH作为进一步工程改造的靶标，并证明C. autoethanogenum作为可持续化学生产的平台。
Acetogenic bacteria use CO and/or CO2 plus H-2 as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogen Clostridium autoethanogenum is known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize the C. autoethanogenum enzymes for lactate and 2,3-butanediol biosynthesis. The putative C. autoethanogenum lactate dehydrogenase was active when expressed in Escherichia coli. The 2,3-butanediol pathway was reconstituted in E. coli by cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resulting E. coli strain produced 1.1 +/- 0.2 mM 2R,3R-butanediol (23 mu M h(-1) optical density unit(-1)), which is comparable to the level produced by C. autoethanogenum during growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, that C. autoethanogenum can act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway from C. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential of C. autoethanogenum as a platform for sustainable chemical production.