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首页> 外文期刊>Applied Microbiology >Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase
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Enantioselective Synthesis of Vicinal (R,R)-Diols by Saccharomyces cerevisiae Butanediol Dehydrogenase

机译：酿酒酵母丁二醇脱氢酶对选择性（R，R）-二元醇的对映选择性合成

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Butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae belongs to the superfamily of the medium-chain dehydrogenases and reductases and converts reversibly R -acetoin and S -acetoin to (2 R ,3 R )-2,3-butanediol and meso -2,3-butanediol, respectively. It is specific for NAD(H) as a coenzyme, and it is the main enzyme involved in the last metabolic step leading to (2 R ,3 R )-2,3-butanediol in yeast. In this study, we have used the activity of Bdh1p in different forms—purified enzyme, yeast extracts, permeabilized yeast cells, and as a fusion protein (with yeast formate dehydrogenase, Fdh1p)—to transform several vicinal diketones to the corresponding diols. We have also developed a new variant of the delitto perfetto methodology to place BDH1 under the control of the GAL1 promoter, resulting in a yeast strain that overexpresses butanediol dehydrogenase and formate dehydrogenase activities in the presence of galactose and regenerates NADH in the presence of formate. While the use of purified Bdh1p allows the synthesis of enantiopure (2 R ,3 R )-2,3-butanediol, (2 R ,3 R )-2,3-pentanediol, (2 R ,3 R )-2,3-hexanediol, and (3 R ,4 R )-3,4-hexanediol, the use of the engineered strain (as an extract or as permeabilized cells) yields mixtures of the diols. The production of pure diol stereoisomers has also been achieved by means of a chimeric fusion protein combining Fdh1p and Bdh1p. Finally, we have determined the selectivity of Bdh1p toward the oxidation/reduction of the hydroxyl/ketone groups from (2 R ,3 R )-2,3-pentanediol/2,3-pentanedione and (2 R ,3 R )-2,3-hexanediol/2,3-hexanedione. In conclusion, Bdh1p is an enzyme with biotechnological interest that can be used to synthesize chiral building blocks. A scheme of the favored pathway with the corresponding intermediates is proposed for the Bdh1p reaction.
机译：酿酒酵母中的丁二醇脱氢酶（Bdh1p）属于中链脱氢酶和还原酶的超家族，可逆地将R-乙酰和S-乙酰转化为（2 R，3 R）-2,3-丁二醇和内消旋-2,3 -丁二醇。它是作为NAD（H）的辅酶而特异的，它是参与酵母中导致（2 R，3 R）-2,3-丁二醇的最后一个代谢步骤的主要酶。在这项研究中，我们使用了不同形式的Bdh1p活性-纯化的酶，酵母提取物，通透的酵母细胞，以及作为融合蛋白（与酵母甲酸盐脱氢酶Fdh1p融合）将几种邻位二酮转化为相应的二醇。我们还开发了一种新的delitto perfetto方法变体，将BDH1置于GAL1启动子的控制之下，从而导致酵母菌株在半乳糖存在的情况下过表达丁二醇脱氢酶和甲酸脱氢酶的活性，并在存在甲酸的情况下再生NADH。使用纯化的Bdh1p可以合成对映纯（2 R，3 R）-2,3-丁二醇，（2 R，3 R）-2,3-戊二醇，（2 R，3 R）-2,3 -己二醇和（3 R，4 R）-3,4-己二醇，使用工程菌株（作为提取物或作为透化细胞）可产生二醇的混合物。也可以通过结合Fdh1p和Bdh1p的嵌合融合蛋白来生产纯二醇立体异构体。最后，我们确定了Bdh1p对（2 R，3 R）-2,3-戊二醇/ 2,3-戊二酮和（2 R，3 R）-2羟基/酮基氧化/还原的选择性，3-己二醇/ 2,3-己二酮。总之，Bdh1p是一种具有生物技术兴趣的酶，可用于合成手性构件。针对Bdh1p反应，提出了具有相应中间体的有利途径的方案。

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