Rodríguez S, Kayser M M, Stewart J D
Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
J Am Chem Soc. 2001 Feb 28;123(8):1547-55. doi: 10.1021/ja0027968.
While whole cells of baker's yeast (Saccharomyces cerevisiae) are a convenient biocatalytic reducing agent for a wide variety of carbonyl compounds, mixtures of stereoisomeric alcohols are often observed since the organism contains a large number of reductase enzymes with overlapping substrate specificities but differing stereoselectivities. We sought to improve the performance of baker's yeast for beta-keto ester reductions by using recombinant DNA techniques to alter the levels of three enzymes known to play important roles in these reactions (fatty acid synthase, Fasp; aldo-keto reductase, Ypr1p; alpha-acetoxy ketone reductase, Gre2p). A complete set of "first-generation" yeast strains that either lack or overexpress each of these three enzymes was created and tested for improvements in stereoselective reductions of a series of beta-keto esters. On the basis of these results, multiply modified ("second-generation") strains were created that combined gene knockout and overexpression in single strains. In some cases, these additional modifications further improved the stereoselectivities of beta-keto ester reductions, thereby making several beta-hydroxy ester building blocks readily available by reactions that can be performed by nonspecialists. This work also revealed that additional yeast proteins participate in reducing beta-keto esters, and further progress using this strategy will require either additional genetic manipulations or the expression of yeast reductases in hosts that lack enzymes with overlapping substrate specificity.
虽然面包酵母（酿酒酵母）的全细胞是用于多种羰基化合物的便捷生物催化还原剂，但由于该生物体含有大量具有重叠底物特异性但立体选择性不同的还原酶，常常会观察到立体异构醇的混合物。我们试图通过使用重组DNA技术改变已知在这些反应中起重要作用的三种酶（脂肪酸合酶，Fasp；醛酮还原酶，Ypr1p；α-乙酰氧基酮还原酶，Gre2p）的水平，来提高面包酵母还原β-酮酯的性能。构建了一套完整的“第一代”酵母菌株，这些菌株要么缺失要么过表达这三种酶中的每一种，并测试其对一系列β-酮酯立体选择性还原的改善情况。基于这些结果，构建了多重修饰的（“第二代”）菌株，这些菌株在单个菌株中结合了基因敲除和过表达。在某些情况下，这些额外的修饰进一步提高了β-酮酯还原的立体选择性，从而使几种β-羟基酯结构单元可通过非专业人员能够进行的反应轻松获得。这项工作还表明，其他酵母蛋白也参与β-酮酯的还原，使用该策略取得进一步进展将需要进行额外的基因操作，或者在缺乏具有重叠底物特异性的酶的宿主中表达酵母还原酶。
