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首页> 外文学位 >Baker's yeast beta-keto ester reductions: Whole cell biocatalysts with improved stereoselectivity by recombinant DNA techniques.
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Baker's yeast beta-keto ester reductions: Whole cell biocatalysts with improved stereoselectivity by recombinant DNA techniques.

机译：贝克酵母的β-酮酸酯减少：通过重组DNA技术提高立体选择性的全细胞生物催化剂。

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摘要

Baker's yeast has been widely used for asymmetric synthesis of alcohols. When a single enzyme dominates the reduction of a particular substrate, whole yeast cells can provide the chiral alcohol in high optical purity. In many cases, however, multiple enzymes with opposing stereoselectivities are involved, thereby diminishing the optical purity of the product. This problem has restricted the utility of baker's yeast for the stereoselective synthesis of beta-hydroxy esters and alpha-substituted beta-hydroxy esters.;Isolated yeast beta-keto ester reductases provide highly stereoselective biocatalysts, however, their utility is hampered by the need for enzyme isolation and cofactor regeneration. On the other hand, whole cells of baker's yeast provide a very handy and inexpensive reagent, but one that often exhibits incomplete stereoselectivity for reductions of these substrates. The optimal biocatalytic solution to production of beta-hydroxy esters would be one that combines the advantages of the isolated enzymes with those of the whole cell system. The goal of this project has been to engineer yeast strains that mirror the stereoselectivities of the individual reductases.;In this work, the stereoselectivities of baker's yeast-catalyzed reductions of beta-keto esters were manipulated by genetic design of yeast strains. Strains in which three beta-keto ester reductases were either knocked out or over-expressed have been constructed. These first-generation strains provided good improvements in stereoselectivity; however, incomplete stereoselectivity was still a common outcome of the reactions. In order to achieve higher levels of diastereo- and enantioselectivities, strains combining overexpression and knock out strategies were designed. These second-generation strains resulted in better improvements in the stereoselectivity. One of these strains furnished S-alcohols in >98% enantiomeric excess for all the tested ketones, nicely illustrating the potential of the engineered yeast strategy. In addition, other yeast strains selectively afforded the optically pure syn and anti diastereomers from reductions of appropriate keto esters.;These studies constitute the first time that a logical approach has been used to improve stereoselectivity in baker's yeast reductions and provided comparable or better improvements than those obtained with previous methodologies. The results obtained during this work also uncovered the participation of additional yeast reductases in reductions of beta-keto esters. Some of these enzymes were identified based on their homology with known reductases and expressed in Escherichia coli. Whole cells of these engineered E. coli strains also acted as useful biocatalysts for the stereoselective reductions of beta-keto esters.
机译：贝克酵母已被广泛用于醇的不对称合成。当单一酶主导特定底物的还原时，整个酵母细胞可以提供高光学纯度的手性醇。然而，在许多情况下，涉及具有相反立体选择性的多种酶，从而降低了产物的光学纯度。这个问题限制了面包酵母在β-羟基酯和α-取代的β-羟基酯的立体选择性合成中的应用。分离的酵母β-酮酯还原酶提供了高度立体选择性的生物催化剂，但是由于需要而受到限制。酶分离和辅因子再生。另一方面，面包酵母的全细胞提供了一种非常方便且便宜的试剂，但是对于减少这些底物，常常表现出不完全的立体选择性。生产β-羟基酯的最佳生物催化解决方案应是将分离的酶的优势与整个细胞系统的优势相结合的解决方案。该项目的目的是设计能够反映单个还原酶立体选择性的酵母菌株。在这项工作中，贝克酵母催化的β-酮酸酯还原反应的立体选择性是通过酵母菌株的基因设计来控制的。已经构建了其中敲除或过表达三种β-酮酯还原酶的菌株。这些第一代菌株在立体选择性方面提供了很好的改进。然而，不完全的立体选择性仍然是反应的常见结果。为了达到更高的非对映选择性和对映选择性，设计了结合过表达和敲除策略的菌株。这些第二代菌株导致更好的立体选择性。这些菌株之一为所有测试的酮提供了超过98％对映体过量的S-醇，很好地说明了工程酵母策略的潜力。此外，其他酵母菌株通过适当的酮酯的还原选择性地提供了光学纯的顺式和反非对映异构体。这些研究首次采用了一种逻辑方法来提高贝克酵母还原中的立体选择性，并提供了可比或更好的改进用以前的方法获得的那些。在这项工作中获得的结果还揭示了额外的酵母还原酶参与了β-酮酯的还原。这些酶中的一些是根据与已知还原酶的同源性鉴定的，并在大肠杆菌中表达。这些工程化的大肠杆菌菌株的全细胞还可以作为有用的生物催化剂，用于立体选择性还原β-酮酸酯。

著录项

  • 作者

    Rodriguez Giordano, Sonia.;

  • 作者单位

    University of Florida.;

  • 授予单位 University of Florida.;
  • 学科 Biochemistry.;Organic chemistry.
  • 学位 Ph.D.
  • 年度 2000
  • 页码 207 p.
  • 总页数 207
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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