Whole-cell bioreduction of aromatic α-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli
Whole-cell bioreduction of aromatic α-keto esters using Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase co-expressed in Escherichia coli
复制标题
DOI：
10.1186/1475-2859-7-37
复制
发表时间：
2008-12-10
影响因子：
6.4
通讯作者：
Nidetzky, Bernd
中科院分区：
文献类型：
--
作者：
Kratzer, Regina;Pukl, Matej;Nidetzky, Bernd
背景：全细胞催化的生物转化是通过对映体选择性还原前体酮来生产手性醇的一种明确的工艺选择。在大肠杆菌中异源表达了多种合成有用的还原酶，并具有很高的活性。因此，该微生物已成为进行不同规模的全细胞生物还原的主要系统。在大肠杆菌中，中心代谢途径的能力有限，通常需要在第二个依赖于NADP(+)或NAD(+)的脱氢酶的共同表达下，通过适当的氧化反应再生NADPH或NADH形式的还原酶辅酶。细嫩假丝酵母木糖还原酶(CtXR)具有较高的Prelog型立体选择性，可促进NADH依赖的芳香族α-酮酸酯的还原。本文描述了一种新的全细胞生物催化剂的开发，该催化剂是基于一株共表达CtXR和来自假丝酵母甲酸脱氢酶(CbFDH)的大肠杆菌。结果：pETDuet-1载体共表达的CtXR和CbFDH活性分别为260和90个单位/g干大肠杆菌细胞质量(g(CDW))。完整细胞和多粘菌素B硫酸盐通透性细胞对4-氰基苯甲酰甲酸乙酯的最大转化率(r(S))相似(2 mmol/g(Cdw)h)，表明CbFDH的活性总体上是部分限速的。底物中的非催化酯水解以及在α-酮酯存在下CtXR和CbFDH的失活是酒精产品产率的主要限制因素。在优化的反应条件下(100mM底物，40g(CDW)/L)，我们得到了对映体过量的R-4-氰基扁桃酸乙酯(E.)收率82%，收率97.2%。但是，通过将底物浓度增加到500 mm，E.可以提高到100%，但代价是产量降低3倍。酿酒酵母重组菌株将100 mM底物转化为45 mM R-4-氰基杏仁酸乙酯，其等电点为99.9%。对重组大肠杆菌的修饰(细胞通透性；添加外源NAD(+))和添加水不相容的溶剂(例如己烷或1-丁基-3-甲基咪唑六氟磷酸盐)是无用的。为了增强系统中NADH再生的整体能力，我们在渗透后的原始生物催化剂中添加了仅表达CbFDH(410U/g(CDW))的通透性大肠杆菌细胞。FDH与XR活性比从2变到20对产率(18%-gt；62%；100 mm底物)的积极影响被相应的产物对映体纯度从86%降至71%所抵消。结论：基于大肠杆菌共表达CtXR和CbFDH的全细胞系统是合成高光学纯度和高产量的R-4-环甘油酸乙酯的强有力的生物催化剂。进一步优化生物催化剂的比生产率的一个明确要求是通过提高胞内FDH活性水平(=10倍)来消除NADH再生的动力学瓶颈。
Background: Whole cell-catalyzed biotransformation is a clear process option for the production of chiral alcohols via enantioselective reduction of precursor ketones. A wide variety of synthetically useful reductases are expressed heterologously in Escherichia coli to a high level of activity. Therefore, this microbe has become a prime system for carrying out whole-cell bioreductions at different scales. The limited capacity of central metabolic pathways in E. coli usually requires that reductase coenzyme in the form of NADPH or NADH be regenerated through a suitable oxidation reaction catalyzed by a second NADP(+) or NAD(+) dependent dehydrogenase that is co-expressed. Candida tenuis xylose reductase (CtXR) was previously shown to promote NADH dependent reduction of aromatic alpha-keto esters with high Prelog-type stereoselectivity. We describe here the development of a new whole-cell biocatalyst that is based on an E. coli strain co-expressing CtXR and formate dehydrogenase from Candida boidinii (CbFDH). The bacterial system was evaluated for the synthesis of ethyl R-4-cyanomandelate under different process conditions and benchmarked against a previously described catalyst derived from Saccharomyces cerevisiae expressing CtXR.Results: Gene co-expression from a pETDuet-1 vector yielded about 260 and 90 units of intracellular CtXR and CbFDH activity per gram of dry E. coli cell mass (g(CDW)). The maximum conversion rate (r(S)) for ethyl 4-cyanobenzoylformate by intact or polymyxin B sulphate-permeabilized cells was similar (2 mmol/g(CDW)h), suggesting that the activity of CbFDH was partly rate-limiting overall. Uncatalyzed ester hydrolysis in substrate as well as inactivation of CtXR and CbFDH in the presence of the alpha-keto ester constituted major restrictions to the yield of alcohol product. Using optimized reaction conditions (100 mM substrate; 40 g(CDW)/L), we obtained ethyl R-4-cyanomandelate with an enantiomeric excess (e. e.) of 97.2% in a yield of 82%. By increasing the substrate concentration to 500 mM, the e. e. could be enhanced to congruent to 100%, however, at the cost of a 3-fold decreased yield. A recombinant strain of S. cerevisiae converted 100 mM substrate to 45 mM ethyl R-4-cyanomandelate with an e. e. of >= 99.9%. Modifications to the recombinant E. coli (cell permeabilisation; addition of exogenous NAD(+)) and addition of a water immiscible solvent (e. g. hexane or 1-butyl-3-methylimidazolium hexafluorophosphate) were not useful. To enhance the overall capacity for NADH regeneration in the system, we supplemented the original biocatalyst after permeabilisation with also permeabilised E. coli cells that expressed solely CbFDH (410 U/g(CDW)). The positive effect on yield (18%-> 62%; 100 mM substrate) caused by a change in the ratio of FDH to XR activity from 2 to 20 was invalidated by a corresponding loss in product enantiomeric purity from 86% to only 71%.Conclusion: A whole-cell system based on E. coli co-expressing CtXR and CbFDH is a powerful and surprisingly robust biocatalyst for the synthesis of ethyl R-4-cyanomandelate in high optical purity and yield. A clear requirement for further optimization of the specific productivity of the biocatalyst is to remove the kinetic bottleneck of NADH regeneration through enhancement (>= 10-fold) of the intracellular level of FDH activity.