A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation.
A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation.
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DOI：
10.1186/s12934-018-1036-2
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发表时间：
2018-12-03
影响因子：
6.4
通讯作者：
Katagihallimath N
中科院分区：
文献类型：
--
作者：
Haq SF;Shanbhag AP;Karthikeyan S;Hassan I;Thanukrishnan K;Ashok A;Sukumaran S;Ramaswamy S;Bharatham N;Datta S;Samant S;Katagihallimath N
化学工业一直在寻找一种快速和环保的方法来生产手性合成物。酮还原酶已被用作催化剂，用于将所需的前手性酮对映选择性转化为相应的醇。我们选择了已报道的属于不同蛋白家族的杂交酮还原酶，并在大肠杆菌中表达它们，以评估它们作为获得具有药用意义的手性醇中间体的全细胞催化剂的能力。除了建立一种方法来生产高价值(S)特异性醇，以前没有评估过，我们提出了一个硅分析程序来预测产品手性。本文比较了来自Sulfolobus sulfotaricus、Zygosaccharomyces rouxii、Hansenula polymorpha、棒状杆菌sp. ST-10、聚球菌sp. PCC 7942和芽孢杆菌sp. ECU0013的六种酶对不同底物的高效活性，以确定该方法的最佳酶。使用过表达这些酶的大肠杆菌对阿瑞吡坦、西格列汀和Dolastatin等药物的酮类中间体进行全细胞催化，产生(S)特异性手性醇。我们解释了这种手性特异性的最佳表现的酶，即Z. rouxii酮还原酶使用在硅模型和MD模拟。这一理论基础应用于另外五种用于合成克唑替尼、MA-20565（一种抗真菌剂）、苏洛培南、利瓦斯汀、塔拉姆和巴尼地平的酮类，并预测了(S)对映体的产率。实验评价与硅分析相吻合，通过生物转化获得了~ 95% (S)特异性醇，化学收率为23-79%。此外，通过将碳源从葡萄糖转换为山梨醇，优化了辅因子的再循环，将化学收率提高到85-99%。在这里，我们提出了一种策略，利用辅助因子平衡的全细胞催化方案，通过酮还原酶合成药学上相关的手性醇，这对工业是有用的。根据这些试验获得的结果，Zygosaccharomyces rouxii酮还原酶被确定为从其各自的酮中获得(S)特异性醇的熟练酶。将全细胞催化剂与以山梨醇为碳源的营养调节相结合，可以获得较高的对映体和化学产率。本文的在线版本（10.1186/s12934-018-1036-2）包含补充材料，授权用户可以使用。
Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole–cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23–79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85–99%. Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole–cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield. The online version of this article (10.1186/s12934-018-1036-2) contains supplementary material, which is available to authorized users.
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影响因子：
5.4
作者：
Kallberg, Yvonne;Oppermann, Udo;Persson, Bengt
通讯作者：
Persson, Bengt
影响因子：
3
作者：
Chen, Qilei;Hu, Youjia;Zhu, Baoquan
通讯作者：
Zhu, Baoquan
影响因子：
5
作者：
Dudzik, A.;Snoch, W.;Szaleniec, M.
通讯作者：
Szaleniec, M.
影响因子：
8
作者：
Kallberg, Y;Oppermann, U;Persson, B
通讯作者：
Persson, B
从hansenii debaryomyces删除了不同短链脱氢酶还原酶的底物多样性。
影响因子：
3.7
作者：
Ghatak A;Bharatham N;Shanbhag AP;Datta S;Venkatraman J
通讯作者：
Venkatraman J