Asymmetric whole-cell bioreduction of (R)-carvone by recombinant Escherichia coli with in situ substrate supply and product removal
Asymmetric whole-cell bioreduction of (R)-carvone by recombinant Escherichia coli with in situ substrate supply and product removal
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DOI：
10.1016/j.bej.2016.10.002
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发表时间：
2017-01-15
影响因子：
3.9
通讯作者：
Weuster-Botz, Dirk
中科院分区：
文献类型：
--
作者：
Castiglione, Kathrin;Fu, Yilei;Weuster-Botz, Dirk
手性结构单元（2 R，5 R）-二氢香芹酮可以通过使用烯-还原酶不对称还原（R）-香芹酮的C=C键来合成。然而，由于底物的毒性、其在水性介质中的低溶解度以及作为副产物的醇的形成，全细胞生物转化具有挑战性。这里，使用过表达来自念珠藻属PCC 7120的烯-还原酶的大肠杆菌生产（2 R，5 R）-二氢香芹酮。母牛分枝杆菌甲酸脱氢酶的NADP（+）接受突变体再生辅因子。而在50 mM（R）-香芹酮的生物转化过程中未检测到作为副产物的醇，观察到宿主细胞介导的产物异构化，导致5 h后81.7%的低非对映体过量（de）。此外，底物的毒性导致27.2%的低转化率。这两种效果都成功地防止，在原位基板进料和产品去除使用水不混溶的离子液体或疏水吸附树脂。优化了双相生物还原反应，并将时空产率最高的反应体系转移到升规模。在优化的条件下（300 mM（R）-香芹酮，400 mM甲酸，36 g L-1生物催化剂，XAD 4，树脂与底物质量比为5，300 mM磷酸盐缓冲液，pH6.3），（2 R; 5 R）-二氢香芹酮在9 h内的转化率为96.8%和96.5%。(C)2016爱思唯尔B. V.保留所有权利。
The chiral building block (2R,5R)-dihydrocarvone can be synthesized by asymmetric reduction of the C=C bond of (R)-carvone using ene-reductases.,However, whole-cell biotransformations are challenging due to the toxicity of the substrate, its low solubility in aqueous media and the formation of alcohols as by-products. Here, (2R,5R)-dihydrocarvone was produced using Escherichia coli overexpressing an ene-reductase from Nostoc sp. PCC 7120. A NADP(+)-accepting mutant of the formate dehydrogenase from Mycobacterium vaccae regenerated cofactors. Whereas alcohols as by-products were not detected during biotransformation of 50 mM (R)-carvone, a host cell-mediated product isomerization was observed leading to a low diastereomeric excess (de) of 81.7% after 5 h. Moreover, the toxicity of the substrate resulted in a low conversion of 27.2%. Both effects were successfully prevented by, in situ substrate feeding and product removal using water-immiscible ionic liquids or hydrophobic adsorbent resins. The biphasic bioreductions were optimized and the reaction system with the highest space-time yield was transferred to the liter scale. Under the optimized conditions (300 mM (R)-carvone, 400 mM formate, 36 g L-1 biOcatalyst, XAD4 at a resin to substrate mass ratio of 5, 300 mM phosphate buffer, pH 6.3), (2R;5R)-dihydrocarvone was obtained with 96.5% de and 96.8% conversion within 9 h. (C) 2016 Elsevier B.V. All rights reserved.