Harnessing Candida tenuis and Pichia stipitis in whole‐cell bioreductions of o‐chloroacetophenone: Stereoselectivity, cell activity, in situ substrate supply and product removal
Harnessing Candida tenuis and Pichia stipitis in whole‐cell bioreductions of o‐chloroacetophenone: Stereoselectivity, cell activity, in situ substrate supply and product removal
复制标题
利用细念珠菌和树干毕赤酵母进行邻氯苯乙酮的全细胞生物还原：立体选择性、细胞活性、原位底物供应和产物去除
作者：
Anne Tscheliessnig;Julita Konrath;Ronald Bates;A. Jungbauer
通常情况下，重组微生物和天然微生物可用作全细胞催化剂。然而，本地寄主的应用避免了菌株构建的多个步骤，从而缩短了过程开发时间。在这里，我们研究了分离的木糖还原酶及其天然宿主细小假丝酵母和树干毕赤酵母对邻氯苯乙酮的NAD(P)H依赖还原反应。将自然宿主与共表达木糖还原酶和脱氢酶的大肠杆菌菌株进行基准比较，以实现辅酶回收。细枝拟青霉和树干拟青霉的木糖培养细胞在无细胞提取液中的邻氯苯乙酮还原酶活性分别为366和90U gCDW-1。以新鲜生物量为原料，葡萄糖为共底物，间歇还原100 mM邻氯苯乙酮。反应在产物浓度约为15 mM时停止，这表明催化剂对形成的产物很敏感。添加40%正己烷的原位底物供应和产物去除提高了催化剂的稳定性。通过对水相的优化，得到了浓度为71 mM的(S)-1-(2-氯苯基)乙醇(ee>； 99.9%)。最终，天然柔嫩葡萄球菌和重组大肠杆菌之间的产量差异是<； 的1.7倍。这种光学纯度的产品是合成一类新的化疗物质(POLO样激酶1抑制剂)所需的关键中间体。
Generally, recombinant and native microorganisms can be employed as whole‐cell catalysts. The application of native hosts, however, shortens the process development time by avoiding multiple steps of strain construction. Herein, we studied the NAD(P)H‐dependent reduction of o‐chloroacetophenone by isolated xylose reductases and their native hosts Candida tenuis and Pichia stipitis. The natural hosts were benchmarked against Escherichia coli strains co‐expressing xylose reductase and a dehydrogenase for co‐enzyme recycling. Xylose‐grown cells of C. tenuis and P. stipitis displayed specific o‐chloroacetophenone reductase activities of 366 and 90 U gCDW–1, respectively, in the cell‐free extracts. Fresh biomass was employed in batch reductions of 100 mM o‐chloroacetophenone using glucose as co‐substrate. Reaction stops at a product concentration of about 15 mM, which suggests sensitivity of the catalyst towards the formed product. In situ substrate supply and product removal by the addition of 40% hexane increased catalyst stability. Optimisation of the aqueous phase led to a (S)‐1‐(2‐chlorophenyl)ethanol concentration of 71 mM (ee > 99.9%) obtained with 44 gCDW L–1 of C. tenuis. The final difference in productivities between native C. tenuis and recombinant E. coli was < 1.7‐fold. The optically pure product is a required key intermediate in the synthesis of a new class of chemotherapeutic substances (polo‐like kinase 1 inhibitors).
影响因子：
3.3
作者：
Kadosh, D;Johnson, AD
通讯作者：
Johnson, AD