Improved Product-Per-Glucose Yields in P450-Dependent Propane Biotransformations Using Engineered Escherichia coli
Improved Product-Per-Glucose Yields in P450-Dependent Propane Biotransformations Using Engineered Escherichia coli
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DOI：
10.1002/bit.22984
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发表时间：
2011-03-01
影响因子：
3.8
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
--
作者：
Fasan, Rudi;Crook, Nathan C.;Arnold, Frances H.
大肠杆菌中依赖于P450的生物转化对于使用温和和可持续的程序选择性氧化有机分子是有吸引力的。然而，这些过程的总体效率依赖于来源于碳源氧化的NAD（P）H辅因子在细胞内如何有效地用于支持异源P450催化的反应。在这项工作中，我们研究了在全细胞反应中使用代谢和蛋白质工程来提高每葡萄糖的产物产率（Y-PPG），所述全细胞反应涉及通过定向进化制备的熟练的NADPH依赖性P450丙烷单加氧酶[P450（PMO）R2; Fasan等人（2007）; Angew Chem Int艾德46：8414-8418]。本研究表明，E.大肠杆菌（W3110）在需氧、非生长条件下仅能够支持适度的丙醇：葡萄糖摩尔比（Y-PPG类似于0.5）。通过改变参与宿主NAD（P）H代谢的关键过程，可以大幅提高该比例。代谢工程E.大肠杆菌菌株的特征在于内源性呼吸链的部分失活（Δ ndh）结合两种发酵途径的去除（Δ adhE，Δ ldh），在所研究的菌株中提供最高的Y-PPG（1.71），使得与天然E.大肠杆菌菌株。使用工程化的P450（PMO）R2变体，其可以以相等的效率利用NADPH和NADH，我们还确定了P450酶的双辅因子特异性可以提供YPPG的明显改善。动力学分析表明，然而，更有利的参数（K-M，k（猫））的NADH驱动的反应需要有效地与宿主的内源性NADH利用酶竞争。总之，本文所述的代谢/蛋白质工程策略对于改善E.杆菌Biotechnol. Bioeng. 2011; 108：500-510。(C)2010 Wiley Periodicals，Inc.
P450-dependent biotransformations in Escherichia coli are attractive for the selective oxidation of organic molecules using mild and sustainable procedures. The overall efficiency of these processes, however, relies on how effectively the NAD(P)H cofactors derived from oxidation of the carbon source are utilized inside the cell to support the heterologous P450-catalyzed reaction. In this work, we investigate the use of metabolic and protein engineering to enhance the product-per-glucose yield (Y-PPG) in whole-cell reactions involving a proficient NADPH-dependent P450 propane monooxygenase prepared by directed evolution [P450(PMO)R2; Fasan et al. (2007); Angew Chem Int Ed 46:8414-8418]. Our studies revealed that the metabolism of E. coli (W3110) is able to support only a modest propanol: glucose molar ratio (Y-PPG similar to 0.5) under aerobic, non-growing conditions. By altering key processes involved in NAD(P) H metabolism of the host, considerable improvements of this ratio could be achieved. A metabolically engineered E. coli strain featuring partial inactivation of the endogenous respiratory chain (Delta ndh) combined with removal of two fermentation pathways (Delta adhE, Delta ldh) provided the highest Y-PPG (1.71) among the strains investigated, enabling a 230% more efficient utilization of the energy source (glucose) in the propane biotransformation compared to the native E. coli strain. Using an engineered P450(PMO)R2 variant which can utilize NADPH and NADH with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in YPPG. Kinetic analyses suggest, however, that much more favorable parameters (K-M, k(cat)) for the NADH-driven reaction are required to effectively compete with the host's endogenous NADH-utilizing enzymes. Overall, the metabolic/protein engineering strategies described here can be of general value for improving the performance of NAD(P)H-dependent whole-cell biotransformations in E. coli. Biotechnol. Bioeng. 2011; 108: 500-510. (C) 2010 Wiley Periodicals, Inc.