1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) nitrilase whole cell catalyst. However, the nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the nitrilase activity compared with the strain containing a single integration of nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.

中文翻译：

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通过增加腈水解酶基因拷贝数和共表达ER氧化还原素1工程化毕赤酵母腈水解酶全细胞催化剂。

1-氰基环己烷乙酸（1-CHAA）是合成抗癫痫药加巴喷丁的关键中间体。以前，我们的小组已经通过大肠杆菌（E. coli）腈水解酶全细胞催化剂催化的生物转化建立了1-CHAA的新型生产路线。然而，在大肠杆菌中表达的腈水解酶具有一些缺点，例如低水平的可重复使用性，这阻碍了其工业应用。在这里，我们调查了使用甲基营养酵母巴斯德毕赤酵母（P. pastoris）生产腈水解酶全细胞催化剂的潜力。为了获得具有高催化活性的菌株，我们研究了启动子选择，表达盒拷贝数和伴侣蛋白共表达对腈水解酶产生的影响。我们的结果表明，该菌株在乙醇氧化酶1（AOX1）启动子的控制下具有腈水解酶基因的多拷贝整合，与ER氧化还原素1（ERO1）的共表达相比，该菌株的腈水解酶活性提高了18倍。含有一个整合的腈水解酶基因，并在3-磷酸甘油醛（GAP）脱氢酶启动子的控制下。与大肠杆菌腈水解酶全细胞催化剂相比，这种优化的巴斯德毕赤酵母菌株显示出可重用性和热稳定性大大提高，同时具有相似的高底物耐受性。