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首页> 外文期刊>Journal of Biotechnology >Improved production of alpha-ketoglutaric acid (alpha-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of L-amino acid deaminase and deletion of the alpha-KG utilization pathway
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Improved production of alpha-ketoglutaric acid (alpha-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of L-amino acid deaminase and deletion of the alpha-KG utilization pathway

机译：枯草芽孢杆菌全细胞生物催化剂通过改造L-氨基酸脱氨酶并删除α-KG利用途径，提高了α-酮戊二酸（α-KG）的产量

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摘要

We previously developed a novel one-step biotransformation process for the production of alpha-ketoglutarate (alpha-KG) from L-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an L-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high alpha-KG degradation. In this study, we further improved alpha-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis alpha-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of L-glutamic acid from 31% to 83.25% and the alpha-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for L-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of alpha-KG production increased from 22.82 mu M min(-1) to 56.7 mu M min(-1). Finally, the sucA gene, encoding alpha-ketodehydrogenase, was deleted to reduce alpha-KG degradation, increasing the alpha-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the alpha-KG degradation pathway in B. subtilis improved alpha-KG production over that of previously developed processes. (C) 2014 Elsevier B.V. All rights reserved.
机译：我们以前开发了一种新型的一步法生物转化工艺，用于通过表达奇异变形杆菌L-氨基酸脱氨酶（pm1）的枯草芽孢杆菌全细胞生物催化剂从L-谷氨酸生产α-酮戊二酸（α-KG）。然而，由于低的底物特异性和高的α-KG降解，该过程的生物转化效率低。在这项研究中，我们进一步改善了蛋白质工程学中的P. mirabilis pm1并删除了枯草芽孢杆菌α-KG降解途径，从而提高了α-KG的产量。我们首先进行了三轮容易出错的聚合酶链反应，并鉴定了影响催化效率的六个位点（F110，A255，E349，R228，T249和I352）的突变。然后，我们在这些位点进行位点饱和诱变，突变体F110I / A255T / E349D / R228C / T249S / I352A将L-谷氨酸的生物转化率从31％提高到83.25％，α-KG滴度从4.65 g / L至10.08 g / L。接下来，分析了该突变体的反应动力学和生化特性。 L-谷氨酸的米氏常数从49.21 mM降低到23.58 mM，α-KG的最大产生速率从22.82μM min（-1）增加到56.7μM min（-1）。最后，删除了编码α-酮脱氢酶的sucA基因，以减少α-KG的降解，从而将α-KG的效价从10.08 g / L提高至12.21 g / L。与以前开发的方法相比，对拟南芥假单胞菌pm1进行蛋白工程改造和枯草芽孢杆菌中α-KG降解途径的缺失提高了α-KG的产量。 （C）2014 Elsevier B.V.保留所有权利。

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