Biotransformation and chiral resolution of d,l-alanine into pyruvate and d-alanine with a whole-cell biocatalyst expressing l-amino acid deaminase.,Biotechnology and Applied Biochemistry

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Biotransformation and chiral resolution of d,l-alanine into pyruvate and d-alanine with a whole-cell biocatalyst expressing l-amino acid deaminase.
Biotechnology and Applied Biochemistry ( IF 3.2 ) Pub Date : 2020-08-21 , DOI: 10.1002/bab.2011
Ke Liu _{1,

2} , Mengyue Gong ₃ , Xueqin Lv _{1,

2} , Jianghua Li _{1,

2} , Guocheng Du _{1,

2} , Long Liu _{1,

2}

Affiliation

Pyruvate is an important pharmaceutical intermediate and is widely used in food, nutraceuticals, and pharmaceuticals. However, high environmental pollution caused by chemical synthesis or complex separation process of microbial fermentation methods constrain the supply of pyruvate. Here, one‐step pyruvate and d‐alanine production from d,l‐alanine by whole‐cell biocatalysis was investigated. First, l‐amino acid deaminase (Pm1) from Proteus mirabilis was expressed in Escherichia coli, resulting in pyruvate titer of 12.01 g/L. Then, N‐terminal coding sequences were introduced to the 5′‐end of the pm1 gene to enhance the expression of Pm1 and the pyruvate titer increased to 15.13 g/L. Next, product utilization by the biocatalyst was prevented by knocking out the pyruvate uptake transporters (cstA, btsT) and the pyruvate metabolic pathway genes pps, poxB, pflB, ldhA, and aceEF using CRISPR/Cas9, yielding 30.88 g/L pyruvate titer. Finally, by optimizing the reaction conditions, the pyruvate titer was further enhanced to 43.50 g/L in 8 H with a 79.99% l‐alanine conversion rate; meanwhile, the resolution of d‐alanine reached 84.0%. This work developed a whole‐cell biocatalyst E. coli strain for high‐yield, high‐efficiency, and low‐pollution pyruvate and d‐alanine production, which has great potential for the commercial application in the future.

中文翻译：

用表达l-氨基酸脱氨酶的全细胞生物催化剂将d，1-丙氨酸生物转化和手性拆分为丙酮酸和d-丙氨酸。

丙酮酸是一种重要的药物中间体，广泛用于食品，保健食品和药物中。然而，由微生物发酵方法的化学合成或复杂的分离过程引起的高环境污染限制了丙酮酸的供应。在这里，研究了通过全细胞生物催化从d，l-丙氨酸一步生产丙酮酸和d-丙氨酸。首先，来自奇异变形杆菌的l-氨基酸脱氨酶（Pm1）在大肠杆菌中表达，丙酮酸滴度为12.01 g / L。然后，将N末端编码序列引入pm1的5'末端该基因增强了Pm1的表达，丙酮酸滴度增加至15.13 g / L。接下来，通过使用CRISPR / Cas9敲除丙酮酸吸收转运蛋白（cstA，btsT）和丙酮酸代谢途径基因pps，poxB，pflB，ldhA和aceEF来防止生物催化剂对产品的利用，产生的丙酮酸滴度为30.88 g / L。最后，通过优化反应条件，丙酮酸滴度在8 H中进一步提高至43.50 g / L，l-丙氨酸转化率为79.99％。同时，d-丙氨酸的分辨率达到84.0％。这项工作开发了一种全细胞生物催化剂大肠杆菌高产，高效，低污染的丙酮酸和d-丙氨酸生产用菌株，在将来的商业应用中具有巨大的潜力。

更新日期：2020-09-10

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