The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China; Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
J Biotechnol. 2023 Feb 20;364:40-49. doi: 10.1016/j.jbiotec.2023.01.010. Epub 2023 Jan 26.
D-Pantothenic acid (D-PA) is an essential vitamin with wide applications. However, the biotechnological production of D-PA is still not competitive with the chemical synthesis in terms of production cost. Ketopantoate hydroxymethyltransferase is a crucial enzyme in the D-PA synthetic pathway in Escherichia coli encoded by the panB gene. Here a hot spots study was applied to a ketopantoate hydroxymethyltransferase from Corynebacterium glutamicum (CgKPHMT) to relieve the product inhibitory effect and thus improve the D-PA production. Compared with the wild type, the double-site variant CgKPHMT-K25A/E189S showed 1.8 times higher enzyme activity and 2.1 times higher catalytic efficiency, 1.88 and 3.32 times higher inhibitory constant of α-ketoisovalerate and D-PA, respectively. The D-PA yield using E. coli W3110 adopted the double-site variant was 41.17 g·L within 48 h, a 9.80 g·L increase. Structural analysis of K25A/E189S revealed the expansion of the entry channel and the change of the electric charge from negative to uncharged due to the substitution from glutamic acid to serine at site 189. Our study emphasized the positive roles of ketopantoate hydroxymethyltransferase in D-PA production and paved the way by analyzing critical enzymes in the synthetic pathway of E. coli to increase the D-PA yield.
D-泛酸（D-PA）是一种具有广泛应用的必需维生素。然而，就生产成本而言，D-PA 的生物工艺生产仍然不如化学合成具有竞争力。酮戊二酸羟甲基转移酶是大肠杆菌中泛酸合成途径中由 panB 基因编码的关键酶。在这里，对来自谷氨酸棒杆菌的酮戊二酸羟甲基转移酶（CgKPHMT）进行了热点研究，以减轻产物抑制作用，从而提高 D-PA 的产量。与野生型相比，双位点变体 CgKPHMT-K25A/E189S 的酶活性提高了 1.8 倍，催化效率提高了 1.8 倍，对α-酮异戊酸和 D-PA 的抑制常数分别提高了 2.1 倍和 3.32 倍。采用双位点变体的大肠杆菌 W3110 的 D-PA 产量在 48 小时内达到 41.17 g·L，增加了 9.80 g·L。K25A/E189S 的结构分析表明，由于第 189 位的谷氨酸被丝氨酸取代，入口通道扩大，电荷从负电荷变为不带电荷。我们的研究强调了酮戊二酸羟甲基转移酶在 D-PA 生产中的积极作用，并通过分析大肠杆菌合成途径中的关键酶，为提高 D-PA 产量铺平了道路。
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