基于级联反应高效制备D-泛酸的生物转化系统构建

Construction of a Biotransformation System for Efficient Preparation of D-Pantothenic Acid Based on Cascade Reactions

摘要:

【目的】D-泛酸（维生素B₅）是一种水溶性维生素，广泛应用于饲料、食品、化工、制药等领域。目前，维生素B₅主要通过化学-酶法生产，但这种生产工艺会产生大量含氰废水，而细胞工厂具有绿色环保的特点。因此，非常有必要开发出一种用低值底物生产D-泛酸的生物转化系统。【方法】 以课题组前期构建的β-丙氨酸合成菌株为出发菌株，通过偶联谷氨酸棒杆菌来源的L-天冬氨酸酶（L-aspartase，AspA）和短芽孢杆菌来源的泛酸合成酶（pantothenate synthetase，PanC）构建三级酶联体系，通过体外验证和优化D-泛酸生物合成途径，而后在细胞内实现重建，并优化3种酶配比和引入ATP循环再生系统，在5 L生物反应器中实现了D-泛酸的体系优化和放大生产。【结果】 在最佳生物转化条件下，通过全细胞催化可将70 g/L的富马酸和 89 g/L的D-泛解酸转化为 128.6 g/L的D-泛酸，摩尔转化率为97.3%。【结论】 成功开发出一种用富马酸和D-泛解酸生产D-泛酸的有效生物转化系统。

Abstract:

[Objective] D-pantothenic acid (vitamin B₅) is a water-soluble vitamin widely used in feed, food, chemical, pharmaceutical and other industries. At present, vitamin B₅ is mainly produced by chemical-enzymatic methods, while this production process generates a large amount of cyanide-containing wastewater. Cell factories have the characteristics of being green and environmentally friendly. Therefore, it is necessary to develop a biotransformation system for producing D-pantothenic acid with low value substrates. [Method] With the β-alanine synthesis strain constructed in the previous study as the starting strain, a three-enzyme system was constructed by coupling L-aspartase from Corynebacterium glutamicum and pantothenate synthase (PanC) from Bacillus subtilis. The D-pantothenic acid biosynthesis pathway was validated and optimized in vitro and then reconstructed in cells. By optimizing the ratio of three enzymes and introducing an ATP cycle system, the system optimization and large-scale production of D-pantothenic acid were finally achieved in a 5 L bioreactor. [Result] Under the optimal biotransformation conditions, 70 g/L of fumaric acid and 89 g/L of D-pantolactone were converted to 128.6 g/L of D-pantothenic acid through whole cell catalysis, with a molar conversion rate of 97.3%. [Conclusion] An effective biotransformation system for producing D-pantothenic acid from fumaric acid and D-pantolactone is successfully developed.