Enhancement of α-ketoisovalerate production by relieving the product inhibition of L-amino acid deaminase from Proteus mirabilis

L-Amino acid deaminase(LAAD) is a key enzyme in the deamination of L-valine(L-val) to produce α-ketoisovalerate(KIV). However, the product inhibition of LAAD is a major hindrance to industrial KIV production.In the present study, a combination strategy of modification of flexible loop regions around the product binding site and the avoidance of dramatic change of main-chain dynamics was reported to reduce the product inhibition.The four mutant PM-LAAD^(M4)(PM-LAAD^(S98A/T105A/S106A/L341A)) achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme. Docking experiments suggested that weakened interactions between the product and enzyme, and the flexibility of the "lid" structure relieved LAAD product inhibition. Finally, the whole-cell biocatalyst PM-LAAD^(M4) has been applied to KIV production,the titer and conversion rate of KIV from L-val were 98.5 g·L^-1 and 99.2% at a 3-L scale, respectively. These results demonstrate that the newly engineered catalyst can significantly reduce the product inhibition, that making KIV a prospective product by bioconversion method, and also provide the understanding of the mechanism of the relieved product inhibition of PM-LAAD.

细胞寿命在大肠杆菌细胞工厂构建中的应用

微生物细胞工厂以可再生资源为原料,为工业化学品的可持续生产提供了一种有前景的替代方案。然而,不适的外界环境显著影响了微生物细胞的存活率,降低了微生物细胞工厂的生产性能。通过延长微生物细胞的时序寿命,可以显著提升微生物细胞工厂的生产性能。首先,基于存活率的变化建立了细胞时序寿命和半时序寿命的评价体系;然后,发现半胱氨酸、肌肽、氨基胍和氨基葡萄糖抗衰老药物可以使大肠杆菌Escherichiacoli细胞的时序寿命分别延长80%、80%、50%和120%;最后,延长E.coli时序寿命可以显著改善E.coli细胞工厂的生产性能,可以用于改善具有本源代谢合成路径的E.coli细胞工厂的生产性能,使乳酸和丙酮酸的得率分别提升30.0%和25.0%,也可以用于改善具有异源代谢合成路径的E. coli细胞工厂的生产性能,使苹果酸的得率提升27.0%。这些研究结果表明延长E. coli细胞寿命提供了一种潜在的改善细胞工厂的生产性能的方法。

酶法转化生产α-酮酸的研究进展

α-酮酸是一种同时含有羧基和酮基的双官能团有机化合物,广泛应用于食品、药品和化妆品等行业。为了满足环境友好、安全高效和可持续发展的社会要求,利用酶转化法生产α-酮酸受到人们的广泛关注。文中从酶的筛选、酶的改造以及酶的转化条件优化3个方面介绍丙酮酸、α-酮戊二酸、酮亮氨酸、酮缬氨酸、苯丙酮酸和酮蛋氨酸酶法合成的研究状况,并展望了α-酮酸进一步高效生产的发展方向。