D-泛酸,作为一种重要的维生素,广泛应用于饲料,医学,化妆品等领域。然而,目前通过微生物发酵生产D-泛酸仍然存在局限性。在本文中,我们通过阻断有机酸途径构建了D-泛酸生产的重组菌株,促进丙酮酸生物合成,缓解乙酰乳酸合酶的反馈抑制,提高葡萄糖摄入能力,并修改代谢途径中的必需基因。此外,获得了由ilvC编码的来自大肠杆菌(EcAHAIR)的新乙酰乳酸异还原酶突变体V412A,以探索其底物混杂性。与野生型相比,变体EcAHAIR-V412A具有减少的空间位阻和增强的分子间力,导致对2-乙酰乳酸的高亲和力。最终,最终菌株DPAN19/trc-ilvCV412A的发酵产量达到4.65g/L,在摇瓶培养中与DPA8菌株相比增加了192.5%,并在5L生物反应器中产生了62.82g/L的D-泛酸。本文所述的代谢工程策略和酶修饰方法为D-泛酸的生物制造提供了一个特殊的视角,支链氨基酸及其衍生物。
D-Pantothenic acid, as a momentous vitamin, is extensively applied to feed, medicine, cosmetics and other fields. However, there are still limitations to produce D-pantothenic acid by microbial fermentation at present. In this paper, we constructed a recombinant strain for D-pantothenic acid production by blocking the organic acid pathway, boosting pyruvate biosynthesis, relieving feedback inhibition of acetolactate synthase, improving glucose intake capacity, and modifying essential genes in the metabolic pathway. In addition, a new acetolactate isomeroreductase mutant V412A origin from Escherichia coli (EcAHAIR) encoded by ilvC was obtained to explore its substrate promiscuity. Compared with the wild type, the variant EcAHAIR-V412A has reduced steric hindrance and enhanced intermolecular forces, resulting in a high affinity for 2-acetolactate. Eventually, the fermentation production of the final strain DPAN19/trc-ilvCV412A reached 4.65 g/L, increased by 192.5% compared with strain DPA8 in shake flask cultivation and produced 62.82 g/L D-pantothenic acid in a 5 L bioreactor. The metabolic engineering strategies and enzyme modification approaches described in this paper provide a particular perspective for the bio-manufacturing of D-pantothenic acid, branched-chain amino acids and its derivates.