State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, People's Republic of China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, People's Republic of China
Appl Environ Microbiol. 2020 Apr 17;86(9). doi: 10.1128/AEM.03041-19.
The cyclodipeptide pulcherriminic acid, produced by , is derived from cyclo(l-Leu-l-Leu) and possesses excellent antibacterial activities. In this study, we achieved the high-level production of pulcherriminic acid via multistep metabolic engineering of DWc9n*. First, we increased leucine (Leu) supply by overexpressing the operon and , involved in Leu biosynthesis, to obtain strain W1, and the engineered strain W2 was further attained by the deletion of gene , encoding a branched-chain α-keto acid dehydrogenase in W1. As a result, the intracellular Leu content and pulcherriminic acid yield of W2 reached 147.4 mg/g DCW (dry cell weight) and 189.9 mg/liter, which were 227.6% and 48.9% higher than those of DWc9n*, respectively. Second, strain W3 was constructed through overexpressing the leucyl-tRNA synthase gene in W2, and it produced 367.7 mg/liter pulcherriminic acid. Third, the original promoter of the pulcherriminic acid synthetase cluster in W3 was replaced with a proven strong promoter, PbacA, to produce the strain W4, and its pulcherriminic acid yield was increased to 507.4 mg/liter. Finally, pulcherriminic acid secretion was strengthened via overexpressing the transporter gene in W4, resulting in the W4/pHY- strain, which yielded 556.1 mg/liter pulcherriminic acid, increased by 337.8% compared to DWc9n*, which is currently the highest pulcherriminic acid yield to the best of our knowledge. Taken together, we provided an efficient strategy for enhancing pulcherriminic acid production, which could apply to the high-level production of other cyclodipeptides. Pulcherriminic acid is a cyclodipeptide derived from cyclo(l-Leu-l-Leu), which shares the same iron chelation group with hydroxamate sidephores. Generally, pulcherriminic acid-producing strains could be the perfect candidates for antibacterial and anti-plant-pathogenic fungal agents. In this study, we obtained the promising W4/pHY- pulcherriminic acid-producing strain via a multistep metabolic modification. The engineered W4/pHY- strain is able to achieve 556.1 mg/liter pulcherriminic acid production, which is the highest yield so far to the best of our knowledge.
由 产生的环二肽 pulcherriminic 酸来源于环（l-Leu-l-Leu），具有优异的抗菌活性。在本研究中，我们通过多步代谢工程对 DWc9n进行改造，实现了 pulcherriminic 酸的高水平生产。首先，我们通过过表达参与亮氨酸（Leu）生物合成的 基因簇和 基因，提高了 Leu 的供应，得到了菌株 W1，然后通过敲除 W1 中的基因 ，进一步得到了工程菌 W2，该基因编码分支链 α-酮酸脱氢酶。结果，W2 的细胞内 Leu 含量和 pulcherriminic 酸产量分别达到 147.4mg/g DCW（干细胞重量）和 189.9mg/L，分别比 DWc9n提高了 227.6%和 48.9%。其次，通过在 W2 中过表达亮氨酰-tRNA 合成酶基因 构建了菌株 W3，其产生 367.7mg/L pulcherriminic 酸。第三，用已证实的强启动子 PbacA 替换 W3 中 pulcherriminic 酸合成酶簇 的原始启动子，得到了菌株 W4，其 pulcherriminic 酸产量增加到 507.4mg/L。最后，通过过表达转运蛋白基因 增强了 pulcherriminic 酸的分泌，得到了 W4/pHY-菌株，其 pulcherriminic 酸产量为 556.1mg/L，比 DWc9n*提高了 337.8%。综上所述，我们提供了一种有效的增强 pulcherriminic 酸生产的策略，该策略可应用于其他环二肽的高水平生产。Pulcherriminic 酸是一种来源于环（l-Leu-l-Leu）的环二肽，与羟肟酸侧链具有相同的铁螯合基团。一般来说，产生 pulcherriminic 酸的菌株可以作为抗菌和抗植物病原真菌剂的理想候选菌株。在本研究中，我们通过多步代谢修饰获得了有前途的 W4/pHY- pulcherriminic 酸生产菌株。经过工程改造的 W4/pHY-菌株能够实现 556.1mg/L 的 pulcherriminic 酸产量，这是迄今为止我们所知的最高产量。
