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Multistep Metabolic Engineering of Bacillus licheniformis To Improve Pulcherriminic Acid Production

机译：芽孢杆菌的MultiSep代谢工程改善耐润滑果酸的生产

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The cyclodipeptide pulcherriminic acid, produced by Bacillus licheniformis , 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 B. licheniformis DWc9n*. First, we increased leucine (Leu) supply by overexpressing the ilvBHC-leuABCD operon and ilvD , involved in Leu biosynthesis, to obtain strain W1, and the engineered strain W2 was further attained by the deletion of gene bkdAB , 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 leuS in W2, and it produced 367.7?mg/liter pulcherriminic acid. Third, the original promoter of the pulcherriminic acid synthetase cluster yvmC-cypX 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 yvmA in W4, resulting in the W4/pHY- yvmA 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.IMPORTANCE 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- yvmA pulcherriminic acid-producing strain via a multistep metabolic modification. The engineered W4/pHY- yvmA 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.
机译：由芽孢杆菌生产的环肽润滑酮钙丙酸衍生自环（L-Leu-L-Leu）并具有优异的抗菌活性。在这项研究中，我们通过B.Licheniformis DWC9N *的MultiSep代谢工程实现了高级别的耐润滑丙酸的生产。首先，我们通过过表达ILVBHC-LEUABCD操纵子和ILVD来增加亮氨酸（LEU）供应，涉及Leu Biosynathesis的ILVD，得到菌株W1，并且通过缺失基因Bkdab来进一步获得工程化菌株W2，编码分支链α -Keto酸脱氢酶在W1中。结果，W2细胞内含量和润润滑酸产率达到147.4mg / g dcw（干细胞重量）和189.9μm？mg /升，分别比dwc9n *高出227.6％和48.9％。其次，通过过表达W2中的白胶-TRNA合成酶基因雷斯来构建菌株W3，并产生367.7μg/升润浆酸。第三，用经过验证的强启动子，PBACA替代W3中的润浆型酸合成酶簇YVMC-CYPX的原始启动子以产生菌株W4，其润滑酸产率升至507.4μmg/毫升/升。最后，通过过表达转运蛋白基因YVMA在W4中过度强化润浆酸分泌，导致W4 / phy- yvma菌株，其产生556.1μmβmg/升润滑浆酸，与DWC​​9N *相比增加337.8％，目前是最高的润滑酸酸屈服于我们所知的最佳。我们一起服用，提供了提高润滑脂酸生产的有效策略，这可能适用于其他环肽的高级别产生。分析润浆酸是衍生自环（L-Leu-L-Leu）的环牛仔肽，其相同铁螯合组与羟肟酸侧翼。通常，耐润滑丙酸产量是抗菌和抗植物 - 致病性真菌剂的完美候选者。在这项研究中，我们通过多步骤代谢改性获得了产生的W4 / phy-yvma润滑酸产量。工程化的W4 / PHY- yvma菌株能够实现556.1毫克/升润滑脂酸生产，这是我们知识的最佳收益率。

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