Institute of Food Science and Biotechnology, Department of Bioprocess Engineering (150K), University of Hohenheim, Fruwirthstraße 12, 70599, Stuttgart, Germany.
Core Facility Hohenheim, Mass Spectrometry Unit, University of Hohenheim, August-von-Hartmann-Str. 3, 70599, Stuttgart, Germany.
Microb Cell Fact. 2020 Nov 10;19(1):205. doi: 10.1186/s12934-020-01468-0.
Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production.
The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10 strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (P) against the strong constitutive promoter P led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (P::P) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition.
This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.
Plipastatin 是一种强效的芽孢杆菌抗菌脂肽，有望替代传统的抗真菌化学物质来控制植物病原体。然而，这种脂肽的应用迄今为止只在少数情况下进行了研究，主要是因为其产量低，生物合成途径的调控机制尚不清楚。枯草芽孢杆菌通过ppsABCDE 操纵子编码的非核糖体肽合成酶合成 plipastatin。在这项研究中，对枯草芽孢杆菌 3NA（一种非产孢菌株）进行了工程改造，以更深入地了解 plipastatin 的单产。
4-磷酸泛酰巯基乙胺转移酶 Sfp 使非核糖体肽合成酶从无活性的脱辅基形式转化为有活性的全酶形式。在 3NA 菌株中，sfp 基因失活。因此，第一步是将修复后的 sfp 版本整合到 3NA 中，构建菌株 BMV9。随后，整合来自枯草芽孢杆菌 DSM10 菌株的完全表达的 degQ 版本（菌株 BMV10）后，plipastatin 的产量增加了一倍，从而确保了 DegU-P 调控途径的刺激，该途径正向控制 ppsABSDE 操纵子。此外，用相对较弱的天然 plipastatin 启动子（P）替代强组成型启动子 P，可使 BMV11 中的 plipastatin 产量增加约五倍，而 BMV9 中的产量则增加了约五倍。有趣的是，同时修复 degQ 的表达和启动子交换（P::P）并没有增加 plipastatin 的产量。之后，通过保留位于 srfAB 内并参与自然感受性发育的调控因子 comS，删除表面活性素（srfAA-AD）操纵子，导致 BMV9 中 plipastatin 产量的丧失，并显著降低了 BMV11 中 plipastatin 的产量。我们还观察到，补充鸟氨酸作为 plipastatin 形成的前体可导致单产菌株中 plipastatin 的产量更高，尽管其 plipastatin 组成模式发生了改变。
本研究提供的证据表明，degQ 可刺激天然 plipastatin 的产生。此外，完全的 plipastatin 生产需要表面活性素合成酶或其某些成分。此外，作为本研究的另一个结论，结果表明提供鸟氨酸是枯草芽孢杆菌 plipastatin 单产菌株生产 plipastatin 的一个不可或缺的组成部分。因此，靶向鸟氨酸代谢通量可能是进一步研究和提高枯草芽孢杆菌 plipastatin 单产菌株 plipastatin 产量的有前途的策略。
