Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD₆₀₀, which was about 121% higher than that of the wild-type strain. Furthermore, LC–MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

中文翻译：

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改善了由ch豆属菌产生的AHL内酯酶AiiO-AIO6的产量。细胞内蛋白酶缺陷型枯草芽孢杆菌中的M231。

群体猝灭（QQ）可以阻止细菌与细胞之间的通信（即群体感应），是一种有前景的抗病原性策略，可通过抑制毒力因子表达和生物膜形成来控制细菌感染。Ochrobactrum sp。的QQ酶AiiO-AIO6 。M231具有一些优异的性能，并显示出对重要的水生细菌病原体的生物治疗潜力。AiiO-AIO6可以通过非经典的分泌途径在枯草芽孢杆菌中表达。为了提高AiiO-AIO6的产量，通过分别敲除胞内蛋白酶编码基因（tepA，ymfH，yrrN 和 ywpE），构建了四个枯草芽孢杆菌1A751的胞内蛋白酶缺失突变体。）。将AiiO-AIO6表达质粒pWB-AIO6BS转化到枯草芽孢杆菌1A751及其四种细胞内蛋白酶缺失衍生物中。结果表明，所有重组细胞内的蛋白酶缺失衍生物（BSΔ TEPA，BSΔ ymfH，BSΔ yrrN和BSΔ ywpE）对AiiO-AIO6生产具有积极的影响。AiiO-AIO6细胞外生产的BSΔ的最高量ywpE在摇瓶中达到1416.47 U /毫升/ OD ₆₀₀，比野生型菌株高约121％。此外，通过纯化AiiO-AIO6对3-oxo-C8-HSL降解产物进行LC-MS / MS分析表明，AiiO-AIO6是一种水解AHLs内酯环的AHL-内酰胺酶。系统发育分析表明，AiiO-AIO6被归类为α/β水解酶家族成员，具有保守的“亲核酸-组氨酸”催化三联体。总之，这项研究表明，胞内蛋白酶是造成异源蛋白质产量降低的原因，并提供了一种有效的策略来增强AHL内酯酶AiiO-AIO6的胞外产生。