Table_1_Functional Cross-Talk of MbtH-Like Proteins During Thaxtomin Biosynthesis in the Potato Common Scab Pathogen Streptomyces scabiei.DOCX

Thaxtomin A is a potent phytotoxin that serves as the principle pathogenicity determinant of the common scab pathogen, Streptomyces scabiei, and is also a promising natural herbicide for agricultural applications. The biosynthesis of thaxtomin A involves the non-ribosomal peptide synthetases (NRPSs) TxtA and TxtB, and an MbtH-like protein (MLP), TxtH, which may function as a chaperone by promoting the proper folding of the two NRPS enzymes in S. scabiei. MLPs are required for the proper function of many NRPS enzymes in bacteria, and they are often capable of interacting with NRPSs from different biosynthetic pathways, though the mechanism by which this occurs is still poorly understood. To gain additional insights into MLP functional cross-talk, we conducted a broad survey of MLPs from diverse phylogenetic lineages to determine if they could functionally replace TxtH. The MLPs were assessed using a protein solubility assay to determine whether they could promote the soluble expression of the TxtA and TxtB adenylation domains. In addition, the MLPs were tested for their ability to restore thaxtomin production in a S. scabiei mutant that lacked TxtH and other endogenous MLPs. Our results showed that the MLPs investigated vary in their ability to exhibit functional cross-talk with TxtH, with two of the MLPs being unable to compensate for the loss of TxtH in the assays performed. The ability of an MLP to serve as a functional partner for the thaxtomin NRPS was not correlated with its overall amino acid similarity with TxtH, but instead with the presence of highly conserved residues. In silico structural analysis of TxtH in association with the TxtA and TxtB adenylation domains revealed that several such residues are situated at the predicted interaction interface, suggesting that they might be critical for promoting functional interactions between MLPs and the thaxtomin NRPS enzymes. Overall, our study provides additional insights into the mechanism of MLP cross-talk, and it enhances our understanding of the thaxtomin biosynthetic machinery. It is anticipated that our findings will have useful applications for both the control of common scab disease and the commercial production of thaxtomin A for agricultural use.

除草素A（Thaxtomin A）是一种强效植物毒素，既是马铃薯疮痂病致病菌疮痂链霉菌（Streptomyces scabiei）的核心致病决定因子，同时也是极具农用潜力的天然除草剂。除草素A的生物合成依赖于非核糖体肽合成酶（non-ribosomal peptide synthetases, NRPSs）TxtA与TxtB，以及MbtH样蛋白（MbtH-like protein, MLP）TxtH。该蛋白可作为分子伴侣，促进疮痂链霉菌中这两种NRPS酶的正确折叠。MLPs对于细菌中多数NRPS酶的正常功能必不可少，且通常能够与不同生物合成途径中的NRPSs发生相互作用，但其具体作用机制目前仍不甚明确。为进一步解析MLP的功能交叉对话机制，我们对不同进化谱系来源的MLPs开展了广泛调研，以验证其是否能够在功能上替代TxtH。我们通过蛋白溶解度实验评估各MLP能否促进TxtA与TxtB腺苷酰化结构域的可溶性表达；同时，在缺失TxtH及其他内源性MLPs的疮痂链霉菌突变株中，检测各MLP恢复除草素A合成的能力。实验结果显示，受试MLPs与TxtH的功能交叉对话能力存在差异，其中两种MLP无法在本次实验中弥补TxtH缺失带来的缺陷。MLP作为除草素A生物合成NRPS系统功能伴侣的能力，与其与TxtH的整体氨基酸相似度并无关联，而是取决于其是否携带高度保守的氨基酸残基。针对TxtH与TxtA、TxtB腺苷酰化结构域复合物的虚拟结构分析表明，此类保守残基中有多个位于预测的相互作用界面上，提示它们可能对MLP与除草素A生物合成NRPS酶之间的功能性相互作用至关重要。综上，本研究为解析MLP功能交叉对话机制提供了新的见解，加深了我们对除草素A生物合成系统的理解。预计本研究成果将在马铃薯疮痂病防控以及农用除草素A的商业化生产中具备重要应用价值。