Type II polyketides are a group of secondary metabolites with various biological activities. In nature, biosynthesis of type II polyketides involves multiple enzymatic steps whereby key enzymes, including ketoacyl-synthase (KS_α), chain length factor (KS_β), and acyl carrier protein (ACP), are utilized to elongate the polyketide chain through a repetitive condensation reaction. During each condensation, the biosynthesis intermediates are covalently attached to KS_α or ACP via a thioester bond and are then cleaved to release an elongated polyketide chain for successive postmodification. Despite its critical role in type II polyketide biosynthesis, the enzyme and its corresponding mechanism for type II polyketide chain release through thioester bond breakage have yet to be determined. Here, kinamycin was used as a model compound to investigate the chain release step of type II polyketide biosynthesis. Using a genetic knockout strategy, we confirmed that AlpS is required for the complete biosynthesis of kinamycins. Further in vitro biochemical assays revealed high hydrolytic activity of AlpS toward a thioester bond in an aromatic polyketide-ACP analog, suggesting its distinct role in offloading the polyketide chain from ACP during the kinamycin biosynthesis. Finally, we successfully utilized AlpS to enhance the heterologous production of dehydrorabelomycin in Escherichia coli by nearly 25-fold, which resulted in 0.50 g/liter dehydrorabelomycin in a simple batch-mode shake flask culture. Taken together, our results provide critical knowledge to gain an insightful understanding of the chain-releasing process during type II polyketide synthesis, which, in turn, lays a solid foundation for future new applications in type II polyketide bioproduction.

中文翻译：

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离散硫酯酶在II型聚酮化合物生物合成中的卸载作用。

II型聚酮化合物是一组具有各种生物活性的次级代谢产物。在自然界中，类型的生物合成II聚酮化合物包括，由此多个酶促步骤的关键酶，包括脂酰合酶（KS _α），链长因子（KS _β），和酰基载体蛋白（ACP），被用来通过以延长的聚酮化合物链重复缩合反应。在每个冷凝，生物合成的中间体共价连接到KS _α或通过硫酯键的ACP，然后裂解以释放细长的聚酮化合物链，以进行连续的后修饰。尽管它在II型聚酮化合物的生物合成中起着关键作用，但该酶及其通过硫酯键断裂释放II型聚酮化合物链的相应机理尚未确定。在这里，金那霉素用作模型化合物，以研究II型聚酮化合物生物合成的链释放步骤。使用基因敲除策略，我们证实AlpS是完整的kinamycins生物合成所必需的。进一步体外生化分析表明，AlpS对芳香族聚酮化合物ACP类似物中的硫酯键具有很高的水解活性，表明其在卡那霉素生物合成过程中从ACP卸载聚酮化合物链方面的独特作用。最后，我们成功地利用AlpS将大肠杆菌中的脱氢雷贝霉素的异源产量提高了近25倍，在简单的分批模式摇瓶培养中产生了0.50 g /升的脱氢雷贝霉素。综上所述，我们的结果提供了重要的知识，可帮助您深入了解II型聚酮化合物合成过程中的链释放过程，从而为II型聚酮化合物生物生产中的新应用奠定了坚实的基础。