We propose here that acylation modification of actinomycete proteins is a restrictive system that limits the excessive synthesis of secondary metabolites, its mechanism has not been clearly elucidated before. We used crotonylation as an example to investigate the acylation effect in the daptomycin biosynthesis by Streptomyces roseosporus. Our experiments revealed abundant crotonylation of numerous secondary metabolic enzymes in Streptomyces roseosporus, a daptomycin producer. DptE, which initiates daptomycin biosynthesis, is crotonylated at K454. We experimentally identified the corresponding DptE crotonyltransferase Kct1 and decrotonylase CobB. Further studies consistently confirmed that decrotonylation increases DptE activity. Decrotonylation functions like loosening a faucet knob, increasing substrate channel throughput and the initial flow of daptomycin biosynthesis. Moreover, DptE catalytic activity was enhanced via K454 and neighboring residues K184 and Q420 mutation, increasing daptomycin yield by 132%; daptomycin biosynthesis related metabolism activities also increased. Substrate channel prediction revealed 38% higher throughput for mutant DptE (K454I/K184Q/Q420N) than crotonylated DptE. Molecular dynamics (MD) simulations revealed significant increases in flexibility and substrate affinity of the mutant. In summary, we elucidated the faucet knob effect of DptE crotonylation on the initial flow of daptomycin biosynthesis and adopted decrotonylation to generate high-yield industrial strains.

中文翻译：

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DptE 巴豆酰化对达托霉素生物合成初始流程的水龙头旋钮效应


我们在这里提出，放线菌蛋白的酰化修饰是一个限制次生代谢物过度合成的限制性系统，其机制以前尚未明确阐明。我们以巴豆酰化为例，研究了玫瑰孢链霉菌在达托霉素生物合成中的酰化作用。我们的实验揭示了达托霉素生产商 Streptomyces roseosporus 中许多次生代谢酶的丰富巴豆酰化。启动达托霉素生物合成的 DptE 在 K454 位点被巴豆酰化。我们通过实验鉴定了相应的 DptE 巴豆酰转移酶 Kct1 和去巴豆酰化酶 CobB。进一步的研究一致证实，去甲酰化会增加 DptE 活性。去甲酰化功能类似于松开水龙头旋钮、增加底物通道通量和达托霉素生物合成的初始流量。此外，通过 K454 和邻近残基 K184 和 Q420 突变增强了 DptE 催化活性，使达托霉素产量提高了 132%;达托霉素生物合成相关的代谢活性也增加。底物通道预测显示，突变体 DptE （K454I/K184Q/Q420N） 的通量比巴豆酰化 DptE 高 38%。分子动力学 （MD） 模拟显示突变体的柔韧性和底物亲和力显著增加。综上所述，我们阐明了 DptE 巴豆酰化对达托霉素生物合成初始流程的水龙头旋钮效应，并采用去巴豆酰化生成高产工业菌株。