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Abstract：
Avilamycins, which possess potent inhibitory activity against Gram-positive bacteria, are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes. Among these structurally related oligosaccharide antibiotics, avilamycin A serves as the main bioactive component in veterinary drugs and animal feed additives, which differs from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. However, the mechanisms underlying assembly and modification of the oligosaccharide chain to diversify individual avilamycins remain poorly understood. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. Remarkably, the ratio of these two components produced by AviZ1 depends on the utilization of specific redox cofactors, namely NADH/NAD+ or NADPH/NADP+. These findings are inspired by gene disruption and complementation experiments and are further supported by in vitro enzymatic activity assays, kinetic analyses, and cofactor affinity studies on AviZ1-catalyzed redox reactions. Additionally, the results from sequence analysis, structure prediction, and site-directed mutagenesis of AviZ1 validate it as an NADH/NAD+-favored aldo-keto reductase that primarily oxidizes avilamycin C to form avilamycin A by utilizing abundant NAD+ in vivo. Building upon the biological function and catalytic activity of AviZ1, overexpressing AviZ1 in S. viridochromogenes is thus effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study represents, to our knowledge, the first characterization of biochemical reactions involved in avilamycin biosynthesis and contributes to the construction of high-performance strains with industrial value.IMPORTANCEAvilamycins are a group of oligosaccharide antibiotics produced by Streptomyces viridochromogenes, which can be used as veterinary drugs and animal feed additives. Avilamycin A is the most bioactive component, differing from avilamycin C only in the redox state of the two-carbon branched-chain of the terminal octose moiety. Currently, the biosynthetic pathway of avilamycins is not clear. Here, we report that AviZ1, an aldo-keto reductase in the avilamycin pathway, can catalyze the redox conversion between avilamycins A and C. More importantly, AviZ1 exhibits a unique NADH/NAD+ preference, allowing it to efficiently catalyze the oxidation of avilamycin C to form avilamycin A using abundant NAD+ in cells. Thus, overexpressing AviZ1 in S. viridochromogenes is effective to improve the yield and proportion of avilamycin A in the fermentation profile of avilamycins. This study serves as an enzymological guide for rational strain design, and the resulting high-performance strains have significant industrial value.
摘要：
阿维拉霉素,对革兰氏阳性细菌具有有效的抑制活性，是由链霉菌产生的一组寡糖抗生素。在这些结构相关的寡糖抗生素中，阿维霉素A作为兽药和动物饲料添加剂中的主要生物活性成分,它与阿维拉霉素C的不同之处仅在于末端辛基部分的两个碳支链的氧化还原状态。然而,寡糖链的组装和修饰使个体阿维拉霉素多样化的潜在机制仍然知之甚少。这里,我们报道了AviZ1，一种阿瓦霉素途径中的aldo-keto还原酶，可以催化阿维拉霉素A和C之间的氧化还原转化，AviZ1产生的这两种成分的比例取决于特定氧化还原辅因子的利用，即NADH/NAD+或NADPH/NADP+。这些发现受到基因破坏和互补实验的启发，并得到体外酶活性测定的进一步支持。动力学分析，以及AviZ1催化的氧化还原反应的辅因子亲和力研究。此外,序列分析的结果，结构预测，AviZ1的定点诱变将其验证为NADH/NAD-偏爱的aldo-keto还原酶，该还原酶通过在体内利用丰富的NAD主要氧化avilamycinC形成avilamycinA。因此，基于AviZ1的生物学功能和催化活性，在病毒嗜铬杆菌中过表达AviZ1可有效提高阿维霉素A在阿维霉素发酵中的产量和比例。这项研究代表，根据我们的知识，参与阿维霉素生物合成的生化反应的首次表征，有助于构建具有工业价值的高效菌株。IMPORTANCEAvilamycins是一组由链霉菌产生的寡糖抗生素，可用作兽药和动物饲料添加剂。阿维拉霉素A是最具生物活性的组分，与阿维拉霉素C的不同之处仅在于末端乳糖部分的两个碳支链的氧化还原状态。目前,阿维拉霉素的生物合成途径尚不清楚。这里,我们报道了AviZ1，一种阿瓦霉素途径中的aldo-keto还原酶，可以催化阿维拉霉素A和C之间的氧化还原转化。更重要的是，AviZ1具有独特的NADH/NAD+偏好，允许它使用细胞中丰富的NAD有效催化阿维拉霉素C的氧化以形成阿维拉霉素A。因此，在嗜绿链球菌中过表达AviZ1可有效提高阿维霉素A在阿维霉素发酵谱中的产量和比例。这项研究为合理的菌株设计提供了酶学指导，所得的高性能菌株具有显著的工业价值。
