比较代谢组学分析阿维拉霉素高产突变株代谢途径

Comparative metabolomics analysis of metabolic pathways in the high-yielding mutant strain of avilamycin

摘要:

阿维拉霉素(avilamycin, AVI)是一种寡糖类抗生素，因其具有较强的革兰氏阳性细菌抑制能力，被广泛应用于畜禽养殖。但传统的育种技术与不成熟的发酵工艺已成为限制其国产化的关键因素。基于已获得的利用核糖体工程技术改造的高产阿维拉霉素突变菌株，本研究采用比较代谢组学技术探究其与出发菌株的胞内代谢差异。利用GC-MS技术对发酵至第4、6、8天的菌丝体进行分析，共检测出112种化合物，经NIST谱库对比后精确匹配到29种胞内代谢物。二维主成分分析(principal component analysis, PCA)表明突变菌株与出发菌株的不同时间点代谢物有明显差异，通过正交偏最小二乘法判别分析(orthogonal partial least squares-discriminant analysis, OPLS-DA)得到11种胞内差异代谢物。KEGG代谢通路富集显示阿维拉霉素的合成主要与碳水化合物代谢和氨基酸代谢密切相关，且进一步筛选出6种关键差异代谢物：l-缬氨酸、l-丝氨酸、l-丙氨酸、d-半乳糖、d-纤维二糖和d-葡萄糖。突变菌株中这些代谢物的上调增强了其代谢流，使其在罐上发酵8 d时，阿维拉霉素产量较出发菌株提高76.86%。本研究的开展为后续阿维拉霉素发酵工艺理性优化提供了参考。

Abstract:

Avilamycin (AVI) is an oligosaccharide antibiotic that has strong inhibitory effect on Gram-positive bacteria. It is widely used in livestock and poultry farming. However, the use of traditional breeding techniques and immature fermentation process have become the key factors limiting its commercialization. In this study, we used comparative metabolomics techniques to examine the difference in intracellular metabolism between a high-yield AVI mutant strain modified by ribosome engineering technology and the parental strain. GC-MS analysis was conducted on mycelia samples taken on days 4, 6, and 8 of fermentation, resulting in the detection of a total of 112 compounds. After comparison with the NIST library, 29 intracellular metabolites were accurately identified. Two-dimensional principal component analysis (PCA) revealed significant differences in metabolites between the mutant strain and the parental strain at different time points. Orthogonal partial least squares-discriminant analysis (OPLS-DA) identified 11 intracellular metabolites that were closely related to AVI biosynthesis. KEGG metabolic pathway enrichment analysis showed that avilamycin synthesis was closely related to carbohydrate metabolism and amino acid metabolism. Six key differential metabolites were selected: l-valine, l-serine, l-alanine, d-galactose, d-cellobiose, and d-glucose. Upregulation of these metabolites in the mutant strain enhanced its metabolic flow for AVI synthesis. After 8 days of fermentation, the mutant strain produced 76.86% more AVI than the parental strain. The findings of this study serve as reference for the future rational optimization of avilamycin fermentation.