Abstract

The ammonia-secreting performance is one of the key factors for determining nitrogen-fixing capacity of the cell. Klebsiella variicola GN02 is an endophytic nitrogen-fixing bacterium isolated from the roots of mature Pennisetum giganteum z.x.lin. In order to reveal the key factor which influences the ammonia secretion in fermentation process of K. variicola, GC-TOF-MS metabolomics was used. The changes of OD₆₀₀, ammonia nitrogen content and intracellular metabolites of bacteria were measured simultaneously during fermentation, and a total of 172 intracellular metabolites were detected. Then some methods were used to analysis these high-throughput data. Principal component analysis (PCA) showed that the substances with more contribution to major component exhibited an order as mannitol, glycerol, glucose-1-phosphate, 2‑hydroxypyridine, glutamic acid, fructose-6-phosphate, aniline, lyxose and norleucine. Orthogonal projection to latent structures (OPLS) results revealed that the most important substances for the secretion of ammonia from K. variicola GN02 were mannitol, Gly-Pro, fructose-6-phosphate, and other 12 substances including glycerol, Glu and diacylglycerol having a significant impact with variable importance plot (VIP) values higher than 1, which can be used as the key metabolites for the secretion of ammonia from bacteria. Heatmap clustering analysis showed that mannitol, Gly-Pro and fructose-6-phosphate were strongly expressed at different fermentation stages as the major metabolites. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that fructose-mannose metabolism and amino acid metabolism are both metabolic pathways with the greatest impact on the secretion of ammonia in bacteria. Mannitol, fructose-6-phosphate and Glu are at the core hub of the metabolic process in bacteria, and Gly-Pro is the major nitrogen-related metabolite synthesized.

中文翻译：

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高效内生固氮细菌发酵水痘克雷伯氏菌GN02中氨分泌的代谢组学分析

摘要

氨的分泌性能是确定电池固氮能力的关键因素之一。水痘克雷伯氏菌GN02是一种从成熟的狼尾草xxlin的根中分离出的内生固氮细菌。为了揭示影响水曲霉发酵过程中氨分泌的关键因素，使用了GC-TOF-MS代谢组学。OD ₆₀₀的变化同时测定发酵过程中细菌的氨氮含量和细胞内代谢产物，共检测到172种细胞内代谢产物。然后使用一些方法来分析这些高通量数据。主成分分析（PCA）显示，对主要成分有较大贡献的物质按甘露醇，甘油，-1-磷酸葡萄糖，2-羟基吡啶，谷氨酸，6-磷酸果糖，苯胺，lyxose和正亮氨酸的顺序排列。正交投影到潜伏结构（OPLS）的结果表明，最重要的物质是从水曲霉中分泌氨的GN02是甘露醇，Gly-Pro，6-磷酸果糖和其他12种物质，包括甘油，Glu和二酰基甘油，其重要影响变量（VIP）值大于1，具有重大影响，可用作关键代谢产物。细菌分泌氨。热图聚类分析表明，甘露醇，Gly-Pro和果糖6-磷酸在不同的发酵阶段均作为主要代谢产物强烈表达。京都基因和基因组百科全书（KEGG）途径分析表明，果糖-甘露糖代谢和氨基酸代谢都是对细菌氨气分泌影响最大的代谢途径。甘露醇，6-磷酸果糖和Glu是细菌代谢过程的核心枢纽，而Gly-Pro是合成的主要氮相关代谢物。