靶向代谢组学分析添加天冬氨酸培养盐单胞菌时四氢嘧啶的代谢通路变化

Targeted metabolomics reveals changes in metabolic pathways related to ectoine in Halomonas cultured with aspartate

摘要:

目的 添加9种不同氨基酸培养坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26，分析菌株的生长情况与胞内四氢嘧啶(ectoine)的积聚量差异，明确最适氨基酸前体对菌株XH26四氢嘧啶的合成代谢通路的影响。方法 在最适盐浓度(1.5 mol/L)条件下，以9种氨基酸(l-谷氨酸钠、l-谷氨酰胺、l-天冬氨酸、l-天冬酰胺、l-组氨酸、l-色氨酸、l-甘氨酸、l-丝氨酸和l-赖氨酸)作为培养基的单一碳/氮源，设置氨基酸浓度梯度范围为20-50 mmol/L (间隔5 mmol/L)，筛选四氢嘧啶积聚量最高的最适氨基酸及其作用浓度。设置l-天冬氨酸低浓度组[low group (L), 20 mmol/L]、中浓度组[medium group (M), 35 mmol/L]和高浓度组[high group (H), 50 mmol/L]进行靶向代谢组学测序与分析。结果 四氢嘧啶合成量随氨基酸浓度梯度先增加后降低，并在最适(30 mmol/L或35 mmol/L)浓度时达到最高。代谢组学分析显示，分别筛选出28个(L vs. M)、27个(L vs. H)和26个(H vs. M)显著差异代谢物，如甘油酸、乳糖、腺苷5′-单磷酸、α-酮戊二酸、葡萄糖-1-磷酸、延胡索酸、柠檬酸等。KEGG代谢通路富集分析发现l-丙氨酸、l-天冬氨酸和l-谷氨酸代谢通路是最显著的富集通路。结论 靶向代谢组学技术分析细菌胞内差异代谢物发现，菌株XH26通过天冬氨酸-丙氨酸轴和精氨酸-脯氨酸代谢轴实现氮稳态与能量供应的再平衡。

Abstract:

Objective We systematically analyzed the growth and compared the ectoine accumulation of Halomonas campaniensis XH26 cultured with nine different amino acids, aiming to clarify the optimal amino acid for ectoine accumulation of strain XH26.Methods Under the optimal salt concentration of 1.5 mol/L, nine amino acids (l-monosodium glutamate, l-glutamine, l-aspartic acid, l-asparagine, l-histidine, l-tryptophan, l-glycine, l-serine, and l-lysine) were selected as the single carbon/nitrogen source of the culture medium and added within the concentration range of 20-50 mmol/L (interval of 5 mmol/L), on the basis of which the optimal concentration and optimal amino acid for ectoine accumulation were screened. l-aspartic acid was selected to culture the cells at low (L, 20 mmol/L), medium (M, 35 mmol/L), and high (H, 50 mmol/L) concentrations for targeted metabolomics sequencing and analysis.Results The amount of ectoine synthesis first increased and then decreased as the amino acid concentration increased and reached the highest at optimum concentration (30/35 mmol/L). Metabolomics analysis screened out 28 (L vs. M), 27 (L vs. H), and 26 (H vs. M) significantly differential metabolites, such as glyceric acid, lactose, adenosine 5′-monophosphate, α-ketoglutaric acid, glucose-1-phosphate, fumaric acid, and citric acid. KEGG metabolic pathway enrichment analysis showed that l-alanine, l-aspartic acid, and l-glutamate metabolic pathways were the most significantly enriched pathways.Conclusion Targeted metabolomics of differential metabolites of bacteria discovers that the strain achieves a rebalance between nitrogen homeostasis and energy supply through the aspartate-alanine axis and the arginine-proline metabolic axis.