一株耐盐碱乳酪短杆菌G20响应盐碱胁迫的差异代谢物分析

Analysis of differential metabolites of Brevibacterium casei G20 in response to saline-alkali stress

摘要:

【目的】探究耐盐碱乳酪短杆菌G20响应盐碱胁迫的代谢物组成以及代谢物合成潜力，为潜在功能分子和盐碱诱导的快速稳定响应逻辑门基因线路的挖掘提供参考。【方法】利用液相色谱-质谱联用技术(LC-MS)检测乳酪短杆菌G20盐碱环境与正常环境下4个生长时期的代谢产物。着重对富含高差异变化倍数代谢物的适应期与指数期进行分析。【结果】乳酪短杆菌G20可以在pH 10.0、9% NaCl环境中正常生长，同时环境pH值会随菌株生长逐步下降。综合正负离子2种模式，乳酪短杆菌G20在盐碱环境下各生长时期间差异代谢物数量分别为正常环境的0.69、0.75和0.81倍。盐碱胁迫诱导下适应期与指数期差异代谢物主要为苯环型化合物、有机酸及其衍生物与有机杂环类化合物。其中上调的有机酸化合物吲哚-3-乙酸、犬尿酸和葡萄糖酸指数期质谱信号强度低于适应期。菌株中可能存在的渗透保护剂有l-瓜氨酸、l-脯氨酸、N-乙酰鸟氨酸和左旋肉碱等。适应期变化倍数较大或质谱信号强度较高的差异化合物有毛果芸香碱、植物鞘氨醇和柠檬酸等，指数期有组胺、l-脯氨酸和硫胺素等。菌株差异代谢通路集中在氨基酸代谢与碳水化合物代谢。菌株代谢物中存在甜菜碱和葫芦巴碱的结构类似物。此外受盐碱胁迫诱导变化幅度较大的代谢物如组胺、l-脯氨酸和胆碱等化合物，能够通过代谢组学与基因组学数据耦合，推测其合成与代谢通路。【结论】菌株会外泌吲哚-3-乙酸、犬尿酸和葡萄糖酸等有机酸化合物降低环境pH值，积累l-瓜氨酸和l-脯氨酸等渗透保护剂维持渗透压平衡。菌株可能具备合成甜菜碱、葫芦巴碱和毛果芸香碱结构类似物的能力，为以原核细菌为底盘构建新的合成路径提供了可能。菌株中组胺、l-脯氨酸和胆碱等化合物合成与代谢通路中酶的编码基因及上下游元件序列，可以为受盐碱理化因子诱导的逻辑门基因菌为底盘构建新的合成路径提供了可能。菌株中组胺、l-脯氨酸和胆碱等化合物合成与代谢通路上酶的编码基因及上下游元件序列，可以为受盐碱理化因子诱导的逻辑门基因线路的梳理提供参考。

Abstract:

[Objective] To explore the metabolite composition and metabolite synthesis potential of Brevibacterium casei G20 in response to saline-alkali stress, and to provide a reference for the mining of potential functional molecules and the rapidly and stably responsive genetic logic gate line induced by saline-alkali. [Methods] We used LC-MS to detect the metabolites of G20 at four growth phases in saline-alkali and normal environments, and focused on the analysis of the lag and logarithmic phases rich in metabolites with high differential fold changes. [Results] B. casei G20 grew well in the environment with pH 10.0 and 9% NaCl, and the pH of the environment gradually decreased with the growth. According to the detection result in positive ion mode and negative ion mode, the numbers of differential metabolites of B. casei G20 during growth in the saline-alkali environment were 0.69, 0.75, and 0.81 times that in the normal environment, respectively. The metabolites that differed between the lag and logarithmic phases under saline-alkali stress were mainly benzenoids, organic acids and their derivatives, and organic heterocyclic compounds. Among them, the MS signal intensity of the organic acids such as indole-3-acetic acid, kynurenic acid, and gluconic acid that were up-regulated in the logarithmic phase was lower than that in the lag phase. The possible osmoprotectants in the strain were l-citrulline, l-proline, N-acetylornithine, l-carnitine, etc. The differential compounds with larger fold changes or higher MS signal intensity in the lag phase were pilocarpine, phytosphingosine, and citrate, and in the logarithmic phase were histamine, l-proline, and thiamine. The differential metabolic pathways of the strain were mainly the amino acid metabolism and carbohydrate metabolism. There were also structural analogs of betaine and trigonelline in the metabolites of the strain. In addition, for metabolites with large changes induced by saline-alkali stress, such as histamine, l-proline, and choline, we could infer their synthetic and metabolic pathways through the coupling of metabolomic and genomic data. [Conclusion] The strain secreted organic acids such as indole-3-acetic acid, kynurenic acid, and Gluconic acid to reduce the pH value of the environment, and accumulated osmoprotectants such as l-citrulline and l-proline to maintain osmotic balance. In addition, the strain may also have the ability to synthesize structural analogs of betaine, trigonelline, and pilocarpine, allowing the construction of a new synthetic pathway based on prokaryotic bacteria. Moreover, the enzyme-coding genes and upstream and downstream element sequences in the synthetic and metabolic pathways of compounds such as histamine, l-proline, and choline in the strain can serve as a reference for sorting out the logic gate gene circuits induced by saline-alkali stress.