转录组和代谢组分析盐度对盐单胞菌合成羟基四氢嘧啶的影响

TRANSCRIPTOMICS AND METABOLOMICS ANALYSIS OF SALINITY ON SYNTHESIS OF HYDROXYECTOINE IN HALOMONAS

为了明确不同盐度对Halomonas campaniensis XH26合成羟基四氢嘧啶(5-hydroxyectoine,5-HE)代谢通路和基础代谢的影响,研究采用无NaCl作为对照组,以12%、14%和16%NaCl浓度梯度作为高盐组。利用转录组测序和液质联用技术(LC-MS)分析了H.campaniensis XH26在不同NaCl浓度条件下的差异表达基因和差异代谢物,并利用qPCR对与Ectoine和5-HE合成密切相关的差异基因的表达量进行了相对定量分析验证。结果显示,5-HE合成量在高盐组间随盐梯度先增加后降低,并在14%NaCl浓度时达到最高。转录组结果显示,在186个KEGG代谢通路中,差异表达基因在ABC转运蛋白、双组分系统、鞭毛组装等通路中高度富集。筛选到与5-HE合成代谢相关的7个关键基因表达存在差异,包括lysC、ectA、ectB、ectC、ectD、doeC和doeD。qPCR验证结果与转录组学分析结果基本一致。代谢组分析共发现了1159个差异代谢物,主要富集在氨基酸代谢、辅因子-维生素代谢和碳水化合物等代谢通路。研究还筛选出了显著差异的相容性溶质,包括四氢嘧啶、羟基四氢嘧啶和甜菜碱。研究分析了菌株H.campaniensis XH26的耐盐特性及盐适应机制,初步明确了5-HE产量及合成基因簇表达随盐度变化的规律,为高产5-HE的野生菌的优化和基因工程菌的构建提供了理论基础。

Hydroxyectoine,a remarkable compatible solute renowned for its effective regulation intra-and extracellular osmotic pressure,undergoes stringent regulation in Halophilic bacteria due to its status as an ectoine derivative.Halomonas campaniensis XH26 produces hydroxyectoine when cultured in a medium containing 3%NaCl.This study aims to elucidate the pathways and key factors influencing hydroxyectoine synthesis in H.campaniensis XH26.Utilizing a control group with 0 NaCl and experimental groups with NaCl gradients of 12%,14%and 16%,we analyzed differentially expressed genes and metabolites across four varying NaCl salinity culture conditions.These analyses were conducted through transcriptomic sequencing and metabolomic profiling using Liquid Chromatograph-Mass Spectrometer(LC-MS).Furthermore,differential gene expression was confirmed through relative quantitative analysis via qPCR.The results showed that the amount of 5-HE synthesis increased and then decreased with the salt gradient and was the highest at 14%NaCl.Transcriptomic analysis identified 186 KEGG metabolic pathways,notably enriched in ABC transporter proteins,the two-component system,and flagellar assembly pathways.Seven key genes implicated in hydroxyectoine biosynthesis-lysC,ectA,ectB,ectC,ectD,doeC,and doeD-exhibited differential expression.Comparative analysis demonstrated that the qPCR validation results were largely consistent with the transcriptomics findings.LC-MS analysis led to the identification of 1159 differential metabolites,among which the compatible solutes ectoine,hydroxyectoine,and betaine showed significant variations.Notably,differences in metabolites were observed in amino acid metabolism,cofactor-vitamin metabolism,and carbohydrate metabolic pathways.In this study,we analyzed the salt tolerance characteristics and salt adaptation mechanism of H.campaniensis XH26.These results preliminarily clarified the patterns of 5-HE production and synthetic gene cluster expression with salinity.In addition,they also provide a theoretical basis for the optimization of wild bacteria with high 5-HE production and the construction of genetically engineered bacteria.