不同浓度L-谷氨酸钠高效促进盐单胞菌XH26胞内合成Ectoine及相关代谢途径与Ectoine合成通路的关联性  

Different concentrations of L-sodium glutamate effectively promoted the biosynthesis of ectoine and the correlation between related metabolic pathways and ectoine synthesis pathway of Halomonas sp.XH26

作　　者：沈国平 张鑫 王智博 舒志万 崔金子 衡珊 汪明香 朱德锐 王嵘 Shen Guoping;Zhang Xin;Wang Zhibo;Shu Zhiwan;Cui Jinzi;Heng Shan;Wang Mingxiang;Zhu Derui;Wang Rong(Basic Medical Research Center,Medical College of Qinghai University,Xining 810016,China)

机构地区：[1]青海大学医学院基础医学研究中心,西宁810016

出　　处：《中国高原医学与生物学杂志》2023年第4期277-284,共8页Journal of Chinese High Altitude Medicine & Biology

基　　金：青海省基础应用研究计划项目(2020ZJ767);高原特色盐湖生物资源的集成研发与转化团队计划项目(2018KYT1)。

摘　　要：目的研究不同浓度L-谷氨酸钠对盐单胞菌XH26胞内四氢嘧啶(Ectoine)的合成影响,并利用基因组测序法分析L-谷氨酸钠代谢途径与Ectoine生物合成通路的关联性,明确相关代谢基因的参与情况,为后续Ectoine的高效生产和系统代谢工程菌株改造提供理论支持。方法设置不同浓度梯度的L-谷氨酸钠培养菌株XH26,采用HPLC法检测胞内Ectoine的合成量。利用PacBio平台完成全基因组测序,基于KEGG数据库整合分析谷氨酸代谢与Ectoine合成途径的关联基因,并利用荧光qRT-PCR法进行关联基因的表达量验证。结果L-谷氨酸钠浓度从15 mM升至30 mM时,主要参与基因davT(4-氨基丁酸转氨酶)、gdhA(谷氨酸脱氢酶)、lysC(天冬氨酸激酶)的相对表达量显著上调。菌株XH26隶属于盐单胞菌科坎帕尼亚盐单胞菌(Halomonas campaniensis),菌株XH26基因组大小为4.11 Mb,编码基因为3927个。通过KEGG代谢通路富集分析显示:氨基酸代谢有277个基因、碳水化合物代谢有221个基因参与。结论30 mM的L-谷氨酸钠能有效提高胞内Ectoine的合成量。Ectoine的合成前体天冬氨酸半醛与天冬氨酸、三羧酸循环代谢中间物、琥珀酸半缩醛、谷氨酸以及谷氨酰胺代谢密切关联。Objective The aim of this study was to clarify the effects of different glutamate concentrations on ectoine accumulations within Halomonas campaniensis strain XH26,and to analyze the correlation,and the related metabolic genes between glutamate metabolism and ectoine biosynthesis pathway based on whole genome data,which may provide theoretical support for the efficient ectoine production,and the metabolic-engineering reconstruction of the strain.Methods Strain XH26 were cultured with the different concentrations of sodium glutamate,and the accumulation of ectoine was detected by HPLC.Whole genome sequencing was performed using a PacBio platform.The associated genes among glutamate metabolism and ectoine synthesis pathway were analyzed based on whole genome data of XH26 strain and KEGG database,and fluorescence qRT-PCR was used to verify the expression level of associated genes.Results Strain XH26 belonged to H.campaniensis within the family Halomonadaceae,with a 4.11 Mb whole genome,and total 3927 coding genes.Enrichment analysis of KEGG metabolic pathway revealed that amino acid metabolism(including 277 genes)and carbohydrate metabolism(including 221 genes)were enriched.Under the sodium glutamate concentration ranged from 15 mM to 30 mM,the relative expression levels of davT(coding 4-aminobutyrate transaminase),gdhA(glutamate dehydrogenase)and lysC(aspartate kinase)were significantly upregulated.Conclusions In the culture medium,the addition of sodium glutamate(30 mM)can increase carbon/nitrogen metabolic flow towards L-aspartic acid or L-aspartate-β-semialdehyde,which may effectively increase the intracellular ectoine accumulation.As a synthetic precursor of ectoine,L-aspartate-β-semialdehyde was closely associated with L-aspartic acid,metabolic intermediates among tricarboxylic acid cycle,succinate semialdehyde,glutamic acid,and glutamine.

关 键 词：L-谷氨酸钠 四氢嘧啶 盐单胞菌属 代谢通路 基因 验证 

分 类 号：R34[医药卫生—基础医学]