基于转录组分析挖掘兽疫链球菌透明质酸分子量调控元件  

Exploration of Regulatory Elements for Hyaluronic Acid Molecular Weight in Streptococcus zooepidemicus via Transcriptome Analysis

作　　者：裴旭娟 狄靖宜 刘浩[1] 高伟霞 PEI Xu-juan;DI Jing-yi;LIU Hao;GAO Wei-xia(Key Laboratory of Industrial Fermentation Microbiology,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457)

机构地区：[1]天津科技大学生物工程学院教育部工业发酵微生物重点实验室,天津300457

出　　处：《生物技术通报》2025年第1期347-356,共10页Biotechnology Bulletin

基　　金：国家重点研发计划(2021YFC2103200)。

摘　　要：【目的】挖掘透明质酸分子量的影响元件,构建生产不同分子量大小的透明质酸兽疫链球菌工程菌。【方法】在利用不同碳源培养兽疫链球菌S12生产透明质酸时,发现10 g/L果糖为碳源发酵的透明质酸分子量为1.48×10^(6) Da,比原始发酵培养基(50 g/L蔗糖为碳源)获得的透明质酸分子量2.10×10^(6) Da降低了29.52%。随后将50 g/L的蔗糖和10 g/L的果糖为碳源的S12发酵液进行转录组学分析,发现除果糖代谢相关基因外,精氨酸脱亚胺酶途径的两个关键基因arcA(编码精氨酸脱亚胺酶)和argF(编码鸟氨酸氨基甲酰转移酶)转录水平分别提升了16.29倍和11.27倍。为了探究这两个基因对HA合成的影响,在S12中分别敲除和过表达基因arcA、argF。【结果】arcA过表达菌株在CDM培养基中合成HA分子量2.96×10^(6) Da,比出发菌1.97×10^(6)Da提高50.25%,另外3个菌株HA分子量变化不大。进一步通过RT-qPCR发现arcA过表达菌株中谷氨酰胺(HA合成中的氨基供体)合成酶编码基因glnA转录水平上调2.0倍,这可能是导致arcA基因过表达HA分子量上升的原因之一。【结论】挖掘到精氨酸代谢相关两个基因对HA分子量具有调控作用,为其他菌株调控HA分子量提供了新靶标。【Objective】The objective of this study is to explore the influencing elements of hyaluronic acid(HA)molecular weight,and to construct Streptococcus zooepidemicus mutants that synthesize HA with different molecular weights.【Method】When Streptococcus zooepidemicus S12 was cultured using different carbon sources to produce HA,it was found that the molecular weight of HA fermented by 10 g/Lfructose as the carbon source was 1.48×10^(6) Da,which was 29.52%lower than that of the original fermentation medium(50 g/L sucrose as the carbon source,2.10×10^(6) Da).Subsequently,transcriptomic sequencing of S12 fermentation broth with 50 g/L sucrose and 10 g/L fructose as carbon sources showed that the transcription levels of the two key genes of arginine deiminase pathway,arcA(encoding arginine deiminase)and argF(encoding ornithine carbamyltransferase),increased by 16.29 and 11.27 times,respectively,in addition to the fructose metabolism-related genes.In order to explore the effects of these two genes on HA synthesis,the genes arcA and argF were knocked out or overexpressed in S12,respectively.【Result】The HA molecular weight of arcA overexpressing strains was 2.96×10^(6) Da in CDM medium,which was 50.25%higher than that of S12(1.97×10^(6) Da),and the molecular weight of HA in the other three mutant strains did not show conspicuous changes.Further RT-qPCR showed that the transcription level of glnA,the synthase-encoding gene of glutamine(amino donor in HA synthesis),was up-regulated by 2.0 times in arcA overexpressing strain,which may be one of the reasons for the increase of the HA molecular weight.【Conclusion】Two genes related to arginine metabolism were found to regulate the molecular weight of HA,which provides new targets for other strains to control the molecular weight of HA.

关 键 词：兽疫链球菌 透明质酸 转录组 分子量 精氨酸代谢 

分 类 号：TQ281[化学工程—有机化工] Q78[生物学—分子生物学]