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

doi:10.13560/j.cnki.biotech.bull.1985.2024-0675

摘要/Abstract

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1.48×10⁶ 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2.10×10⁶ 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2.96×10⁶ Da，比出发菌1.97×10⁶ 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。

关键词: 兽疫链球菌, 透明质酸, 转录组, 分子量, 精氨酸代谢

Abstract:

【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 1.48×10⁶ Da, which was 29.52% lower than that of the original fermentation medium（50 g/L sucrose as the carbon source, 2.10×10⁶Da）. 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 2.96×10⁶ Da in CDM medium, which was 50.25% higher than that of S12（1.97×10⁶ 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.

Key words: Streptococcus zooepidemicus, hyaluronic acid, transcriptome, molecular weight, arginine metabolism

裴旭娟, 狄靖宜, 刘浩, 高伟霞. 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[J]. 生物技术通报, 2025, 41(1): 347-356.

PEI Xu-juan, DI Jing-yi, LIU Hao, GAO Wei-xia. Exploration of Regulatory Elements for Hyaluronic Acid Molecular Weight in Streptococcus zooepidemicus via Transcriptome Analysis[J]. Biotechnology Bulletin, 2025, 41(1): 347-356.

图/表 10

表1 实验所用质粒及菌株

Table 1 Plasmids and strains used in this study

Type	Laboratory number	Genotype/Phenotype	Description	Source
Plasmid	pLH67	pSET4s::sacB	Temperature sensitive vector	Our Laboratory
	pLH421	pDL278	Streptococcus shuttle vector, Specr	Our Laboratory
	pXJ00	pLH421::PR22	pDL278 carrying PR22	Our Laboratory
	pXJ01	pSET4s::sacB::arcA LR	arcA markerless deletion vector	This work
	pXJ02	pLH421::PR22::arcA	pDL278 carrying PR22::arcA gene	This work
	pXJ03	pSET4s::sacB::argF LR	argF markerless deletion vector	This work
	pXJ04	pLH421::PR22::argF	pDL278 carrying PR22::argF gene	This work
Strains	S12	ATCC 39920	Wild-type strain	Our Laboratory
	S3896	ΔarcA	arcA markerless deletion mutant	This work
	S3897	arcA/OP	S12 containing plasmid pXJ02	This work
	S3898	ΔargF	argF markerless deletion mutant	This work
	S3899	argF/OP	S12 containing plasmid pXJ04	This work

图1 基因敲除菌株筛选流程图（以arcA为例）

Fig. 1 Screening schematics of the gene-deleted strains（Having arcA gene as an example）

图2 野生型菌株在不同碳源下产HA的表征 A：野生型菌株摇瓶生长情况；B：野生型菌株摇瓶发酵24 h HA的产量；C：24 h HA分子量凝胶图，1：原培养基（50 g/L蔗糖）；2：10 g/L葡萄糖；3：10 g/L果糖；4：10 g/L蔗糖； D： HPGPC 检测不同碳源下发酵S12菌株 HA分子量；图中50 g/L S（原）：50 g/L 蔗糖（原始培养基）；10 g/L G：10 g/L 葡萄糖；10 g/L F: 10 g/L 果糖；10 g/L S: 10 g/L 蔗糖

Fig. 2 Characterization of HA production by wild-type strains under different carbon sources A: Growth of wild-type strain in shaking bottle. B: Yield of hyaluronic acid from wild-type strain in shaking flask for 24 h. C: Molecular weight gel map of 24 h HA, 1: original medium（50 g/L sucrose）; 2: 10 g/L glucose; 3: 10 g/L fructose; 4: 10 g/L sucrose）. D: HPGPC to detect HA molecular weight of fermented S12 strain under different carbon sources. In the figure, 50 g/L S（original medium）: 50 g/L sucrose（original medium）; 10 g/L G: 10 g/L glucose; 10 g/L F: 10 g/L fructose; 10 g/L S: 10 g/L sucrose

图3 差异基因火山图

Fig. 3 Differential gene volcano map

表2 转录组差异明显的关键基因

Table 2 Key genes with distinct transcriptome differences

Gene	Gene ID	对数期（FPKM）		Regulate
Gene	Gene ID	S12（50 g /L蔗糖）S12（50 g/L sucrose）	S12（10 g /L果糖）S12（10 g/L fructose）	Regulate
arcA	SeseC_RS02770	102	1 700	Up
argF	SeseC _RS02780	160	1 900	Up
scrA	SeseC _RS01805	1 907.5	20	Down
fruR2	SeseC _RS05815	1.72	175	Up
SeseC _RS05805	SeseC _RS05805	20.25	364.3	Up
SeseC _RS01810	SeseC _RS01810	344	8.2	Down

图4 GO 富集P-adjust 柱状图

Fig. 4 GO enrichment P-adjust histogram

图5 差异基因 KEGG 富集散点图

Fig. 5 KEGG enrichment scatter plot of differential genes

图6 兽疫链球菌中蔗糖代谢途径相关基因的转录水平倍数示意图红色表示基因转录水平升高，蓝色表示基因转录水平降低

Fig. 6 Schematic diagram of transcriptional levels of genes related to sucrose metabolism pathway in Streptococcus zooepi-demicus Red indicates elevated gene transcription levels, and blue indicates decreased gene transcription levels

图7 四个构建菌株与野生型菌株在CDM培养基中产HA的表征 A：各菌株摇瓶生长情况；B：各菌株摇瓶发酵24 h 的HA产量； C：24 h 的HA分子量凝胶图（1：兽疫链球菌野生型 S12；2：ΔarcA菌株；3：arcA/OP菌株；4：ΔargF菌株；5：argF/OP菌株）；D：HPGPC 检测各菌株产HA分子量

Fig. 7 Characterization of HA in CDM medium of four constructed strains and wild-type strains A: Growth of each strain in shaking flask. B: HA production of each strain in shaking flask for 24 h. C: Molecular weight gel map of HA at 24 h (1: Streptococcus zooepidemicus wild-type S12; 2: ΔarcA strain; 3: arcA/OP strain; 4: ΔargF strain; 5: argF/OP strain). D: HPGPC to detect the molecular weight of HA produced by each strain

图8 S12、S3896、S3897 中 glnA和 gdhA 基因转录水平

Fig. 8 Transcription levels of glnA and gdhA gene in S12, S3896, and S3897

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