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

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

Abstract

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

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.

Figures/Tables 10

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

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

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

Fig. 3 Differential gene volcano map

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

Fig. 4 GO enrichment P-adjust histogram

Fig. 5 KEGG enrichment scatter plot of differential genes

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

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

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

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