Optimization Of Hyaluronic Acid Synthesis In Streptococcus Zooepidemicus Based On Omics Analysis

Hyaluronic acid is an acidic linear mucopolysaccharide composed of alternating residues ofβ-D-（1-3）glucuronic acid andβ-D-（1-4）-N-acetylglucosamine,widely present in animal tissues and the capsular layer of some bacteria.Hyaluronic acid has a variety of physiological functions such as moisturizing,lubrication and anti-inflammatory,and has a wide range of application prospects in the yield of food,cosmetics and medicine.Streptococcus Zooepidemicus（S.zooepidemicus）can naturally synthesize hyaluronic acid,and has become the main strain of hyaluronic acid industrial production.There are many advantages of hyaluronic acid fermentation using S.zooepidemicus,such as short fermentation cycle,low fermentation cost,and simple product separation and purification operation.However,the unclear genetic background and the lack of genetic manipulation system limit the further improvement of hyaluronic acid yield.Based on the genome-wide sequencing and transcriptome sequencing result,this study optimized the culture medium components and hyaluronic acid metabolism pathways of S.zooepidemicus WSH-24,and achieved to increase the yield of hyaluronic acid.The main conclusions of this paper are as follows:（1）Basic physiological characteristics and omics analysis.By basic physiological characteristics,it was found that S.zooepidemicus WSH-24 entered the stable growth phase at10 h.The product hyaluronic acid was growth-coupled typea and the yield was 0.8 g·L^-1in shaker flask.By the genome-wide sequencing,the basic genomic information of S.zooepidemicus WSH-24 was determined:the complete genome size was 2,147,053 bp,the GC content was 41.75%,and there were 1969 coding sequences（CDSs）in total.Through comparative genomic analysis,it was found that S.zooepidemicus WSH-24 was similar to S.zooepidemicus ATCC 35246.Analysing the central metabolic pathway,the strain had an efficient glycolytic pathway and lactic acid synthesis capabilities,as well as an incomplete tricarboxylic acid cycle.（2）Fermentation optimization based on omics analysis.Combined with the analysis of genome-wide sequencing result,this strain was determined to be deficient in four amino acids,including valine,glutamine,arginine,and tryptophan.Combined with transcriptome sequencing result,the transcription level of vitamin B5,B7,and B12-related synthetic genes was determined to be low.And the yield of hyaluronic acid separately increased 26.79%and21.33%by exogenous addition of corresponding amino acids and vitamins.（3）Establishment of genetic manipulation system.It was clarified that S.zooepidemicus WSH-24 was sensitive to antibiotics chloramphenicol,and chloramphenicol was selected as the resistance gene element to successfully construct the plasmid expression system.The conditions for competent cells preparation and electroporation were successfully optimized.During the competent cell preparation,when the bacteria was cultured to OD₅₃₀=0.35,hyaluronidase of1.25×10⁵ U·m L^-1 was added for 30 min.And during the electroporation,the electric field strength was 2.5 k V and the post-incubation time was 2 h.（4）Development of metabolic regulatory elements and optimization of hyaluronic acid synthesis pathway.Combined with transcriptome sequencing result,promoter elements were established in S.zooepidemicus WSH-24,including inducible promoter P_nisin and 66endogenous and exogenous constitutive promoters.The endogenous promoter P_has was the strongest.P_has was used to overexpress hyaluronic acid synthesis pathway gene,and the recombinant strain of the overexpression pathway gene glm M had the highest increase in hyaluronic acid yield,which reached 1.28 g·L^-1in shaker flask.Then,through the combined overexpression of hyaluronic acid synthesis pathway gene,the recombinant strain S.zooepidemicus WSH-24/p NZ8148has-glm S-glm M produced 1.37 g·L^-1 hyaluronic acid in shaker flask.Finally,combined with the fermentation optimization,the production capacity of recombinant strain reached 5.75 g·L^-1 in a 3 L fermenter.