| Hyaluronic acid(HA),a linear glycosaminoglycan,is comprised of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid linked via alternating beta-1,4 and beta-1,3 glycosidic bonds.The unique biological and physicochemical characters of HA,such as moisturizing,lubricating and wound healing accelerating,etc.,have facilitated HA to be used widely in medical fields and cosmetics.Currently,HA is produced through extraction from animal tissues and microbial fermentation.However,animal tissue extraction method has unavoidable disadvantages,such as shortage of raw materials,high-cost purification and susceptibility to contamination.Microbial fermentation method has unique advantages compared with animal tissue extraction method,such as high HA yields,high quality and low cost.In the 1980s,Shiseido company produced large scale HA via fermentation by Streptococcus zooepidemicus for the first time.In the early stages,the yield of HA from fermentation by S.zooepidemicus was upregulated by optimization of fermentation process and modification of the metabolic pathway.Although the yield of HA was improved,the promotion by those conventional methods have reached the upper limit.With the further disclosing of the regulatory mechanism and metabolic network of S.zooepidemicus HA biosynthetic pathway,engineering global transcription factor to reconstruct the metabolic network will be a promising and effective way to lower fermentation costs and improve the quality of HA.Based on the above background,the main contents,results and conclusions of this study are as follows:(1)Construction of Streptococcus zooepidemic cvcc 2336ΔhasA.First,We optimized the transformation efficiencies in Streptococcus zooepidemic cvcc 2336 through investigating culture age,cell concentration,wall weakener,electroporation parameter,plasmids size,plasmids concentration and osmoprotection agents.In the present study,S.zooepidemicus cvcc 2336 cells were grown in THY medium containing 1%glycine to the early log phase(OD600 0.2～0.3)and then used to prepare the competent cells.Competent cells were washed free of media,suspended in electroporation buffers containing 0.5 mol/L of sucrose,electroporated parameters were set to 2.5 kV,25 μF and 200 Ω.After electroporated,cells were further recovered in THY medium containing 0.5 mol/L of sucrose,the transformation efficiency was 2×102cfu/μg plasmid DNA,which was tenfold higher compared with that of pre-optimized.The electrotransformation method reported here laid the foundation for extensive genetic manipulation and metabolic engineering of this industrially important bacterium S.zooepidemicus.Subsequently,the endogenous CRISPR-Cas9(Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9)system was reprogrammed to achieve hasA gene deletion in S.zooepidemicus cvcc 2336.S.zooepidemicus cvcc 2336 was transformation with plasmid containing single guide RNA(sgRNA)expression cassette targeting the hasA gene,HA concentration of transformants,determined by cetyltrimethylammonium bromide(CTAB)turbidity analysis,was used to select the transformants.And finally,the S.zooepidemicus cvcc 2336 Δ hasA which show 4-bp insertion within hasA gene was obtained,and the ΔhasA mutant appeared a definitely decressing HA production ability than that of the wild type strain.Taken together,we developed a stable and efficient electrotransformation protocol for S.zooepidemic cvcc 2336 strains and obtained a HAS-deficient strain by using endogenous CRISPR-Cas9 system successfully.(2)RNA sequencing identifies the Mga as a regulator of HA biosynthesis.RNA-seq on wild-type S.zooepidemic cvcc 2336 and S.zooepidemic cvcc 2336 Δ hasA was performed to investigate the potential regulator of HA biosynthesis.117 genes displayed differential expression in response to hasA deficiency,and KEGG analyses suggested these genes play a role in pathways associated with HA synthesis,including microbial metabolism in diverse environments,pentose and glucuronate interconversions,citrate cycle and pyruvate metabolism.After analysis of the function of each gene differentially expressed in the wild-type and the ΔhasA strain,we surprisingly found that although ΔhasA strain failed to synthesis HA,the level of global transcription regulator Mga and hasA,hasB and hasC in has operon(hyaluronic acid synthesis operon)were elevated significantly.Moreover,the elevation of those genes was further comfirmed by RT-PCR.Conclusively,we assumed that in S.zooepidemicus,Mga was the regulatory activator of HA synthesis.(3)The regulation role of the global regulator Mga in HA synthesis in S.zooepidemicus cvcc 2336.Through mga knockout and complementation experiment,it was found that the deletion of mga resulted in a decrease in HA production and mRNA expression of the hasA gene,while the mga complementation in the mga-deficient strain resulted in an increase in HA production and mRNA expression of the hasA gene,indicating that the transcription regulatory protein Mga activates the expression of the hasA gene.Results of electrophoretic mobility shift assays(EMSA)suggested that purified Mga showed high-affinity binding to the promoter of has operon(PhasA).Moreover,the binding site was localized by DNase I protection assays to 42 bp fragment containing the TSS(transcription start site)and-10 box.In summary,Mga in S.zooepidemicus functions as a transcriptional regulator activating the expression of the has gene through its promoter PhasA.(4)Construction of Mga based promoter library in Escherichia coli.It has been confirmed that the streptococcal transcriptional regulator Mga was also a regulatory activator of PhasA in Escherichia coli.Firstly,green fluorescent protein(GFP)was used as reporter gene to constructed a gradient strength constitutive promoter library based on Mga sensored promoter PhasA,because that the promoter PhasA is a strong constitutive promoter for GFP and Mga,activating transcription factor of PhasA,could promote the expression of GFP.Subsequently,we attempted to use our self-constructed Mga-related PhasA promoter library to build and optimize a glucaric acid(GA)metabolic pathway in E.coli.GA sensor was coupled to the transcriptional activator Mga to establish GA signal positive feedback amplifier,leading to an increase of GA production with the weakest PhasA mutant promoter.Taken together,Mga sensored PhasA promoter library provided an effective genetic circuit for gene networks in E.coli. |
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