Study On The Transformation Of The Metabolic Pathway Of Bacillus Subtilis To Synthesize Hyaluronic Acid

Hyaluronic acid(HA)is based on two monosaccharides,uridine diphosphate-acetylglucosamine(UDP-GlcNAc)and uridine diphosphate-glucuronic acid(UDP-GlcUA).?-1,3 and ?-1,4 glycosidic bonds are alternately connected to form a disaccharide unit,which is a glycosaminoglycan composed of a repeating structural unit.HA is widely used in cosmetics and pharmaceuticals.The biological function of HA is directly related to its molecular weight(MW).Large-molecular-weight hyaluronic acid has the effects of space filling,anti-angiogenesis,and immunosuppression;low-molecular-weight hyaluronic acid has unique biological activity,which can stimulate fibroblast proliferation and Collagen synthesis.The traditional preparation method of HA is mainly extracted from animal tissues,which has the risk of infection with cross-species viruses.Therefore,currently commercialized HA production mainly relies on the fermentation of certain attenuated strains of the genus Streptococcus.The production cost of this process is low and the environmental pollution generated is also less.However,streptococcus species have a certain risk of pathogenicity,coupled with a complex genetic background,there are fewer genetic manipulation tools available for genetic modification,which limits the development of this method.Therefore,it is necessary to construct HA-producing strains with high biological safety and clear genetic background.B.subtilis,generally considered a safe strain,an ideal cell factory.The predecessors have successfully transferred the Hyaluronan synthase(HAS)gene into Bacillus subtilis 168,and have carried out Fermentation production of HA.First,we constructed a CRISPR / Cas9 dual vector system in Bacillus subtilis.Secondly,we cloned the synthetic hyaluronic acid-related gene from Bacillus subtilis168,ligated it with the cloning vector PUC119,and transformed it into E.coli for subsequent gene-related operations.Then,we used overlapping extension PCRtechnology to ligate the genes on the GlcUA operon,GlcNAc operon,and tetM operon,respectively,to construct three plasmids that can replicate in E.coli in large quantities,and to integrate three operons Expression unit was transformed into Bacillus subtilis.Finally,in the shake flask culture,we tested the yield and molecular weight of HA.Through the comparison of whether theophylline small molecules were added to the medium,it was proved that adding theophylline small molecules to the medium can increase the yield of HA and make HA The yield reached 1.81 g / L;meanwhile,the molecular weight of HA was reduced,and the molecular weight was reduced to 1.01 MDa.It was proved that theophylline small molecules can regulate the transcription of GlcUA pathway.It shows that the overexpression of all genes of the GlcUA branch pathway(pgcA,gtaB and tuaD)is inversely related to the molecular weight of HA and positively related to the yield of HA.The upstream of the GlcUA operon contains the P43 promoter and theophylline nuclear switch.The P43 promoter can enhance gene expression.When small theophylline molecules are added to the culture medium,theophylline binds to the nuclear switch,inhibits the formation of terminator structure,and performs transcription.Increased the synthesis of UDP-GlcUA,enabling Bacillus subtilis to efficiently synthesize low molecular weight HA.The upstream of the GlcNAc operon contains the P43 promoter and the tetracycline nuclear switch.When a small molecule of tetracycline is added to the culture medium,the tetracycline binds to the nuclear switch,inhibits the formation of terminator structure,performs transcription,increases the synthesis of UDP-GlcNAc,and makes Bacillus subtilis Can efficiently synthesize high molecular weight HA.The upstream of the tetM operon contains the P43 promoter and the tetracycline resistance gene,allowing Bacillus subtilis to grow normally in the presence of tetracycline in the medium.Based on synthetic biology,this paper constructs a metabolic pathway in Bacillus subtilis that can regulate the production of specific HA molecular mass,which provides the possibility for subsequent small molecules to regulate the metabolic pathway of Bacillus subtilis and the mass production of hyaluronic acid with a specific molecular weight.