Optimization Of Hyaluronic Acid Metabolic Pathway And Synthesis Of Hyaluronic Acid Oligosaccharides

The market demand of Hyaluranic acid?HA?which is widely applied in casomestics,food and medicine field is increasing nowadays,and it is expected to reach$9.85 billion in2019.The biological activity of HA depends on its molecular weight?Mw?.Compared with large molecular weight HA,HA oligosaccharides have important application prospects in the medical field due to their important physiological activities and special physiological functions.At present,the preparation methods of HA oligosaccharide are generally concentrated on animal tissue extraction,microbial fermentation,and followed by physical method,chemical method and enzymatic method.However,these preparation methods are complicated in process and economical,which could not satisfy the market demand of HA oligosaccharide.With major advances in the fields of metabolic engineering and molecular biology,the way to obtain HA or HA oligosaccharides by one-step fermentation of engineered strains is promising and attractive.Streptococcus zooepidemicus and Bacillus subtilis have advantages in biosynthesis of HA oligosaccharides.More than 90%of the HA presented on the market are produced by these bacteria,which are the platform for research and application in bio-fermentation of HA or HA oligosaccharides.In this paper,Streptococcus zooepidemicus and Bacillus subtilis were used as research objects and combined various strategies to optimize the metabolic pathway of HA oligosaccharides,which was achieved high yield of HA oligosaccharides.The main conclusions are as follows:?1?Overexpression of HasA with the control of the nisA promoter in Streptococcus zooepidemicus WSH-24,and the final concentration of nisin at 80 g·L^-11 glucose concentration was determined to be 40 ng·m L^-1.The LHAase was secreted and expressed in Streptococcus zooepidemicus by LHAase N-terminal fusion of six His-tag and RBS sequence optimization strategies,which the recombinant LHAase could achieve the maximum activity of 11113.2U·mL^-1.Meanwhile,overexpression and optimization of LHAase derived from leech can solve the problem of dissolved oxygen in the fermentation process and efficiently synthesis HA oligosaccharides.HA oligosaccharides accumulated to 0.97 g·L^-11 when the recombinant strain was shaken at 24 h,which was 182.0%higher than that of wild bacteria.And HA oligosaccharides accumulated to 7.06 g·L^-11 after fermentation at 24 h in 3 L fermenter,which was 112.4%higher than that of wild bacteria.?2?HA yield reached 0.49 g·L^-11 when the shake flask was fermented at 24 h,though optimizing the HA oligosaccharide metabolic pathway in Bacillus subtilis 168,and expressing szHasA and pmHasA in Bacillus subtilis by promoter engineering and expression isoenzyme strategy.Enhancement of the HA precursor was achieved by genomically integrating the pathway genes glmM,glmS,glmU,tuaD overexpressing with the P₄₃3 promoter.The efficient synthesis of HA oligosaccharides was achieved by integrating the expression of the leech hyaluronidase gene LHyal regulated by the P₄₃3 promoter on the genome.The autolysis-related genes lytH,lytG,lytD,and lytC were knocked out to enhance the biomass of recombinant bacteria and increase the HA oligosaccharide production.Finally,the yield of HA oligosaccharides reached 1.54 g·L^-1at 24 h of shake flask fermentation.?3?Construction of Bsr E/sr5,a post-transcriptional regulatory system of Bacillus subtilis,was proved to have good applicability and reliability though its application to the regulation of green fluorescent protein GFP and the cleavage initiation protein FtsZ.The regulatory system bsrE/sr5 was applied to reduce the expression of genes involved in the alternative pathway.To speak specifically,the expression of sr5 was down-regulated by the weaker promoter PsigW of the endogenous class IV constitutively to down-regulate the expression of the alternative pathways zwf,pfkA,mnaA,murAA,murAB.At the same level,the HA oligosaccharide production was improved while maintaining the normal physiological activities of the cells.The OD600 was basically maintained at about 9,while the HA oligosaccharide production was greatly increased,and the shake flask fermentation reached2.51 g·L^-11 at 24 h.?4?High-efficiency biosynthesis of HA oligosaccharides was achieved by batch fed fermentation in a 3 L fermentor.The yield of HA oligosaccharide reached 12.6 g·L^-11 and the production intensity was 315.0 mg·?L·h?^-1at 40 h of the recombinant strain BSha34 fed-batch fermentation.The constructed recombinant bacteria have obvious advantages in the production of HA oligosaccharides.