| Hyaluronic acid(HA)is a negatively charged glycosaminoglycan comprising repeating Uridine diphosphate glucuronate(UDP-Glc A)and Uridine diphosphate N-acetylglucosamine(UDP-Glc NAc)disaccharide units,widely present in animal tissues and some bacterial capsules.HA's unique molecular structure and physicochemical properties make it have many physiological functions,such as lubrication,moisturizing,and wound healing promotion,so it is widely used in medical,cosmetics,food and other fields.At present,HA on the market is mainly obtained by animal tissue extraction and Streptococcus zooepidemicus fermentation.Animal tissue extraction method has many disadvantages such as insufficient raw material supply and complex extraction process,and the microbial fermentation method has probems of pathogenicity and endotoxin.Therefore,there is an urgent need to construct a safe and high-efficient HA producing strain.This article constructed the HA synthesis pathway in food-grade host Corynebacterium glutamicum,and the effects of HA substrate synthesis pathway,competitive pathway(extracellular polysaccharide),capsular layer on the synthesis of HA were also studied.The main conclusions of this paper are as follows:(1)Construction and optimization of HA synthesis pathway in C.glutamicum.Comparison of HA synthesizing capacity of four HA synthases Sp Has A,Se Has A,Su Has A and Pm Has A,and screening out the best source of hyaluronan synthase Sp Has A.Further systematically analyzed the influence of HA precursor pathway genes on the production of HA in 7 hosts including S.zooepidemicus and C.glutamicum,and the rate-limiting steps of HA synthesis were clarified.By overexpressing key genes(cgugd A2 and ptglm S),the yield of the recombinant strain HA shake flask reached 5.4 g·L-1(2)Weakened metabolic pathways of byproducts(extracellular polysaccharides).Through the study of C.glutamicum extracellular polysaccharides,it was found that Cg0420 and Cg0424are the key glycosyltransferases for the synthesis of Mannan and Arabic-Mannan Through gene knockout,the total amount of extracellular polysaccharides decreased by 28.5%and the production of HA is increased by 14.9%up to 6.4 g·L-1 and 5 L fermentor level HA production reach 34.2 g·L-1.(3)The formation of HA capsule and its effect on mass transfer.By studying the fermentation process of recombinant Corynebacterium glutamicum and Streptococcus zooepidemicus,it is clear that HA is first attached to the cell surface to form a capsular layer,and the capsular layer will inhibit the cell from taking nutrients from the external environment Affects cell metabolism.Based on this,the strategy of using HA hydrolase to reduce the molecular weight of HA and weaken the inhibitory effect of macromolecular HA on cell metabolism,thereby promoting the efficient synthesis of low molecular weight HA Through this strategy,the recombinant strain Cgsp H-7 has the highest HA production of 74.1 g·L-1,molecular weight 53.3±4.2 k Da at the level of 5 L fermentor. |
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