Geaetic Engineering To Produce Heparosan

Heparin is an anticoagulant and is widely used in the medical field.However,most of the heparin drugs currently on the market are extracted from animal tissues,which have potential insecurity and supply hazards.In order to solve this problem,the preparation of non-animal heparin has become a hot research topic.Heparosan's polysaccharide skeleton is similar to heparin,except that glucuronic acid is not isomerized to iduronic acid,and there is no sulfation modification,so heparin drugs can be prepared by enzymatic chemical modification.After analyzing the synthetic pathway of heparosan,this paper selected the food-grade microbial Corynebacterium glutamicum rich in glutamine as the host,and constructed the recombinant strain producing heparosan,which laid a material foundation for the subsequent preparation of heparin drugs.The main findings of this paper are as follows:(1)By introducing the exogenous genes KfiA and KfiC into Corynebacterium glutamicum,the recombinant strain AC producing heparosan was constructed,and the synthesized heparosan was an intracellular product.The intracellular heparosan yield was up to 331.2 mg/L at 36 h after fermentation.(2)Based on the AC strain,a recombinant strain galU with a shake flask yield of 1 g/L was constructed by overexpressing the hasB and galU genes and exogenously adding KfiB and KfiD genes.On the basis of the BDH strain,the glycolytic pathway was weakened,and the yield of the best weakened strain A3 was 17.53%higher than that of the BDH strain.(3)The optimal induction conditions for the recombinant strain were determined by induction with 0.8 mM IPTG and induction at 30? for 40 h.The fermenter culture was then carried out under optimal induction conditions,and the intracellular heparosan yield could reach 2.93 g/L.(4)The molecular weight of the recombinant strain was studied by the addition of the lyase gene Elma,and the product size was degraded from 139 kDa(75%)and 5 kDa(25%)to 37 kDa(47%)and 5 kDa(53%).