“One Pot” Synthesis Of Chondroitin Sulfate A By Engineered Pichia Pastoris

Chondroitin sulfate(CS)is an important glycosaminoglycan(Glycosaminoglycans,GAGs).Consisting of disaccharide sequences glucuronic acid(Glc A)and N-acetamidogalactose(Gal NAc)alternately by β-1,3 and β-1,4 glycoside bonds,sulfonylation reactions occur with different sulfonyltransferases at different locations.Because chondroitin sulfate has physiological functions such as repairing cartilage tissue,antioxidant,anti-inflammatory and so on,chondroitin sulfate is widely used in medical,health care and other fields.At the same time,the synthesis mode and transformation efficiency of chondroitin sulfate have gradually become the focus.The traditional way of obtaining chondroitin sulfate mainly depends on animal tissue extraction.This way not only has the risk of destroying the ecological balance,but also has the risk of immune safety.At present,the modified method is not only suitable for the industrialized production of chondroitin sulfate A(CSA),but also a more efficient and controllable method for the large-scale synthesis of CSA.(1)Chondroitin synthesis pathway was constructed in Pichia pastoris GS115 to achieve chondroitin synthesis.Using plasmid p GAPZB as the vector,the "multi-cistransgenerate" expression frame was simulated to integrate the chondroitin synthase coding genes kfo C,kfo A and UDP-glucose dehydrogenase coding genes tua D into the P.pastoris genome.Finally,this subsection achieved the synthesis of 105 mg·L-1chondroitin.(2)Through the optimization of chondroitin synthesis pathway,the titer of chondroitin was increased.Firstly,glm S,gpa T,pag M and gal U genes from P.pastoris and Saccharomyces cerevisiae were overexpressed respectively.It was found that increasing the synthetic flux of metabolic intermediates UDP-glucose、Glc N-6-P、Glc NAc-6-P and Glc NAc-1-P could increase the titer of chondroitin,and the recombinant strain Pp00-pgal U was obtained to increase the titer of chondroitin to 170mg·L-1.Then,through the combined overexpression of chondroitin synthesis pathway genes,the chondroitin production was further increased to 495 mg·L-1.Finally,the large-scale production capacity of the optimal strain Pp00-DSM was verified in a 3 L fermentor.The highest chondroitin titer was 2.6 g·L-1 after fermentation in BSM medium for 144 h.(3)Optimization of 3?-phosphoadenosine-5?-phosphoryl sulfate(PAPS)production conditions and C4 ST storage conditions.In order to construct the sulfonation modification system in vitro,the culture condition of Escherichia coli BL21(DE3)strain expressing the bifunctional enzyme gene for PAPS synthesis were optimized to improve the titer of PAPS.By optimizing the shaking flask conditions,it was found that when the culture temperature was 30℃,the inducer concentration was0.5 m M,the culture time was 15 h and the induction OD600 was 1.0,the productivity of PAPS was the highest,which was 5 times that of the control group.Subsequently,in order to increase the storage time of C4 ST and reduce the production cost of CSA,it was found that the residual enzyme activity of C4 ST crude enzyme solution with 10%BSA was still up to 80% after being stored at refrigerator for 20 days.(4)Establishment and optimization of "one pot" synthesis system of chondroitin sulfate A.Chondroitin-producing engineered strains were fermented and collected,and the supernatant obtained by breaking the cells was added with PAPS and C4 ST crude enzyme solution prepared in advance,and placed at 30 °C for 12 h.CSA was detected,and the conversion rate was 5%.In order to improve the conversion of CSA,the reaction time and temperature were optimized successively.It was found that the conversion of CSA increased to 15% after 48 hours at 37℃.In addition to the optimization of reaction time and temperature,this study tried to change the ratio of C4 ST to PAPS in the "one-pot" system to further improve the conversion of CSA,and finally achieved the synthesis of CSA with different sulfonation levels of 0-40%.