Construction And Optimization Of Chondroitin Sulfate A Biosynthesis System

Glycosaminoglycans(GAGs)are long-chain linear polysaccharides composed of repeating disaccharide units and modified by sulfation and isomerization.They are widely distributed on the surface of vertebrate cells and extracellular matrix.According to the difference of its monomers and sulfation positions,GAGs can be divided into five categories,namely hyaluronic acid(HA),chondroitin sulfate(CS),dermatan sulfate(DS),heparin(HP)and keratan sulfate(KS).GAGs participate in a variety of metabolic and physiological functions and have a wide range of biological functions,including regulating cell proliferation,signal transduction and interacting with various cytokines,growth factors and pathogens.So,they have been applied in a variety of products and widely used medical,health products,cosmetics and food fields.Among them,CS is a drug that is widely used to prevent and treat osteoarthritis.Almost all of the currently commercialized CS is extracted from animal tissues.The extraction process has many drawbacks,such as the need for high-temperature treatment,consumption of a large amount of acid and alkali,potential pollution sources and pathogenic factors in animal tissues,and the mixing of other types of GAGs in the raw materials.Therefore,it is necessary to develop a green and environmentally friendly method that can synthesize CS with a specific structure.In this study,the efficient synthesis of CSA was achieved by using the enzymatic method and microbial de novo biosynthesis.Firstly,the synthesis of chondroitin was achieved in Pichia pastoris by introducing 3 foreign genes.Then the sulfonation-modification system was optimized and the enzymatically efficient synthesis of CSA was achieved in vitro through bifunctional protein and scaffold protein strategy.Then,C4 ST was optimized and active expressed in the P.pastoris,laying the foundation for the de novo biosynthesis of CSA.By combining the C4 ST expression module and the chondroitin synthesis module,the de novo biosynthesis of CSA in eukaryotic microorganisms was achieved for the first time.Through the enhancement of metabolic flux and the screening of high-copy strains,the production of chondroitin sulfate and the sulfation level have been improved.The main findings are as follows:(1)Constructing and optimizing the chondroitin synthesis pathway in P.pastoris.By introducing genes eckfo C,eckfo A from E.coli,and gene bstua D from B.subtilis,the successful synthesis of chondroitin was initially achieved with the yield of 5.5 mg/L.Then chondroitin production increased from 5.5 mg/L to 53.3 mg/L by optimizing the codon of bstua D.Additionally,the coding sequences of eckfo C and eckfo A were also optimized according to the codon preference of P.pastoris,which increased the concentration of chondroitin to 102.5 mg/L.Then 2A peptides were used to construct the polycistronic expression cassettes,and the highest chondroitin yield reached 189.8 mg/L,which was 34.5 times the initial yield.Then the recombinant strain was cultured in the 5-L fermenter to evaluate CSA biosynthesis.The highest yield of chondroitin reached 3.1 g/L(26 mg/g DCW),and the maximum dry cell weight reached127.5 g/L,which was 16.3 times the shake flask.(2)Optimization of CSA enzymatic synthesis system and large-scale preparation.Firstly,the influence of different stabilizers in the catalytic system was optimized,and it was found that glycerol had a positive effect on the enzyme-catalyzed reaction.Further,the concentration of glycerol,PNPS and PAP in the reaction system was evaluated.The conversion of chondroitin sulfate reached 76.8%,which increased 46.0% compared to the control(52.6%).To further improve the conversion efficiency of chondroitin sulfate,14 different kinds of linkers were designed to construct C4ST-linker-AST IV bifunctional proteins.The bifunctional protein d with(G4S)5 had higher C4 ST and AST IV enzyme activities.The bifunctional protein was used to catalyze the synthesis of CSA,and the conversion reached 94.0%,which was 22.4% higher than the conversion of the free reaction.The scaffold protein SH3-PDZ was assembled with C4 ST and AST IV which fused ligand protein at the C-terminus,respectively.This scaffold strategy was carried out to synthesize CSA and its 24 h conversion reached 97.2%.Finally,this reaction system was scaled up to 1-L,and the conversion efficiency reached 98.0%.(3)Construction and optimization of the de novo synthesis pathway of CSA.Firstly,six different Kozak sequences were designed and tested.It was found that different Kozak sequences had a significant effect on the expression of e GFP.When the K4 sequence(CAAACG)was used,the expression level of e GFP was much higher than that of the other groups.It was 20.2 times that of the K1 which had no Kozak sequence.Then 10 kinds of endogenous promoters of P.pastoris were further screened and compared.When the inducible promoter PAOX was used,C4 ST had the highest expression level.The combination of the best Kozak sequence(K4)and best promoter PAOX,C4 ST was successfully expressed in P.pastoris with the highest enzyme activity reaching 41.3 U/L.By integrating the chondroitin synthesis module and the C4 ST expression module,and optimizing the concentration of(NH4)2SO4 in the medium,the de novo synthesis of CSA was successfully achieved,with a yield of 182.0mg/L and a maximum sulfation level of 1.1%.In the 3-L fermenter,the output and the sulfation level of CSA were increased to 2.1 g/L and 4.0%,respectively,which were 11.3 times and 1.4times that of the shake flask level.(4)Increase the sulfation level of chondroitin sulfate A.Then the PAPS synthesis pathway genes derived from S.cerevisiae and P.pastoris were strengthened.The accumulation of PAPS in yeast cells was 0.64 and 0.75 ?mol/g DCW,respectively.When the PAPS synthesis pathway genes derived from P.pastoris were strengthened,the sulfation level of engineered strain C4CAD2 P was increased from 1.1% to 2.8%,and the corresponding yield was 185.6 mg/L.Then screening of multi-copy strains through the r DNA strategy increased the copy number of C4 ST in the cell and the sulfation level of CSA was further increased to 6.1%?13.1% by screening strains with different copy numbers.