Whole-Cell Catalytic Synthesis Of Chondroitin Sulfate A Via Multi-Enzyme Cascade Pathway

Chondroitin sulfate A（CSA）is an important glycosaminoglycan,which is widely used in food,medicine,biomaterials and other fields.Compared with traditional physical extraction and chemical synthesis methods,the enzyme conversion method has the advantages of mild conversion conditions,a high conversion rate,and uniform product quality.At present,it is one of the most promising biological preparation methods.However,there are many problems with the key carbohydrate sulfotransferase 11（CHST11）,including difficult expression,poor thermostability,and low enzyme activity.It is noteworthy that adenosine 3’-phosphate5’-phosphosulfate（PAPS）,one of the substrates,is extremely expensive.In addition,the enzymatic conversion also brings high-cost separation and purification steps and costs,which limit the large-scale production of CSA.In this study,we developed a method to realize the supply of PAPS with cheaper ATP as the substrate and coupled the sulfonyl transfer pathway to synthesize CSA.We integrated cofactor engineering,protein engineering and surface display strategies to significantly improve the catalytic efficiency of whole-cell synthesis of CSA.The major results are described as follows:（1）Design,construction and validation of the one-pot production pathway of chondroitin sulfate A.First,the ATP sulfatase（Kl ATPS）from Kluyveromyces lactis and the adenosine 5’-phosphate sulfate kinase（Pn APSK）from Penicillium chrysogenum were identified as the best enzymes in the pathway through literature research;CHST11（Hs CHST11）derived from Homo sapiens was determined as the best enzyme based on the enzyme activity.The purified enzyme-catalyzed cascade reaction of three enzymes was carried out in vitro.The generation of CSA(10 g·L^-1substrate,degree of sulfation:38.8%)was determined by mass spectrometry,which verified the feasibility of the tri-enzyme cascade pathway.The optimal enzyme activity ratio of Kl ATPS:Pc APSK:Hs CHST11 was determined to be 1:1.5:3 and the degree of sulfation was 42.3%in vitro.Finally,the pathway enzymes’specific activity and conversion rate were determined,and Hs CHST11 was determined as the key rate-limiting enzyme.（2）Protein engineering of CHST11 to improve its thermostability and catalytic efficiency.Based on the 3D model built by Alpha Fold2,five mutants M1,M2,M3,M4 and M5 were predicted.M2 and M5 increased the half-life and T_mvalues to 10.5 h,10.0 h and66.0^oC,65.5^oC respectively.After integrating the beneficial sites in M2 and M5,the mutant M6 was obtained.Its half-life,T₅₀^2hand T_mvalues were increased to 11.5 h,47.4^oC and68.5^oC.The results showed that the sites in M6 optimized surface charge,increased enzyme hydrophilicity,and contributed energy to stability.Based on M6,the open and closed mechanism of the key"door ring"structure that controls the entry and exit of the substrate PAP（S）was studied through MD simulation,and two mutation strategies were proposed based on this.The best mutant M12 was obtained through multiple rounds of mutation,and the sulfation degree of chondroitin sulfate A reached 68.1%.The kinetic parameters showed that the specific enzyme activity of M12 increased to 132.4 U·L^-1and was 2.1 times that of wild type（WT）.K_mwas 4.21 m M,which was 0.64 of WT.And k_cat/K_mincreased from 12.45s^-1·M^-1of the wild type to 15.91 s^-1·M^-1.Molecular dynamics showed that in the mutant M12,the site Y304S increased the bottleneck of the PAPS entry channel from the original 4.5?to8?,while Y298S,A305G and T316A increased the RMSF value by 1.1?compared with WT,thus increasing the flexibility of the"door ring".（3）Double cofactor engineering to improve the supply of PAPS.Through literature research,it was determined that pyrophosphatase（Ec PPA）from Escherichia coli and polyphosphate kinase（Rs PPK）from Rhodobacter sphaeroides were used to realize the recycling of ATP.Aryl sulfotransferase IV（Rn ASTIV）from Rattus norvegicus was determined to realize the recycling regeneration of PAPS.Under the condition of purified enzyme catalysis in vitro,the conversion rate of 75%was achieved in the combination of the ATP cycle stage with the CSA synthesis path,75.3%was achieved in the combination of the PAPS cycle stage with the CSA synthesis path,and 82.7%was achieved in the combination of both stages with the CSA synthesis path(Kl ATPS:Pc APSK:Ec PPA:Rs PPK:Hs CHST11^M12:Rn ASTIV was 1:1.5:1:2:1.4:1.4).（4）Whole-cell catalytic synthesis of chondroitin sulfate A.The fusion expression of ice nucleation protein and Hs CHST11^M12in Escherichia coli Rosetta（DE3）realized the accurate subcellular localization of the target enzyme.The successful anchoring of Hs CHST11^M12was verified by laser confocal and flow cytometry analysis.Subsequently,all pathway enzymes were linked into E.coli Rosetta（DE3）to construct strain P6,which could achieve 38.3%conversion rate(10 g·L^-1chondroitin and 20 g·L^-1wet cells).Through the strategy of increasing the induction time and adding the cell membrane permeability agent,it was found that the conversion rate reached 79.8%when 0.8 g·L^-1tralatone was added at 16^oC for 24hours.The optimal conversion conditions were optimized as follows:10 g·L^-1chondroitin,20g·L^-1wet cells,0.8 g·L^-1Triton X-100,5 m M protamine,5 m M Mn Cl₂,60 m M ATP,20 m M Mg Cl₂,11 m M poly P6,200 m M Na₂SO₄,2 m M dithiothreitol and 120 m M p NPS,p H 6.8,37^oC reaction for 24 hours.The final conversion rate（sulfation level）was 89.5%in a 100 m L system.