Study On Protein Engineering Of Pyruvate Decarboxylase To Enhance Tyrosol Yield

Tyrosol is widely used in the fields of medicine and chemical industry as aromatic alcohols with pharmacological activity.Tyrosol can be synthesized by the four-enzyme cascade pathway previously constructed in our laboratory,however,the specific activity of pyruvate decarboxylase from Candida tropicalis（CtPDC）that catalyzed the decarboxylation of 4-hydroxyphenylpyruvate（4-HPP）was low,which made it a rate-limiting enzyme in the cascade of tyrosol synthesis.In this study,the crystal structure of CtPDC was analyzed for the first time,and its catalytic mechanism was analyzed.Based on the structure and mechanism,we conducted protein engineering on this allosteric enzyme.The obtained optimal mutant increased the enzyme activity and decarboxylation conversion,the mechanism of mutant performance improvement was analyzed by molecular dynamics（MD）simulation and other means.The optimal mutant of CtPDC was applied to the production of tyrosol,the yield and space time yield（STY）of tyrosol were further improved through subsequent conditions optimization,and the preparation of tyrosol in the amplification system of 3 L fermentor was realized.The main results are as follows:（1）Determination of rate-limiting enzyme in cascade pathway of synthetic tyrosol.First,the whole cell transformation experiment of L-tyrosine was carried out at different substrate concentrations,and the amount of each substance in the reaction solution was determined.It was found that the decarboxylated substrate 4-HPP catalyzed by CtPDC accumulated gradually with the increase of substrate concentration.Then,the transformation experiment of pure enzyme in vitro was carried out,and the accumulation of 4-HPP was alleviated by increasing the amount of CtPDC in vitro.Coupled with the determination of the kinetic parameters of four pure enzymes,it was found that the catalytic efficiency of CtPDC was the lowest.Finally,the rate-limiting enzyme CtPDC was determined by the above results.（2）Protein crystallization and analysis of structure and mechanism of CtPDC.First,the recombinant strain E.coli-p ET28a-CtPDC was isolated and purified,and the crystallization conditions were screened using commercial crystal screening kit,and then the crystallization conditions were determined and optimized.Under the optimal conditions,we determined the structure of apo-CtPDC and the cofactors(Th DP,Mg²⁺)-CtPDC complex via X-ray diffraction with 3.05?（PDB ID:8HP2）and 2.52?resolution（PDB ID:8HP4）,respectively.Then,the subunit polymerization and secondary structure of obtained crystal structure was analyzed.Subsequently,through the mutation verification after molecular docking,the key residues of catalytic region and allosteric region were analyzed.Finally,the substrate allosteric activation mechanism and decarboxylation mechanism of CtPDC were speculated.（3）Protein engineering of CtPDC to increase decarboxylation efficiency.To promote the decarboxylation efficiency of the catalytic region,protein engineering was carried out based on the allosteric regulation caused by substrate binding in the allosteric region,and the optimal mutant Mu₅was obtained.Compared with the wild type,the specific activity and decarboxylation conversion of Mu₅increased by 10.1-fold and 120.9%,respectively.Then,through MD simulation and MM-PBSA calculation,it was found that the model of Mu₅was more stable,and the two key distances(D_{（O-G415/S415）-H4’}and D_C2H-N4’)related to the formation of the ylide center in the catalytic region（C2 deprotonation）were shortened.Subsequently,we found that the nucleophilic attack distance was shortened from 3.8?to 3.0?.It was further found that the whole allosteric transmission pathway was shortened from 20.4?to18.6?.The loops on both sides of the allosteric transmission pathway also became more flexible.（4）Application of CtPDC^Mu5in tyrosol synthesis.Replacing the CtPDC in the original cascade with CtPDC^Mu5,it was found that the accumulation of 4-HPP in the pathway was reduced,but the effect was not significant.Subsequently,unbalance of protein expression was found by SDS-PAGE analysis.So the two plasmid vectors were combinatorial optimized.And then the transformation conditions were optimized,which shortened the original reaction time and reduced the addition of Th DP.Finally,the reaction system was expanded to 3 L fermenter.After 24 hours of reaction,the titer of tyrosol reached 38.0 g·L^-1with 99.6%conversion and 1.6 g·L^-1·h^-1space time yield（STY）.