Efficient Production Of Salvianic Acid A From L-DOPA Through A Tri-enzyme Cascade

Salvianolic acid（SAA）is widely used to treat cardiovascular and cerebrovascular diseases,and has a variety of pharmacological effects,such as anti-inflammatory,anti-tumor,anti-aging,and so on,which can effectively bring patients’physical condition to health.So far,the synthesis methods of SAA mainly include chemical method and enzyme conversion method.The chemical method requires a complicated and multi-step process,high production cost,and it is difficult to obtain SAA with optical purity.In contrast,the enzyme conversion method has a lower cost,but the low substrate load makes the spatio-temporal yield lower.Therefore,it is vital to develop an efficient synthesis process of SAA.In this study,we constructed a three-enzyme cascade pathway using L-dihydroxyphenylalanine（L-DOPA）as substrate to synthesize SAA through the catalytic action of L-amino acid deaminase（PmLAAD）from Proteus mirabilis,phenylpyruvate reductase（LaPPR）from Lactobacillus sp.CGMCC9967 and formate dehydrogenase（CbFDH）from Candida boidinii.Protein engineering was used to improve the catalytic activity of LaPPR and optimize the expression level of pathway enzymes,and finally improve the efficiency of SAA synthesis by this cascade pathway.The main study results are described as following:（1）Design,construction and verification of the synthesis pathway of SAA.First of all,based on literature review and database analysis,L-amino acid deaminase mutants PmLAAD^M2（H295S/V437S）from Proteus mirabilis and formate dehydrogenase（CbFDH）from Candida boidinii and phenylpyruvate reductase（LaPPR）from Lactobacillus sp.CGMCC9967 with high catalytic activity were selected as the pathway enzymes of this cascade reaction,the synthesis of SAA was verified by the reaction catalyzed by pure enzyme in vitro,and further LaPPR was determined as the rate-limiting enzyme of this cascade reaction by in vitro purified enzyme conversion.（2）Structural characteristics and catalytic mechanism of LaPPR.First,the crystal structure of LaPPR apo protein was analyzed.Based on the crystal structure,the docking model of wild type LaPPR-NADH-DHPPA was constructed and preliminary analysis was conducted,and the catalytic mechanism of LaPPR was speculated.The results of MD simulation of LaPPR-NADH-DHPPA complex and its representative snapshot indicated that the long hydride transfer distance d₁ and proton transfer distance d₂are the key factors for the low catalytic activity of LaPPR.（3）Protein engineering to improve the catalytic activity of LaPPR.The optimal mutant LaPPR^Mu2（H89M/H143D/P256C）was obtained through iterative saturation mutation and combination mutation,and its specific activity and catalytic efficiency(K_m/k_cat)were 2.8times and 9.3 times higher than that of the wild type,respectively.Representative snapshots of LaPPR-NADH-DHPPA and LaPPR^Mu2-NADH-DHPPA complexes were compared and analyzed by MD simulation results,it was found that the hydride transfer distance d₁ and proton transfer distance d₂ decreased from 3.97（?）and 4.12（?）to 3.51（?）and 4.01（?）,respectively,and the proportion of catalytic active conformations in the total conformation increased significantly（from 42/25000 to 610/25000）.Thus,the catalytic efficiency of LaPPR was improved.（4）Three enzymes were combined in vivo to synthesis SAA.PmLAAD^M2,LaPPR^Mu2and CbFDH were constructed into the same strain,and the in vivo activity ratio of the three enzymes was regulated by different copy number plasmids and RBS sequences,respectively,E.coli YJH11 strain was obtained,the activity ratio of three enzymes in vivo was close to the optimal activity ratio in vitro.Finally,the fermentation enzyme production conditions and transformation conditions were optimized.On the basis of the optimal conditions,90 g·L^-1 L-DOPA was converted into 82.6 g·L^-1 SAA by batch feeding,with the conversion rate of 91.3%and ee>99%.