Production Of Hydroxytyrosol From L-DOPA By Multienzyme Cascade Transformation

Hydroxytyrosol,a phenolic compound derived from olives,is conducive to human health and is widely used in food additives and cosmetic industries due to its significant antioxidant activity and cancer prevention properties.While traditional plant extraction and chemical synthesis methods have many limitations,biotransformation methods have the advantages of green sustainability,simple reaction process,high substrate specificity,and undependence on metal catalysts,and gradually become favorable tools for the synthesis of the natural products hydroxytyrosol.In this study,we developed a novel four-enzymes cascade system to co-express genes of L-amino acid deaminase（LAAD）,α-keto acid decarboxylase（Pm KDC）,aldehyde reductase（Yah K）,and glucose dehydrogenation（GDH）in Escherichia coli BL21（DE3）.Therefore,which sequentially convert 3,4-dihydroxyphenyl-L-ananine（L-DOPA）into hydroxytyrosol through deamination,decarboxylation,and reduction reactions.GDH catalyzes the dehydrogenation of glucose to regenerate NADH,which is an essential hydrogen donor for the accumulation of hydroxytyrosol catalyzed by Yah K.Subsequently,the strategy of different copy-number plasmid combination was adopted to coordinate the expression of pathway enzymes to achieve better cooperativity in the whole-cell muti-enzyme cascade reactions.The main research contents and conclusion of this paper are divided into the following aspects:（1）Selection and in vitro validation of multi-enzyme cascade pathway enzymes.The LAAD form Cosenzaea myxofaciens,Pm KDC from Proteus mirabilis JN458,Yah K from Escherichia coli BL21 and GDH from Bacillus subtilis ATCC 13952 were selected for the cascade pathway in our laboratory for previous screening.Firstly,LAAD,Pm KDC,Yah K and GDH enzyme solutions were used for the vitro cascade reaction,and the production was detected by HPLC,which proved the feasibility of this cascade pathway.（2）Construction of recombinant E.coli and optimization of enzymes expression levels.The aad L,pmkdc,yahk,and gdh were integrated into E.coli with the two-plasmid co-expression strategy.Subsequently,three different copy number compatible plasmids were combined to express these four genes to regulate the relative activity of the enzyme as well as the equilibrium reaction rate.Nine recombinant strains were successfully reconstructed and Pm KDC was determined to be the most important rate-limiting enzyme in this pathway.Among these nine strains,the coordination between each enzyme of HTG7（E.coli BL21（DE3）/p RSF-aad L-pmkdc-yahk/p ET-gdh）was the best,and the yield of hydroxytyrosol catalyzed by the whole-cell catalyst was the highest at 36.40 mmol·L^-1.（3）Serial optimization of culture and transformation conditions for recombinant strains.The optimum medium was determined as follows:15 g·L^-1 glycerol,25 g·L^-1 yeast extract,20g·L^-1 peptone,1.5 g·L^-1 magnesium sulfate,2.2 g·L^-1 potassium dihydrogen phosphate,and 9.4g·L^-1 potassium dihydrogen phosphate;The optimal conditions for enzyme induction were determined as follows:when the bacterial concentration at the beginning of induction was controlled at OD₆₀₀=0.8,the concentration of IPTG was controlled at 0.4 mmol·L^-1,and the induced temperature was controlled at 20°C;The optimal whole-cell biotransformation conditions were determined as follows:NAD⁺concentration 0.3 g·L^-1,temperature 30°C,p H7.0,wet cell concentration 35 g·L^-1,and rotation speed 200 r·min^-1.After series optimization,the yield of hydroxytyrosol was increased to 45.27 mmol·L^-1.According to the whole cell optimized biotransformation conditions,the production of hydroxytyrosol was expanded in a 5L fermentor,and finally 56.85 mmol·L^-1(8.75 g·L^-1)hydroxytyrosol was synthesized from60.91 mmol·L^-1 L-DOPA in 8 h.And,the spatiotemporal productivity of hydroxytyrosol was1.1 g·L^-1·h^-1.Finally,the hydroxytyrosol with high chromatographic purity was obtained by multiple extraction of the reaction solution with ethyl acetate and distillation of the organic phase by a rotary evaporator,and the extraction rate of hydroxytyrosol by ethyl acetate was as high as 95.9%.