Synthesis Of Hydroxytyrosol From L-DOPA Catalyzed By Recombinant Escherichia Coli Whole Cells

Hydroxytyrosol?HT?is a natural polyphenolic compound mainly found in olives.It has a variety of beneficial biological and pharmacological activities,such as antioxidation,anticancer,antibacterial,anti-inflammatory,prevention of cardiovascular and cerebrovascular diseases and diabetes,protection of retina and bone,and has great potential applications in medicine,food and cosmetics.In view of the shortcomings of current HT synthesis methods,a method for the production of hydroxytyrosol from L-DOPA?L-3,4-dihydroxyphenylalanine?using engineered E.coli whole cells is proposed in this study,which is economical,simple and efficient,and is expected to pave the way for the industrial production and practical application of HT.The main contents and results are as follows:?1?The synthesis pathway of HT was proposed on the basis of aromatic amino acid transaminase?TyrB?,?-keto acid decarboxylase?PmKDC?,aldehyde reductase?YahK?and L-glutamate dehydrogenase?GluDH?screened in the previous work of our laboratory.In this pathway,L-DOPA is converted to 3,4-dihydroxyphenylpyruvic acid by the transamination of TyrB,which is further converted to 3,4-dihydroxyphenylacetaldehyde by the decarboxylation of PmKDC,and finally,3,4-dihydroxyphenylacetaldehyde is reduced to HT by Yah K.GluDH is used to couple the transamination and reduction to achieve cofactor regeneration.The feasibility of this pathway was successfully verified by the mixed catalysis of four enzymes.?2?A series of co-expression strains were constructed by co-expression of the four enzymes in two plasmids.The relative activities of the four enzymes were regulated by using three plasmids with different copy numbers:pRSFDuet-1?high copy number?,pETDuet-1?medium copy number?and pCDFDuet-1?low copy number?,to balance the reaction rate of each enzyme and resolve the bottleneck of the reaction pathway.The HT production ability of each strain was compared,and the best HT synthesis strain?E.coli BL21?DE3?/pRSF-yahK-tyrB/pCDF-gludh-Pmkdc?was obtained.?3?The culture medium was optimized,including carbon source,nitrogen source,inorganic salt and proportion of different components in the culture medium.The optimum culture medium was peptone 15 g·L^-1,yeast extract 30 g·L^-1,glucose 10 g·L^-1,MgSO₄ 2.5mmol·L^-1,KH₂PO₄ 17 mmol·L^-1,K₂HPO₄ 72 mmol·L^-1.The yield at 1 h(6.21 mmol·L^-1)after medium optimization was 81.05%higher than that of unoptimized LB medium?3.43mmol·L-¹?.?4?The induction conditions including induction timing,IPTG concentration,induction temperature and induction duration were optimized.The optimum induction conditions were as follows:induced when OD₆₀₀ is 0.8,IPTG concentration 0.4 mmol·L^-1,induction temperature 20?,induction time 18 h.The yield at 1 h(7.45 mmol·L^-1)after induction conditions optimization was 19.97%higher than that before optimization.?5?The whole-cell catalysis conditions including pH,temperature and rotational speed were optimized.The optimum whole-cell catalysis conditions were as follows:pH 7.5,temperature 35?,rotational speed 50 r·min^-1.The yield at 1 h(9.60 mmol·L^-1)after whole-cell catalysis conditions optimization was 28.86%higher than that before optimization.?6?When the optimal strain was used to synthesize HT under the optimal conditions,the yield of HT reached the highest after 7 h,which was 39.79 mmol·L^-1(6.13 g·L^-1).The conversion rate was 97.98%,and the comprehensive space-time yield of 7 h was 0.88g·L^-1·h^-1,which is better than the studies of biosynthesis of HT reported in the literatures.?7?The HT obtained by the reaction was preliminarily purified by adsorption of macroporous adsorption resin and extraction of ethyl acetate,and the HPLC detection results showed that the purity calculated by peak area normalization method was 99.6%under 280nm.