One-Pot Cascade Biotransformation For Efficient Synthesis Of Hydroxytyrosol And Intermediates From Biobased L-Tyrosine

Hydroxytyrosol(HT)is one of the most powerful antioxidants,with low toxicity and antioxidant capacity.HT has been used for the prevention and treatment of cardiovascular diseases,breast cancer,gout and other diseases.HT synthesis has also become one of the hot spot in scientific research.Currently,commercial HT is mainly obtained from extraction of natural hosts or by chemical synthesis.While the processes have drawbacks,such as low yield,strong acid aqueous steam,long duration and unsustainability.In many synthetic processes,Multienzyme cascade biotransformation is a highly versatile and powerful method for HT synthesis which has the advantages of mild reaction conditions,inexpensive,simple process flow and high yield.Therefore,this study established a multi-enzyme cascade biotransformation pathway using Ltyrosine as a substrate to efficiently synthesize HT.We first attempted to use dopamine-mediated route for HT production.At the same time,we also detected the production of dopamine,which is often used clinically to treat diseases such as shock,Parkinson’s disease,and Alzheimer’s disease.In this study,the native hydroxylase(HpaBC)from Escherichia coli and L-DOPA decarboxylase(DODC)from Pseudomonas putida could efficiently convert L-tyrosine into dopamine with conversion above 90%.When 200 μM iron and 50 μM vitamin B6 were supplemented into the reaction system,the dopamine yield reached more than 99%,suggesting the activity of HpaBC and DODC require iron supplementation and vitamin B6.However,further incorporation of monoamine oxidase(MAO)from Micrococcus luteus and phenylacetaldehyde reductase(PAR)from Solanum lycopersicum only resulted in 69.4%conversion of L-tyrosine into HT.Next,we designed a second enzyme cascade that comprises L-amino acid deaminase from Proteus mirabilis(LAAD)and HpaBC from E.coli,α-keto-acid decarboxylase(ARO10)from Saccharomyces cerevisiae and PAR from S.lycopersicum.The second enzymatic cascade allowed more efficient production of HT,with 97.1%of L-tyrosine(25 mM)converted into HT(24.27 mM).It represents the most efficient HT biosynthetic system reported to date.In summary,we have developed a high-yielded dopamine and HT biosynthesis platform.