Construction Of Escherichia Coli Cell Factory For Efficient Synthesis Of Tyrosol And Hydroxytyrosol

Tyrosol and its derivatives are phenolic phytochemicals derived from olives.Among which tyrosol and hydroxytyrosol have natural antioxidant properties,so they are widely used in functional food ingredients and nutritional supplements.In recent years,more and more researches on the microbial synthesis of tyrosol and hydroxytyrosol have been conducted.In our previous studies,we have constructed.a series of microbial synthetic strains of tyrosol and hydroxytyrosol.Among them,in YMGRD1H1,hydroxytyrosol synthetic strain,Hpa BC could not completely transformed tyrosol.Hydroxylase Hpa BC and phenylpyruvate decarboxylase ARO10,as key enzymes in metabolic pathway,have not been reformed,and their catalytic efficiency needs to be further improved.Therefore,based on these strains,combined with metabolic engineering,directed evolution of enzymes and semi-rational modification strategies,this study aims to obtain high-yielding strains of tyrosol and hydroxytyrosol.The main findings are as follows:（1）Using protein engineering strategy,constructed the whole cell catalyst by the directed evolution of Hpa B and screening,a key enzyme in the synthesis of hydroxytyrosol.The hydroxylase Hpa B mutant library was screened by high-throughput screening workstation and hydroxytyrosol-sodium periodate color reaction.The fermentation results showed that YMG5A*R-Hpa B^TLEHC strain had a yied of 48.2 mmol·L^-1(7.43 g·L^-1)with the addition of 50mmol·L^-1 tyrosol,double the catalytic efficiency compared to the wild-type.The Hpa B^TLEHmutant had higher catalytic efficiency on the substrate tyrosol by the fermentation results.The recombinant strain BL21（DE3）p KK223-Hpa B^TLEHC was obtained by optimizing the chassis cells and expression vectors.The shaking yied of hydroxytyrosol was 59.8 mmol·L^-1(9.22g·L^-1)under the condition of adding 60 mmol·L^-1 tyrosol.（2）Construction recombinant strain with dual pathway for synthesis of hydroxytyrosol based on metabolic engineering.Exogenous tyramine oxidase（TYO）and tyrosine decarboxylase（DDC）were selected according to the literature.The fermentation results showed that the production of recombinant strain YMGRD1H1 p KK223-TYO-DDC hydroxytyrosol yied was 2.08 g·L^-1,with YMGRD1H1 of 1.81 g·L^-1,which increased by 14.9%.（3）Based on the computer simulation strategy,the key enzyme ARO10 in the synthesis of tyrosol was semi-rationally designed and screened,and the high-yielding strain of tyrosol was constructed.First,based on the computer simulation analysis of the substrate binding site of phenylpyruvate decarboxylase ARO10,the ARO10 hot-spot saturated mutant library was constructed,and a rapid color screening method was established to screen high-yielding tyrosol mutants.Finally,three recombinant strains YMGR p KK223-ARO10^D331S,YMGR p KK223-ARO10^D331V and YMGR p KK223-ARO10^D331C,with yield of tyrosol reached 1.91 g·L^-1,2.02g·L^-1,2.04 g·L^-1,respectively.Compared with the wild type yield 1.34 g·L^-1,the yield increased by 42.5%,50.7%,52.2%,respectively.By adopting protein engineering and metabolic engineering strategies,the conversion efficiency of Hpa BC hydroxylase and ARO10 of phenylpyruvate decarboxylase is improved,and a series of strains are obtained,which lays a foundation for the construction of hydroxytyrosol cell factory with industrial application potential.