Production Of Tyrosol And Its Derivatives Salidroside By Cell Factories Of Saccharomyces Cerevisiae

Saccharomyces cerevisiae as a long history of living production strain,is closely related to human production activities.Because of its clear genetic background,easy cultivation,biosafety and other advantages,it is widely used in biosynthesis of high value-added metabolites.Aromatic compounds are a cyclic compounds that contain at least one delocalized bond in the molecule,and are indispensable in the field of medicine and chemical industry.Among aromatic compounds,the demand of tyrosol and its derivatives-salidroside increasing because of their function,such as anti-inflammatory,anti-tumor,nerve,protect cardiovascular health and other medical effects etc.,However,tyrosol and salidroside production with plant extraction and chemical synthesis were limited due to material shortage,environment pollution.Therefore,biological synthesis was focused because of its green sustainable features.The metabolic engineering of microbial cells with synthetic biology is benefit for improving tyrosol and salidroside production.In this study,to further increase the production of tyrosol and salidroside in S.cerevisiae,some rational metabolic engineering strategies were developed and applied in.Using the tyrosol-producing strain DA-1 as the chassis cells,which previously constructed by our team.Because of the enzymes play key roles in shikimate pathway are under the feedback inhibition of aromatic amino acids,the mutants ARO4K229L and ARO7G141S were introduced into the aro3 locus of DA-1 to release the feedback inhibition of aromatic amino acids in S.cerevisiae.The tyrosol yield of DAY-1 was increased by 65.29%.Gcr2 is a transcriptional regulator of glycolysis,and the deletion of GCR2 led to the upregulation of PPP genes.To enhance the pentose phosphate pathway,the is a global transcriptional factor gene GCR2 of S.cerevisiae was disrupted in DAY-1.The results showed that neither the biomass nor tyrosol yield of strain DAY-2-22 changed significantly after the deletion of GCR2.In order to redirect the carbon flow towards the biosynthesis of ethanol to the biosynthesis of tyrosol,the overexpression of ADH2 into the adhl locus of DAY-1,and the tyrosol yield of DAY-2-24 was increased by 15.41%.The introduction of ADH2 was an effective strategy to improve tyrosol biosynthesis of S.cerevisiae.In order to reduce the production of by-products of biosynthesis of aromatic amino acids,the O-aminobenzoate synthase gene TRP3(DAY-2-26)and prephenate dehydrase gene PHA2(DAY-2-27)were knocked out in strain DAY-1.The tyrosol production of DAY-2-27 achieved reached 1.05±0.05 g/L after 96 h of shake flask cultivation,which resulted in an increase of 49.08%in tyrosol production.In order to increase the supply of E4P,an exogenous E4P biosynthesis pathway based on phosphoketolase was was introduced into pha2 site of strain DAY-1.The tyrosol yield of strain DAY-2-29 was increased by 107.87%to 1.41±0.03 g/L after 96 h of shake-flask fermentation.It was reported that U8GT33 from Rhodidum rosea had a high catalytic efficiency.In order to introduce salidroside biosynthesis pathway into S.cerevisiae,RrU8GT33 was overexpression into trp3 locus of tyrosol-producing strain DAY-2-29.The yield of salidroside reached 2.77 g/L after 96 h of shaking fermentation.Using the surface system of "SSS" cassette of S.cerevisiae,RrU8GT33 was displayed on the cell surface of strain DAY-1 to catalyze the glycosylation of extractyrosol.The results showed that DAY-2-12 strain displayed on U8GT33 was seriously flocculated during shaking flask fermentation,but 1.25 g/L salidroside was generated after 96 h of shaking flask fermentation,indicating that the catalytic efficiency of RrU8GT33 was high on cell surface.