Design And Construction Of An Engineered Strain Of Saccharomyces Cerevisiae That Produces High Levels Of Tyrosol And Its Derivative

Tyrosol,hydroxytyrosol and salidroside have been widely used in medicine,food,and cosmetics as their good pharmaceutical and nutritional activities.Currently,these three bioactive molecules are mainly extracted from plants,yet the low extraction efficiency and heavily plants dependency hamper their applications.Saccharomyces cerevisiae is a promising platform for synthesizing value-added compounds from simple carbon sources with several advantages.It is environmental friendly,besides,its production is sustainable and low-cost.These advantages draw our interest in harnessing S.cerevisiae to produce tyrosol,hydroxytyrosol and salidroside.Here we constructed tyrosol,hydroxytyrosol and salidroside overproducing S.cerevisiae strains via pathway engineering and protein engineering.The strategies presented here would provide some insights for the synthesis of other aromatic amino acid derivates.The tyrosol biosynthetic pathway in S.cerevisiae has many restrictions so that tyrosol is naturally synthesized with a very low yield.Hence,four strategies were performed to overcome these limitations.Firstly,the reported feedback-insensitive enzymes were overexpressed to unlock the carbon flux into the _L-tyrosine pathway.Secondly,the glycolysis and the pentose phosphate pathway were reconstituted to balance the precursors of the shikimate pathway.Thirdly,the rate-limiting enzymes of the shikimate pathway and the _L-tyrosine branch were overexpressed to channel the flux into tyrosol.Fourthly,the competing pathways were blocked to divert carbon flux entering tyrosol and restrict flux into competitive compounds.The titer of tyrosol was greatly increased through combining the above-mentioned strategies,reaching 9.9 g/L in tyrosol-producing S.cerevisiae strain through fed-batch fermentation in a 5 L fermenter.This is the highest yield in microbial cell factories ever reported.To enhance tyrosol production,we sought to explore novel strategies through three different perspectives.Firstly,one of the potential bottlenecks of the shikimate pathway is the feedback inhibition of _L-phenylalanine on the critical enzyme of DAHP synthase Aro3.We confirmed a feedback resistant mutant of Aro3^D154N via in vitro enzyme activity and plate assay with 5 m M _L-phenylalanine.Secondly,4-hydroxyphenylacetate biosynthetic pathway was explored in which Ald4 was majorly responsible for the conversion of 4-hydroxyphenylacetaldehyde to 4-hydroxyphenylacetate.Thirdly,down-regulation of TRP2（encoding anthranilate synthase）was performed to restrict carbon flux towards _L-tryptophan biosynthetic pathway during tyrosol production.Based on these endeavors,the tyrosol titer achieved 1350.5 mg/L while the titer of by-product tryptophol was only 10.5mg/L in our best-performing tyrosol-producing S.cerevisiae strain.Hydroxytyrosol（3,4-dihydroxyphenylethanol）,the hydroxylation derivate of tyrosol,could be directly synthesized from tyrosol through the catalyzation of 4-HPA 3-hydroxylase（Hpa B/Hpa C）.Thus,two different heterologous Hpa B and three different heterologous Hpa C were orthogonally overexpressed in the tyrosol-overproduction strain.As a result,overexpression of Pa Hpa B from Pseudomonas aeruginosa and Ec Hpa C from Escherichia coli could efficiently convert tyrosol to hydroxytyrosol in the tyrosoloverproducing S.cerevisiae.In addition,down-regulation of TRP2 facilitated flux into hydroxytyrosol.Moreover,Hpa BC would eliminate NADH during catalyzation,and introduction of feedback insensitive mutant of Tyr A^M53IA354V from Escherichia coli into S.cerevisiae could restore the pools of NADH and partially balance the NADH/NAD⁺ ratio as it converts NAD⁺ to NADH.The highest titer of 1120.0 mg/L hydroxytyrosol from simple carbon source（glucose）was achieved in our final hydroxytyrosol producing strain.Eventually,the crystal structure of Pa Hpa B and the complex of Pa Hpa B with FAD were elucidated via X-ray crystallography,which laid the foundation for rational protein engineering of Pa Hpa B to increase the catalytical activity and to improve the substrates’ specificity.Some uridine diphosphate glycosyltransferases（UGTs）could directly catalyze the glycosylation of tyrosol to salidroside.Three different heterologous UGTs were selected to synthesize salidroside in tyrosol producing strains,among which Rr U8GT33 from Rhodiola rosea outperformed Yji C from Bacillus subtilis and UGT72B14 from Rhodiola sachalinensis in our study.The highest titer reached 2396.0 mg/L salidroside in flask shakes,and the titer of salidroside reached 26.6 g/L in the 5 L fermenter,which were the highest reported titers in microbial cell factories so far.