Studies On Metabolic Engineering Of Saccharomyces Cerevisiae For The Production Of Tyrosol And Salidroside

Natural products are excellent sources of drugs and food additives,and are widely used in the food,health and beauty industries as highly valuable raw materials.Nowadays,plant extraction and chemical synthesis are the main methods commonly used in the industrial production of natural products.The traditional method of extracting natural products from plant tissues is low efficiency and requires complicated separation process.Moreover,the plants biomass is susceptible to the quality of soil,climate and other external factors.Chemical synthesis is difficult to achieve de novo synthesis,and requires harsh and complicated reaction conditions,which is energy consuming and environmental polluted.Differed from the methods described above,the biosynthesis method is biosynthesizing high-value-added natural products using cheap carbon sources as raw materials via microbial cell factories,which is low-cost,high efficiency,environmentally friendly,and sustainable,and has received increasing attention in recent years.Under the guidance of the disciplines of synthetic biology and metabolic engineering,systematic metabolic engineering of microorganisms for the efficient production of natural products with the biotechnologies of molecular biology is a feasible strategy to meet the growing demand for natural products in the marketplace.So far,the production of aromatic compounds using microorganisms remains a major challenge for industrial production.Tyrosol,an aromatic compound derived from olive,has anti-oxidant and anti-inflammatory effects,which is a direct precursor of salidroside.Salidroside is an important cardiovascular drug widely used in pharmaceutical,food and cosmetics industries.However,the production of tyrosol and salidroside obtained is still less than 2 g/L via metabolic engineered Saccharomyces cerevisiae strain to date.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 S.cerevisiae for improving the production tyrosol and salidroside.Ultimately,the production of tyrosol and salidroside in the engineered industrial S.cerevisiae strain was significantly improved.The main contents of this thesis were described as follows:(1)Metabolic engineering of Saccharomyces cerevisiae for efficient production of tyrosolThe two exogenous biosynthetic pathways(the TDC-TYO pathway and the PcAAS pathway)from tyrosine to tyrosol were separately introduced into S.cerevisiae for improving tyrosol production.The results indicated that the PcAAS pathway was more conducive to the tyrosol biosynthesis in S.cerevisiae than the TDC-TYO pathway.The tyrosol production of the engineered strain with the PcAAS pathway introduced was about 117 times higher than that of the control strain.A large amount of ethanol is produced by S.cerevisia as a by-product when glucose is used as a sole carbon source due to the Crabtree effects.Therefore,in order to redirect the carbon flow towards the biosynthesis of ethanol to the biosynthesis of tyrosol.The main pyruvate decarboxylase gene PDC1 involved in the ethanol pathway of S.cerevisiae was disrupted,which resulted in an increase of 18.53%in tyrosol production.In addition,to enhance the carbon flux from the shikimic acid pathway product choristmate to tyrosol biosynthesis,a bifunctional NAD+-dependent fused chorismate mutase/prephenate dehydrogenase gene from E.coli(EctyrA)and its tyrosine-feedback insensitive mutant EctyrAM531/A354V were heterologously expressed in S.cerevisiae and increased tyrosol production by 33.59%and 35.18%.To promote the tyrosol production from the lab scale to the industrial scale,and develop a suitable industrial S.cerevisiae strain as a chassis for efficient production of tyrosol and its derivatives from glucose.Four haploid strains were separated from two diploid industrial S.cerevisiae strains and screened for tyrosol production based on the tyrosol level.A haploid industrial strain HLF-Da with high tyrosol production was obtained.Compared with the laboratory strain BY4741 used in the previous study,industrial S.cerevisiae strain is more robust and exhibiting higher fermentation performance.Afterwards,the metabolic engineering strategies developed in BY4741 were applied in the metabolic engineering of HLF-Da and generated the tyrosol-producing strain DA-1.The tyrosol production of DA-1 achieved reached 742.02±22.45 mg/L after 72 h of shake flask cultivation in the complex medium YPD,which was 11 times higher than that of HLF-D?.DA-1 was served as a platform strain for further improving tyrosol production via metabolic engineering.Subsequently,we optimized the biosynthetic pathway of erythrose-4-phosphate(E4P),which is one of the precursors of tyrosol biosynthesis in S.cerevisiae.First,to enhance the endogenous E4P biosynthetic pathway-the pentose phosphate pathway(PP pathway),the glucose-6-phosphate dehydrogenase gene ZWF1 was overexpressed in DA-1.The results showed that neither tyrosol yield nor the biomass changed significantly after the overexpression of ZWF1.We speculate that,it is difficult to improve the production of tyrosol by modifying the endogenous E4P biosynthetic pathway due to the complexity of the pentose phosphate pathway for biosynthesizing E4P from glucose-6-phosphate,and many known and unknown rate-limiting reaction steps existed in the PP pathway,Therefore,an exogenous E4P biosynthesis pathway based on phosphoketolase(Xfpk)-the Xfpk-based pathway was taken into consideration.With the thermodynamic analysis of the two pathways,the Xfpk-based pathway was more suitable for biosynthesizing E4P from glucose than the PP pathway.The Xfpk-based pathway was consisted of only four steps,and its maximum reaction driving force was about twice that of the PP pathway.Furthermore,the Xfpk-based pathway required no cofactors and energy molecules,and its theoretical molar yield was twice that of the PP pathway.The data of the pathway thermodynamic analysis supported that the Xfpk-based pathway might be a more conducive pathway for the precursor E4P supply for the shikimate pathway,which could further facilitate the biosynthesis of aromatic amino acids and the production of tyrosol in S.cerevisiae.By expressing the codon-optimized phosphatase gene from Bifidobacterium adolescentis(Baxfpk)at different loci(PHA2,XKS1,GRE3)in the chromosomes of S.cerevisiae,we found that expressing Baxfpk at the PHA2 locus was more beneficial for improving tyrosol yield than that of the XKS1 or GRE3 locus.In addition,Xfpk enzymes from different organisms was selected for improving E4P biosynthesis.The phosphatase genes from Bifidobacterium breve(Bbxfpk)and from Bifidobacterium dentium(Bdxfpk)were expressed at the PHA2 locus,respectively.It was found that the heterologous expression of Bbxfpk in S.cerevisiae preferentially increased tyrosol biosynthesis compared with Baxfpk and Bdxfpk.These results indicated that the introduction of the Xfpk-based pathway was an effective strategy to increase the tyrosol yield of S.cerevisiae.Xfpk can convert the substrate one molecule of fructose 6-phosphate(F6P)into o E4P and acetyl-phosphate(AcP).In S.cerevisiae,AcP can be catalyzed into acetic acid by the endogenous glyceryl phosphatases GPPlp and GPP2p,and resulted in the accumulation of acetic acid.The excessive acetic acid accumulation in S.cerevisiae cells can affect the fermentation performance negatively.AcP can be catalyzed into acetyl-CoA reversibly by phosphotransacetylase(Pta).The introduction of Pta into S.cerevisiae might be a feasible strategy to reduce the accumulation of acetic acid caused by the introduction of Xfpk.Thus,the codon-optimized phosphotransacetylase gene from Clostridium kluyveri(Ckpta)was heterologously expressed at the GPP1 locus by disrupting the major glyceryl phosphatase gene GPP1 in S.cerevisiae.It was found that the introduction of Ckpta did not alleviate the accumulation of acetic acid,instead,decreased the tyrosol production of the Xfpk overexpressed S.cerevisiae strain.These results indicated that the simultaneous heterologous expression of Xfpk and Pta in S.cerevisiae exerted a negative effect on tyrosol production.The enzymes(AR03p,AR04p,AR07p)play key roles in shikimate pathway are under the feedback inhibition of aromatic amino acids.The feedback inhibition existed limit the further improvement of the aromatic compounds production.Thus,the mutants ARO4K229L and ARO7G141S were constructed and expressed at the AR03 locus to release the feedback inhibition of aromatic amino acids in S.cerevisiae.Tyrosol production increased by 27.54%after introducing these mutants.The results confirmed that the expression of the mutants AR04K229L and AR07G141S at the AR03 locus was an effective strategy to improve tyrosol production by release the feedback-inhibition of the shikimate pathway.Finally,the tyrosol production reached 8.37 g/L via glucose fed-batch fermentation,which was 6-fold higher than the highest titer ever reported in yeast.(2)Metabolic engineering of Saccharomyces cerevisiae for efficient production of salidrosideTyrosol is a direct precursor of salidroside and hydroxytyrosol,which are important cardiovascular drugs widely used in the pharmaceutical,food and cosmetics industries.We intended to construct an engineered S.cerevisiae strain for efficient salidroside production through further metabolic engineering of the tyrosol high-producing strain DA-9.A salidroside producing strain named DA-91 was constructed by heterogeneously expressing the codon-optimized glucosyltransferase gene from Arabidopsis thaliana ugt85a1 at the TRP3 locus and establishing the reaction from tyrosol to salidroside.The production of tyrosol and salidroside reached 8.47 g/L and 1.82 g/L respectively at 216 hours of glucose fed-batch fermentation.The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached a level suitable for large-scale production.