Metabolic Engineering Of Saccharomyces Cerevisiae For ?-Phenylethanol Production

2-Phenylethanol(2-PE)is an important aromatic alcohol with potential economic value for its rose-like fragrance and superior property,therefore being widely used in food,perfume,cosmetic industries.Nowadays 2-PE is mainly produced by seriously polluted chemical synthesis or highly costed physical extraction which immensely limited its applied range.Hence,the research of microbial synthesis for natural 2-PE production gradually became attractive for the advantages of low energy cost and environment friendly.In this work,Saccharomyces cerevisiae CICC 31906 is used to establish a platform for 2-PE production,employing synthetic biology strategies such as de novo pathway from glucose,efficient synthetic route optimization for complement of amino-receptor by heterotopic reconstruction.Based on the omics technique,the related regulation mechanism and key genes of 2-PE stress and producing has been analyzed.Major results achieved with this study are listed below.(1)De novo synthetic pathway for aromatic alcohol productions from glucose was constructed in S.cerevisiae.Based on shikimate pathway,the synthesis pathway for 4-hydroxy-phenylethanol(4HPEA)and 2-PE production was established by the methods such as alleviation of key enzymes feedback inhibition and flux bottleneck.(1)The aromatic alcohols overproducing strain was constructed by expressing feedback inhibition resistant 3-deoxy-D-arabinoheptulosonate-7-phosphate(DAHP)synthase(aro G^D146N),chorismite mutase/prephenate dehydratase(CM/PDT)(phe A^fbr).(2)For release flux bottleneck inside penta-functional enzyme Aro1p,shikimate kinase(aro L)and shikimate hydrogenase(ydi B)were introduced into S.cerevisiae.(3)Meanwhile,relative gene knockout also contributed to aromatic alcohol accumulation,such as aromatic amino acid transaminase(ARO9)deleted to reduce phenylpyruvate lost and phenylpyruvate decarboxylase(ARO10)expressed.(4)Along with ketoaldehyde transferase(TKL1)expressed,the recombinant strain JM26 produced543 mg/L 4HPEA and 641 mg/L 2-PE.(2)Efficient synthetic pathway for 2-PE production from L-phenylalanine was constructed in S.cerevisiae.Multivariate elements metabolic engineering was introduced to optimize 2-PE synthesis,such as reinforce of heterogenous key enzymes and supplement of precursor.(1)Aromatic amino acid transaminase(Tyr B)from Escherichia coli,phenylpyruvate decarboxylase(Kdc A)from Lactococcus lactis and acetaldehyde dehydrogenase(Adh2)from Saccharomyces cerevisiae were chose to construct a fusion protein.Together with L-glutamate oxidase from Streptomycete sp.X-119-6 successfully overexpressed in order for amino-receptor supplement,leading to 2-PE titers up to 3.13 g/L.The deletion of gene PDC5 led to an improvement of2-PE.The optimum strain RM26 was capable of producing 3.59 g/L 2-PE with a yield of 0.72 mol/mol.(3)Analysis of regulatory mechanism to improve 2-PE production in RM22 was studied by transcriptome sequencing analysis.The yield of 2-PE in mutant strain RM22(pdc5?)with L-phenylalanine as substrate was 1.12-fold higher than that of the control strain.Transcriptome sequencing analysis of the original strain WT-A and the mutant RM22 showed that 87 genes involved in oxidation-reduction,stress response etc.were differentially expressed(DEGs),among which 60 genes were significantly up-regulated and 27 genes were significantly down-regulated.There are27 pathways involved,including intracellular metabolism,biosynthesis of secondary metabolites and biosynthesis of amino acids.By analyzing DEGs that may be related to the synthesis of 2-PE,it was determined that the up-regulation of ARO9,which encoding aromatic amino acid transaminase,was the main reason for the increase of2-PE synthesis in strain RM22.(4)Key genes for 2-PE tolerance and production were found by re-sequencing.The enhancement of 2-PE tolerance contributed to improve the fermentation performance of yeast growth and 2-PE synthesis.Therefore,screened by adaptive revolution method and synthesis pathway optimized,the 2-PE resistant strain AD032showed superior 2-PE synthetic capacity which was significantly higher than the control strain.Based on strain AD032,a recombinant strain KM032(AD032 pdc5?PTPI-tyr B-kdc A-ADH2 PTPI-LGOX)was obtained,resulting the 2-PE titer of 4.26 g/L and the yield of 0.86 mol/mol with 6.7 g/L L-phenylalanine.The genomes of AD032were re-sequencing and showing 113 genes and ORFs involved in biological process,cellular process etc.were influenced by the 191 mutations.By assemble and annotate methods,the possible genes improved 2-PE were discussed.The overexpression of hexose transporters(HXTs),ubiquitins(UBI4)and regulatory factors(SWI1-SNF2)in the mutant gene could significantly improve the ability of 2-PE.However,the putative aryl-acetaldehyde dehydrogenase gene AAD15 was not detected with the activity of reducing phenylacetaldehyde to 2-PE.