Reconstruction Of Metabolic Pathway To Produce 24-epi-ergosterol And Its Derivatives In Saccharomyces Cerevisiae

Brassinolide(BL)is the most biologically active compound among natural brassinosteroids.Since the 1930s,the researchers of the U.S.Department of Agriculture have begun to explore the phytohormone components in pollen,and it was not until 1979 that BL was extracted and purified from pollen and its chemical structure was elucidated.However,the agricultural applications are limited by the extremely low abundance in plants and the scarcity of synthetic precursors.Recently,the rapid development of synthetic biology enables microbial cell factories for scalable production of complex natural product(e.g.,BL or its precursors).Saccharomyces cerevisiae(S.cerevisiae)has become one of the best chassis cells for synthetic biology,as this yeast is generally regarded as safe(GRAS status)and has been engineered with a variety of genetic tools.In this study,by comparing the existing chem-synthesis pathways of BL analogues and the side chains of their precursors(such as ergosterol,stigmasterol,and crinosterol),we propose 24-epiergosterol as an alternative precursor for BL synthesis and for the purpose of the agricultural applications of BL,S.cerevisiae was used as chassis cell for scalable preparation of 24-epi-ergosterol.Firstly,design of an artificial pathway for de novo production of 24-epiergosterol in yeast.DWF1 genes(encoding Δ24(28)sterol reductase in plants)from different plants were introduced into ERG4(encoding Δ24(28)sterol reductase in yeast)inactivated yeast by CRISPR/Cas9 genome editing technology.A new peak was found in HPLC analysis.And then,this peak was attributed to 24-epi-ergosterol based on the analysis of mass spectrum(MS)and nuclear magnetic resonance(NMR).The strain harboring DWF1 from Ajuga reptans(Ar)produced higher titer of 24-epi-ergosterol,13.79 mg/L.Secondly,directed evolution has been applied for the enhanced enzymatic activity of DWF1.The high-throughput screening(HTS)method was developed based on the phenomenon that ergosterol-deficient yeast exhibited pleiotropic defects.And after pre-test,YPD/Hyg+0.025%SDS medium was chosen as the HTS medium.ArDWF1 mutants from error-prone PCR and linearized expression vectors were co-transformed into erg4 Δ yeast,and the mutant library was constructed by plasmid assembly using homologous recombination.Positive mutations were obtained based on HTS method and confirmed by HPLC analysis.DNA shuffling was performed to construct a combinatorial library and the best mutant Ar207(V143G/S306P/Y338H)was obtained,whose 24-epi-ergosterol was 3.46-fold higher than that of the wild-type.Subsequently,sterol homeostasis was manipulated for strengthened sterol flow to downstream and thus,enhanced 24-epi-ergosterol production.The genes of two sterol acyltransferases(ARE1 and ARE2)and three steryl ester hydrolases(YEH1,YEH2,and TGL1)were manipulated(deletion,overexpression or combinatorial overexpression)to analyze the effect on sterol production and intracellular sterol homeostasis.A synergistic effect was observed by simultaneous overexpression of acyltransferase genes(ARE2)and hydrolase genes(YEH1 and/or YEH2).More specifically,strain YQE717 overexpressing ARE2,YEH1,and YEH2 produced 71.04 mg/L 24-epi-ergosterol and 220.07 mg/L late sterols,which was 1.65-and 2.09-fold higher than that of un-manipulated strain,respectivelyFinally,pathway gene expression was strengthened to achieve increased production of 24-epi-ergosterol in high-purity.Dynamic regulation of Ar207 using inducible promoter PGAL1 to enhance the transcription level of Ar207 in the middle and late stages of fermentation(feeding ethanol).Increasing the expression level of ACC1,a key enzyme in the fatty acid synthesis pathway,was used to increase sterol storage pool.What’s more,the expression of Ar207 driven by PGAL1 caused the accumulation of 24-epi-ergosta-5,7-dien-3β-ol,another precursor in the 24-epiergosterol biosynthetic pathway.Thus,we constructed YQE734 by overexpressing ERG5,encoding the C-22 sterol desaturase,which decreased the accumulation of 24epi-ergosta-5,7-dien-3β-ol.In the fed-batch fermentation of YQE734,2.76 g/L 24-epiergosterol was produced and the ratio of 24-epi-ergosterol to total late sterols reached to 84.2%.In addition,based on BL biosynthesis pathway,artificial pathway was constructed in 24-epi-ergosterol producing yeast to synthesize the sterol with(22R,23R)-22,23dihydroxyl side-chain.Sterol C-22 hydroxylase,sterol C-23 hydroxylase and P450s reductase,three enzymes originated from Arabidopsis thaliana,were introduced into erg5Δ strain’s genome to obtain the strain producing(22R,23R)-22,23-dihydroxy-Δ7canpesterol(22,23-diOH-Δ7-CR),which was confirmed by HPLC analysis and LCMS.Then,the transcription level of genes involving in the artificial pathway were optimized and truncated AtCPR1 was employed to increase the titer of 22,23-diOHΔ7-CR.At last,using fed-batch fermentation,we were able to produce 100.60 mg/L 22,23-diOH-Δ7-CR.In this thesis,based on sterol biosynthetic pathway in S.cerevisiae,we constructed an artificial pathway,followed by protein engineering and sterol homeostasis engineering,for the production of 24-epi-ergosterol,promising as a synthetic precursor for scalable production of BL.After that,the development of yeast platform cell factories for the synthesis of dihydroxyl side-chain sterols was explored.