Synthetic Process Development And Heterologous Synthesis Of Important Precursors For 4-acetylantroquinonol B

4-Acetylantroquinonol B(4-AAQB)is a ubiquinone that has been shown to have a variety of anticancer activities and is only found in the rare medicinal fungus Antrodia cinnamomea(A.cinnamomea),which is unique to Taiwan,China.However,the single host,long production cycle and low 4-AAQB content in A.cinnamomea have limited the large-scale production and application of 4-AAQB.In this thesis,through transcriptomics analysis,precursor addition and synthetic biology;the mechanism of promoting 4-AAQB synthesis by fructose was analyzed;the metabolic synthesis pathway of 4-AAQB was proposed and validated;accordingly,a comprehensive fermentation process for high yield of 4-AAQB in A.cinnamomea was developed;and a heterologous synthesis system of 4-AAQB precursor orsellinic acid(OA)was constructed based on the results of transcriptomics analysis.The research results are as follows:(1)Strain screening and transcriptomics analysis of 4-AAQB synthesis in A.cinnamomea.A vigorous growing strain with good 4-AAQB yield was obtained from the original fruiting bodies;the energy supply pathway and mode of the new strain in the synthesis of 4-AAQB were explored and it was found that fructose could effectively promote the synthesis of 4-AAQB;the important time points for the synthesis of 4-AAQB in A.cinnamomea were identified.Transcriptomics analysis showed that fructose was able to upregulate the expression of key enzymes in the glyoxylate pathway(isocitrate lyase,ACU7,malate synthase,ACUE)and acetate-Co A ligase ACS in A.cinnamomea and regulate key enzymes in the TCA cycle(citrate synthase CS,isocitrate dehydrogenase IDH),thus promoting the accumulation of acetyl coenzyme A(Ace-Co A),to provide precursor Ace-Co A for the synthesis of 4-AAQB precursor OA and farnesyl pyrophosphate with ends lactonization(FPPB);when fructose is present,the 4-AAQB chain precursor GPP was able to accumulate through up-regulation of geranyl pyrophosphate synthase GPS and down-regulation of other GPP consumption pathway related enzymes;meanwhile,fructose can up-regulate polyisoprene transferase and enzymes related to the 4-AAQB ring precursor modification pathway,thereby promoting the synthesis of 4-AAQB.(2)Proposal and validation of the 4-AAQB metabolic synthesis pathway.Based on the transcriptomics analysis,through precursor addition and related gene expression analysis during the synthesis process of 4-AAQB,a new 4-AAQB metabolic synthesis pathway was proposed and validated: 4-AAQB ring precursor OA is formed by condensation of Ace-Co A with malonyl coenzyme A(Mal-Co A)under the action of polyketide synthase(PKS63787);the chain precursor of 4-AAQB is FPPB,was formed by the condensation of geranyl pyrophosphate(GPP)with lactonized dimethylallyl pyrophosphate(DMAPPB)or isopentenyl pyrophosphate(IPP)with geranyl pyrophosphate with ends lactonization(GPPB);OA and FPPB form 3-farnesyl pyrophosphate B-orsellinic acid(FOAB)by the action of polyisoprene transferase PPT,and then the ring structure of FOAB is modified to form 4-AAQB.The intermediate metabolite addition experiments demonstrated that OA is the main constraint to the synthesis of 4-AAQB in A.cinnamomea,and that the phenolic acids protocatechuic acid can effectively replace OA as a ring precursor of 4-AAQB.(3)Design and optimisation of fermentation strategies for efficient production of 4-AAQB.Guided by transcriptomics analysis and metabolic pathway analysis,a liquid deep fermentation process with high yield of 4-AAQB was developed through medium screening,condition optimisation,traditional Chinese medicine and precursor substance addition.The content of4-AAQB reached the highest reported level of 30.71 mg/g,and the yield of 4-AAQB reached 214.02 mg/L,shortening the fermentation time from 28 days to 20 days.The production of 4-AAQB in 500 L scale liquid deep fermented A.cinnamomea was achieved and the yield of 4-AAQB was stabilized at about31.36 mg/L after 13 days of incubation.On this basis,the extraction,isolation and purification process of 4-AAQB was established,and the extraction rate of 4-AAQB reached 97.13%,the purity of 4-AAQB reached 92.01% and the4-AAQB recovery rate was 65.35%.(4)Construction of the heterologous synthesis system for 4-AAQB precursor OA.OA is not only an essential precursor for the synthesis of 4-AAQB,but also an important drug platform compound and a widespread precursor for a variety of secondary metabolites in fungi.OA is mainly formed by the condensation of Ace-Co A and Mal-Co A catalyzed by orsellinic acid synthase(OAS).In this thesis,Yarrowia lipolytica was used as the chassis strain to construct a strain Yali3 with low expression of both fatty acid synthase and citrate synthase.Through expressed and screened different kinds of OAS and the regulation of fermentation process,the yield of OA reached16.55 mg/L,which realized the heterologous synthesis of OA and laid the foundation for the heterologous synthesis of OA downstream products such as4-AAQB.In summary,this thesis identified the key enzymes and synthesis steps for4-AAQB synthesis;proposed and validated the metabolic synthesis pathway of 4-AAQB;designed and optimized the 4-AAQB fermentation process;constructed a heterologous synthesis system of 4-AAQB precursor OA,which laid the foundation for the scale-up of 4-AAQB production process by A.cinnamomea fermentation and the research of heterologous and efficient synthesis of 4-AAQB.