Optimized Biosynthesis Of Santalenes And Their Derivatives In Saccharomvces Cerevisiae

One of the important contents of synthetic biology is to utilize microbial cell factory to synthesize high-value rare compounds,including drugs,food additives,spices and health products.Its basic idea is to reconstruct and optimizing express metabolic pathways in suitable hosts and to synthesize target compounds using intermediates from host metabolic processes.Basing on the"push-pull-inhibit-detoxification"strategy and using S.cerevisiae as host,the metabolic pathways of Glucose→Santatalenes→Santalols→Santalol glycosides and their corresponding engineering strains were constructed,respectively in this paper.More than 30 genes and element sequences from multi species（Santalum album,Arabidopsis thaliana,S.cerevisiae,Staphylococcus aureus）and artificial sources were applied.In addition,some key enzymes in the pathway were analyzed by bioinformatics.The feasibility of using protein engineering to optimize a series of bottleneck enzymes that limit the efficiency of synthesis were explored.The synthesis pathways of santalenes and santalols were optimized and integrated in S.cerevisiae.Santalols were transformed into new compound santalol glycosides by integrated glycosyltransferase genes in the synthetic pathway of santalols for the first time.It will lay the foundation stone for efficient biosynthesis of flavor and flavor precursors from S.album.The main results are as follows.1.Three biosynthetic pathways（small,medium and big synthetic pathway）of santalenes were constructed and integrated atδsite by gene editing tool p Di-CRISPR in S.cerevisiae.The recombinant engineered strains obtained were all capable of producing four productsα-santalene,α-bergamotene,epi-β-santalene andβ-santalene.Among them,titer of four products produced by the strain BC11 were 28.49 mg/L,13.97 mg/L,4.08 mg/L and,20.54 mg/L,respectively,after 7 days of shaking flask fermentation.Meanwhile,titer of by-product squalene and farnesol were 25.66 mg/L and 24.26 mg/L,respectively.In addition,it was found that HMGR gene from S.aureus had the greatest effect on the titer of the product.Whereas erg20 gene from S.cerevisiae had no obvious effect,but it could increase the titer of squalene and farnesol.The titer of squalene was increased by three times with the help of p Di-CRISPR tool.2.Two P450 genes（Sa-CYP76F39v1,Sa-CYP736A167）and two CPR genes（Sa CPR2,t46ATR1）were randomly combined to form chimeras.The differences of their oxidation abilities on santalenes were systematically verified.The differences of expression between Sa-CYP76F39v1 gene in multiple copies and Sa CPR2 and46t ATR1 gene in single copy were also compared.Meanwhile,the activity of Sa-CYP76F39v1-Sa CPR2 chimera with linker was verified.Four recombinant strains MC4-P2A,MC4-P2C,BC11-P2A and,BC11-P2C were screened to produce santalol and its isomers,which of them were identified as（Z）-α-santalol,（Z）-α-bergamotal and（Z）-β-santalol by GC-MS.The total titer of the three products fermented by the BC11-P2A strain in the shake flask reached 23.34 mg/L.It was confirmed that only Sa-CYP736A167 and two CPR（Sa CPR2,t46ATR1）chimeras could oxidized santalenes into santalols in vivo.This is a key step for the synthesis of santalenes into santalols in S.cerevisiae.3.A flexible and open platform for expression of plasmid was constructed.It was compatible with the expression of glycosyltransferase（GTS）and the down-regulation function of ERG9.The GTS and P_ERG9 could be changed arbitrarily to verify the functions of different GTS,and the down-regulation efficiency of different promoters to ERG9 was tested by this platform.When the two promoters P_HXT1 and P_ERG1 were integrated into the BC11 strain to down regulate ERG9,the titer of squalene decreased from 28.37 mg/L to 12.97 mg/L and 18.87 mg/L;down regulated by 54.3%and 33.5%,respectively.While the titer of santalenes increased from 94.65 mg/L to 164.71 mg/L and 106.99 mg/L;increased by 74.0%and 13.3%,respectively.When the two promoters P_HXT1 and P_ERG1 were integrated into the BC11-P2A strain to down regulate ERG9,the titer of squalene decreased from 38.04 mg/L to 18.54 mg/L and 26.58 mg/L;decreased by 51.3%and 30.1%,respectively.While the titer of santalols increased from 29.56 mg/L to 68.81 mg/L and 37.12 mg/L,up regulated by 132.8%and 25.6%,respectively.The experimental results confirmed that ERG9 down-regulation significantly increases the titer of target product and decreases that of by-product.In addition,three representative terpenoid glycosyltransferases UGT73C1,UGT73C5 and UGT75D1 from A.thaliana were selected for activity test by integration of them into the BC11-P2A strain.The UPLC-MS assay of fermentation product showed that UGT73C1 could transformed santalols to corresponding glycoside compounds.4.The online databases and analysis tools were used to model the three-dimensional protein structure of the santalene synthase Sa SS,which of structure was compared with several known sesquiterpene synthase activity sites.It was speculated that the 293Trp and 545Phe sites of Sa SS would be more conservative and may be potential sites for directional mutagenesis.By comparing the crystal structures of Sa SS protein（PDB No:5ZZJ）and sesquisabinene synthase（PDB No:6A2C）with the ligand FPP and Mg²⁺complexes,it was found that the two enzymes form hydrophilic/hydrophobic bonds with Pi and Mg²⁺with the same side chain amino acids.The 288 Tyr,289 Ala,312 Ile,417 Ile and 422 Leu sites of sesquisabinene synthase form hydrophobic bonds with FPP hydrocarbon chains,while Sa SS also has the same amino acid sites as 417 Ile and 422 Leu,which of these 2 sites may be potential active ones.In addition,online tools such as Prot Scale,Phobius,PSORT,i PSORT and Signal P-5.0 Server were used to analyze CYP76F39v1,CYP736A167,CPR1,CPR2and cytochrome P450 reductases（ATR1、ATR2）from A.thaliana.The results showed that both P450s had N-terminal signal peptide sequences.However,the subcellular localization of CYP76F39v1 was unclear.It might exist in endoplasmic reticulum,plasma membrane or Golgi body.Whereas the subcellular localization of CYP736A167 was clearly located in endoplasmic reticulum.That might be why CYP76F39v1 did not show activity in S.cerevisiae.The results were also confirmed by the experiment.On the other hand,the results of comparative analysis of the four CPRs showed that all of them possess a clear N-terminal trans-membrane signal peptide.The truncated 46t ATR1 exhibited better catalytic activity during the experiment.That means the truncated chimeras could eliminate the trans-membrane signal peptide and facilitate the active domains to be close to each other.In summary,the entire biosynthetic pathway of santalenes/santalols and their derivatives has been completely constructed in S.cerevisiae.It will lay the foundation for subsequent optimization of target compounds yield.