Functional Genes Study On The Formation Of 1-phenethylisoquinoline Scaffold In The Synthetic Pathway Of Cephalotaxus Alkaloids In Cephalotaxus Hainanensis

Cephalotaxus alkaloids is a class of plant active ingredients with anticancer activity isolated from the Cephalotaxus plant,and is a clinical first-line drug widely used in the treatment of a variety of leukemias.The content of Cephalotaxus alkaloids in plants is only about one hundred thousandth of the dry weight.At present,it mainly depends on the semi-synthetic method that consumes the resources of Cephalotaxus.It is far from meeting the clinical needs,and it has long been faced with pharmaceutical raw materials shortage and biodiversity conservation.The use of synthetic biotechnology to produce plant-derived natural products in microbial cells has become a hot spot in the field of pharmaceutical biotechnology.However,there are still many difficulties in realizing the heterologous synthesis of Cephalotaxus alkaloids from plants or microorganisms.First,a clear and complete synthesis pathway is required.Second,the discovery,functional expression and verification of these enzyme genes is a huge and challenging engineering.Isotope tracing experiments have proved that the synthesis of Cephalotaxus alkaloids originated from a molecule of phenylalanine and a molecule of tyrosine,and the unique1-phenylethyl-1,2,3,4-tetrahydroisoquinolie skeleton is derived into the Cephalotaxus alkaloid core.Based on this strong chemical evidence,this study used Cephalotaxus hainanensis plant,through full-length transcriptome sequencing and comparative transcriptomics analysis,study on the relevant genes in the synthetic pathway of 1-phenethyl isoquinoline,including extensively mining,cloning,functional verification and catalytic mechanism.The main findings are as follows:1.The third-generation single-molecule real-time sequencing technology was used to sequence the full-length transcriptome of the roots,stems,and leaves of Cephalotaxus hainanensis,and directly obtained 16,598,625 subreads sequences,totaling 18.17 Gb of original offline data;through de-redundancy,correction of consensus sequences,and clustering,313,640 high-quality full-length transcripts were finally obtained,and 652,097 Unigene obtained functional annotations of seven major database genes;combined with the comparative transcriptome analysis results of the Me JA treatment group and the control group,the expression levels of differential genes in phenylalanine-tyrosine biosynthetic pathway,phenylalanine metabolic pathway,and isoquinoline alkaloid biosynthetic pathway.A total of 309 transcripts were discovered related to the phenethylisoquinoline skeleton synthesis pathway,which belong to which belong to phenylalanine ammonia-lyase PAL gene family,cinnamic acid-4-hydroxylase C4 H gene family,4-coumarate-Co A ligase 4CL gene family,cinnamoyl-Co A reductase CCR gene family,2-alkene double bond reductase[NADP(+)dependence] DBR gene family,tyrosine decarboxylase Ty DC gene family,polyphenol oxidase PPO gene family,plant disease-related protein PR10/Bet v I gene family(PSS).Further delete incomplete,repeated redundant and unexpressed sequences,finally confirmed that 30 candidate genes are closely related to 1-phenethylisoquinoline biosynthesis,including 4 PAL gene members,4 C4 H gene members,2 4CL gene members,10 CCR gene member,5 DBR gene members,2 Ty DC gene members,2 PPO gene members and 1 PSS gene member.2.All candidate gene members were cloned and constructed into the pET-28 a or pET-29 a prokaryotic expression vector.By optimizing the expression conditions of the foreign host bacteria to induce expression,separating and purifying to obtain a large number of high-purity recombinant proteins,using high-performance liquid chromatography-mass spectrometry HPLC-PDA-ESI-MS detects and analyzes the target product.The analysis of the results showed that the 4 PAL enzymes catalyze the deamination of L-phenylalanine to cinnamic acid.The high Kcat/Km value indicates that ChPAL4 has a higher catalytic efficiency.Among the 4 C4 H enzymes,only ChC4H3 can catalyze the C-4 hydroxylation of cinnamic acid to produce p-coumaric acid,the activity of converting phenylpropyl aldehyde to 4-hydroxyphenylpropanal(4-HDCA)was not detected;Among the two 4CL enzymes only Ch4CL2 can transfer Co A to produce p-Coumaroyl-Co A;Among the 10 CCR enzymes only ChCCR1 can catalyze the reduction of Co A sulfide of p-coumaroyl-Co A to produce p-coumaroyl aldehyde;Among the five DBR enzymes,ChDBR2 and ChDBR3 can catalyze the reduction alkenal double bond of p-coumaroyl aldehyde to 4-HDCA,the high Kcat/Km value indicates that ChDBR3 has a high catalytic efficiency,and the activity of converting cinnamaldehyde to phenylpropyl aldehyde is not detected;Among the two Ty DC enzymes,ChTy DC2 can catalyze the decarboxylation of L-tyrosine or L-dopa to generate tyramine or dopamine,the high Kcat/Km value indicates a preference for the substrate L-tyrosine;Among the two PPO enzymes only ChPPO1 can catalyze can catalyze the C-3 hydroxylation of L-tyrosine or tyramine to produce L-DOPA or dopamine,respectively.3.Recombinantly construct pACYCDuet-ChPAL4+ChC4H3::ATR,p CDFDuetCh4CL2+ChCCR1 and pET28a-ChTy DC2+ChPPO1 co-expression or fusion expression vector in microbial cells,use HPLC-PDA-ESI-MS to detect and analyze the target product.The results showed that the multifunctional enzyme ChPAL4+ChC4H3::ATR can convert L-phenylalanine to produce the intermediate product cinnamic acid and the final product p-coumaric acid;the multifunctional enzyme Ch4CL2+ChCCR1 can convert cinnamic acid or p-coumaric acid to produce cinnamaldehyde or p-coumaroyl aldehyde,respectively;the multifunctional enzyme ChTy DC2+ChPPO1 can convert L-tyrosine to produce intermediate products tyramine,L-DOPA and final product dopamine.4.Using Alphafold 2 algorithm to predicted the three-dimensional structure model of the ChPSS of Cephalotaxus hainanensis phenethylisoquinoline synthetase,and simulate the interaction between ChPSS protein macromolecules and dopamine and 4-HDCA or 4-HPAA small molecules interaction relationship by molecular docking,predicted and identified the potential binding sites of proteins and small molecules,mainly composed of 4 amino acid residues glutamic acid Clu-70,tyrosine Tyr-86,glutamic acid Clu-91 and aspartic acid Asp-122 is responsible for the Picket-Spengler directional cyclization reaction;using site-directed mutagenesis to mutate 4 amino acids into alanine(Ala),compared with the wild-type of ChPSS,the catalytic activity of Clu-70-Ala and Tyr-86-Ala mutants are reduced by 74% and34%,respectively;the activity of Clu-91-Ala and Asp-122-Ala mutants increased by 87%and 128%,respectively;Asp-122-Glu mutants increased by 212%;multiple Lewis acids such as Clu-91 and Asp-12 are dispersed in the ChPSS active cavity,causing small molecules to be "pulled each other" by the charges.This may be the main reason why the catalytic activity of Clu-91 and Asp-122 is increased after mutation.Therefore,it can be inferred that Tyr-86 is responsible for the role of small molecule hydrogen bond donors,Clu-70,Clu-91 and Asp-122 are responsible for the exchange of protons between small molecules,multiple small molecules interact to form one molecule by covalent bonding.This study clarified the biosynthetic pathway of 1-phenethylisoquinoline in Cephalotaxus hainanensis,provided a reference for further study on the "downstream synthesis steps" of the phenethylisoquinoline skeleton synthesis pathway,in the future used genetic engineering to realize industrial production of Cephalotaxus hainanensis.It will lay the foundation for the industrial production of Cephalotaxus hainanensis 1-phenethylisoquinoline and even Cephalotaxus alkaloids by genetic engineering in the future.