Cloning And Functional Identification Of Enzymes Involved In The Biosynthesis Of Curcumin, Quinolone Alkaloids, And 2-(2-phenylethyl) Chromon

Using modern molecular biology method to reveal the biosynthetic pathway and regulatory mechanism of pharmaceutical componds can solve the difficulties of the utilization of active ingredients extracted in traditional medicine plant caused by the low content and complex chemical synthetic.The utility of gene recombination technology to regulate the expression levels of related genes or the construction of engineering bacteria to synthetic a large number of target products and their derivatives become a hot topic.Ginger(Zingiber officinale Roscoe.)contains curcumin,gingerol,flavonoids,volatile oil and other ingredients.Due to its many important pharmaceutical properties,the biosynthesis of curcumin has attracted much attention.The biosynthetic pathway of curcumin is regulated by a variety of enzymes,while the genes involved in ginger are not clearly elucideted.Curcumin synthase(CURS)and diketide-CoA synthase(DCS)are participated in the biosynthetic pathway of curcumin and analogs in turmeric.And the expression level of CURS in different tissues from giner,and the substrate selectivity may have difference with the reported CURS.Euodia(Euodia rutaecarpa(Juss.)Benth.)is used in a large number of prescriptions for clinical prescriptions.It has a large number of quinolone alkaloids and its 2 position often replaced by alkyl.The structure has important influence on its biological activity.The QNS expressed in plant produces N-methyl-4-hydroxy-quinolone from one n-butyryl-CoA and one malonyl-CoA.Studies of such compounds’ biosynthetic pathway are only present in microbes.In response to biotic or abiotic stress,such as worm bite,lightning strike,axe cut or fungal infection,some resin-impregnated heartwoods are slowly forming around wounds of the trunk and branches of Aquilaria sinensis(Lour.)Gilg.Those resinous heartwoods are called Aquilariae Lignum Resinatum(ALR),which has been used for a long time pharmaceutically in traditional Chinese medicine.As one of the precious Chinese medicinal materials,ALR has effects on moving qi to stop pain,warming the middle-eneregizer to bring down rebellion qi and grasping qi to relieve pain.The extremely long growing cycle and increased consumption probably are the main reasons of over-exploitation and serious depletion of ALR,leading to rarity of its wild resource.2-(2-Phenylethyl)chromones are the characteristic components existing in ALR,and a key factor in determining the quality of ALR.The biosynthetic pathway of which 2-(2-Phenylethyl)chromones also remains unclear.In addition,it is of great difficulties to chemically synthesize structurally-varied 2-(2-phenylethyl)chromones,especially the highly-oxygenated tetrahydrochromones(ATC-type 2-(2-phenylethyl)chromones)and complicated stereo-configuration.The C-6,C-7,C-8,C-3’and C-4’ positions of its 2-(2-phenylethyl)chromone compounds are more likely to be substituted by hydroxyl or methoxy groups on its nucleus.Studies on biosynthesis of 2-(2-phenylethyl)chromones will also be meaningful to synthetic biology research of them.Based on the above study,we extracted the RNA from the fresh rhizome and leaf of ginger,fresh leaf and fruit of Euodia and callus of ALR induced by 150 mM NaCl.Through analysis of ginger’s EST library,the Euodia core sequences obtained by type Ⅲ PKS degenerate primers and LAR transcript datas,we obtained the related enzymes involved in the three compounds combined with the technology of race ampification.The main research results were obtained:Firstly,Through analysis ginger’s EST library and NCBI library,four new genes,diketide-CoA synthase(ZoDCS),curcuminase synthase(ZoCURS),type Ⅲ polyketide synthase(ZoPKS)and carbon bond reductase(ZoReductase)were cloned and expressed from ginger by homologous cloning.ZoDCS,ZoCURS and ZoReductase are involved in the biosynthesis of curcuminoids and their analogues in ginger,which is the first to mentioned the curcumin biosynthetic pathways.DCS catalyzes the condensation of feruloyl-CoA and malonyl-CoA to produce feruloyldiketide-CoA.Then CURS catalyzes the conversion of feruloyldiketide-CoA to β-keto acid and sequentially condenses with another molecule of feruloyl-CoA to produce curcumin.ZoReductase,which is cloned from ginger,can specifically reduce the double bonds in curcumin and its analogue.Though ZoPKS has the similar sequence of amino acids with CURSs cloning from ginger and turmeric,it does not have the function of CURSs.It reveals that the change of vital amino acid sites has significant effects on the catalytic function of enzyme.Secondly,a curcuminoid-producing unnatural fusion protein diketide-CoA synthase::curcumin synthase(DCS::CURS)was constructed by introduced three amino acids(Gly-Ser-Gly)in the open reading frame.And the non-natural fusion protein DCS::CURS was successfully expressed in Escherichia coli heterologous expression system.Comparing to ZoCURS,DCS::CURS indicated similar substrate specificities and catalytic potentials to catalyze the formation of various curcuminoids,however,the yield of curcuminoids produced by DCS::CURS obviously increased and the yield of benzylacetone significantly reduced,which would be useful for metabolic engineering biosynthesis of curcuminoid analogs.Thirdly,the non-natural fusion protein DCS::CURS was introduced into the biosynthetic metabolism of curcumin firstly.And the biosynthetic metabolism system of curcumin and its analogues with sorbitol as carbon source and ferulic acid as substrate was sucessfully constructed.We explored and optimized of Escherichia coli induced expression system and fermentation conditions.The results showed that the yield of curcumin increased to 386.8 mg/L,which was much higher than reported in the previous literature.Lastly,novel quinolone synthase genes,E.r QNS1 and E.r QNS2,were cloned from the traditional Chinese medicine Euodia.E.r QNS1 can accept long chain β-keto acid as substrate to produce quinolone alkaloids that 2 position replaced by alkyl,indicating that E.r QNS1 is the key enzyme for biosynthesis of such compounds.Although Er QNS1 and Er QNS2 have high homology,Er QNS2 can only catalyze the formation of simple quinolone alkaloids.In addition,two typical chalcone synthase genes,ErCHS1 and Er CHS2,were cloned,which can catalyze the formation of chalcone by three molecules malonyl-CoA and p-hydroxycinnamoyl-CoA.Last but not the last,through the analysis of ALR callus’ transcriptome datas,we found that there were a lot of methyltransferase genes of which methyltransferase A.s OMT1 and A.s OMT2 genes expression levels changed significantly after salt stress.Therefore,we cloned and expressed A.s OMT1 and A.s OMT2 from ALR callus by recombination technology.The A.s OMT1 and A.s OMT2 were 744 bp and 1212 bp,encoding 247 and 403 amino acids,respectively.We gained 28 KDa and 44 KDa recombinant proteins by heterologous expression in E.coli.For their catalytic function analysis,we found that the two methyltransferases catalytic the methylation reaction of simple 2-(2-phenylethyl)chromones,and two enzymes position selective of the methylation is different.Beyond that A.s OMT1 and A.s OMT2 can catalyzes the methylation reaction of flavonoids,flavonoid glycosides,coumarins.In addition,OMT1 can catalyzed the substrate of caffeic acid.They have a broad substrate selectivity.