Functional Study Of Two Biosynthesis Key Enzymes

Natural products are secondary metabolites produced by organisms.Although they are not essential for basic survival,but play an important role in disease defense and environmental adaptation.Now,numerous natural products have become a precious library of drug source molecules.Natural products are synthesized by orderly catalysis of enzymes in a cluster in vivo.Key enzymes in some steps are usual the points of biosynthesis research.With key enzymes research,it is helpful to understand the catalysis feature and mechanism,which lays the foundation for the application of catalysis in vitro.On the other hand,catalysis in vitro can conversely be a proof in key enzymes research.It has potential application value as a kind of environmental friendly and efficient manner of synthesis.With laccase from a mantis endophyte Daldinia eschscholzii IFB-TL01,Dirigent Protein from sesame,halogenase from an ant endophyte Spiromastix sp.MY-1 as materials,the enantioselective synthesis of bioactive plant lignans by fungal laccase together with plant Dirigent Protein and positioning of gene cluster involved in halogenated biosynthesis pathway were studied respectively.As follows:The first chapter is a brief introduction and mini review of the key enzymes and related compounds in this thesis.The second chapter is about the enantioselective synthesis of(+)-pinoresinol,a bioactive plant lignin,by fungal laccase together with plant Dirigent Protein.Laccase from Daldinia eschscholzii IFB-TL01 was extracted by ammonium sulfate precipitation.Dirigent Protein was heterologous expressed in Pichia pastoris GS115.The dimer of coniferyl alcohol catalyzed by laccase was separated,purified and structure identified.After adding Dirigent Protein to catalyze,the changes of chiral proportion was analyzed.The results not only verified the enantioselective effect of Dirigent Protein,but also provided another method to enantioselective synthesize(+)-pinoresinol in vitro.The third chapter is about the positioning of the halogenation gene cluster of Spiromastix sp.MY-1.First we constructed a genome fosmid library of Spiromastix sp.MY-1.Then,according to four halogenase gene from related species,we designed four degenerate primers to obtain a small part of sequence of this fungi,which was the basis for specific primer probe designing.However,the latter part of the cluster was unobserved,and twice Genome Walking was done to obtain the whole cluster.As the position of halogense was more near to the PKS than oxidase,we conferred that the halogenation occurred prior to radical coupling.To further explore the mechanism,we expressed and obtained the soluble protein of halogenase in E.coli BL21.