Study On The Biosynthesis Mechanism Of Anthraquinone Dimer Skyrin

Natural products are one of the types of compounds with the highest medicinal properties.Due to the repeated discovery of a large number of compounds,the medicinal properties of natural products are getting lower and lower.The polymerization of natural product molecules can significantly expand its structural diversity and enhance biological activity,and even produce new biological activities.The analysis of its polymerization mechanism is conducive to the use of synthetic biology and other strategies to produce new natural product polymers.Discovery of new drugs is of great significance.Skyrin is a homosymmetric anthraquinone dimer formed by the coupling of two emodin molecules through carbon-carbon bonds(C-C).It has biological activities such as antibacterial,anti-tumor and inducing cell apoptosis.Although skryin has been found in filamentous fungi,lichens,plants and other organisms,its biosynthetic pathway has not yet been resolved.This article mainly describes the biosynthesis of skyrin,a secondary metabolite from the endophytic fungus Talaromyces sp.YE 3016.Through genome sequencing,bioinformatics analysis,gene knockout,and identification of heterologous expression of Aspergillus oryzae,the biosynthetic gene cluster of skyrin is identified for the first time,and a series of intermediate compounds are isolated.On this basis,its complete biosynthesis is speculated.This article is divided into six parts,the main contents are as follows:The first part is a brief overview of the research status of natural products,anthraquinone compounds and anthraquinone dimer skyrin,and expounds the purpose and significance of studying skyrin biosynthesis mechanism.The second part is to optimize the medium of Talaromyces sp.YE 3016,and select the most suitable medium for large-scale fermentation.The fermentation products are preliminary separated,purified and structured.Finally,6 compounds,including skyrin(1)、flavomannin A(2)、flavomannin B(3)、rugulosin A(4)、wortmannilactones E(5)、wortmannilactones F(6)have been obtained.The third part is to sequence the whole genome of Talaromyces sp.YE 3016,speculate the biosynthetic gene cluster of skyrin through bioinformatics analysis,and predict the functions of related enzymes.The results show that the gene cluster that may be related to skyrin biosynthesis in this bacterium is composed of the genes of pks12 and its upstream and downstream genes.The fourth part is to confirm the location and scope of skyrin biosynthesis gene cluster by gene knockout method.The results show that skyA,skyB,skyD,skyE,skyF,and skyG are related to skyrin biosynthesis,while skyH,which is predicted to be aldehyde ketone reductase,and skyC,which is predicted to be α-glucoside permease,has nothing to do with skyrin biosynthesis.The fifth part is to explore the catalytic function of non-reducing polyketide synthase Sky A and β-lactamase SkyB in the skyrin biosynthetic gene cluster through the preliminary study of the heterologous expression of functional genes in Aspergillus oryzae NASR1.Among them,through the separation of the fermentation products of the A.oryzae NASRl/skyA+skyB expression strain and the LC-MS mass spectrometry identification,a total of 6 compounds were finally obtained:endocrocin(7),atrochrysone(8),endocrocin anthrone homodimer(9、10),emodin(11)and emodin anthrone(12).Through comprehensive bioinformatics enzyme function prediction,gene knockout,heterologous expression of functional genes,and structural analysis of compounds,this study inferres the complete biosynthetic pathway of skyrin.The specific approach is that the starting unit acetyl-CoA and the seven-molecule extension unit malonyl-CoA are catalyzed by the non-reducing polyketide synthase SkyA to generate ACP-linked atrochrysone carboxylic acid,and β-lactamase SkyB catalyzes atrochrysone carboxylic acid to be hydrolyzed from ACP,and then decarboxylated to produce atrochrysone monomer under the catalysis of decarboxylase SkyF。Then atrochrysone is spontaneously dehydrated to produce emodin anthrone.Emodin anthrone is oxidized spontaneously or under the action of anthrone oxidase SkyE to produce emodin.Finally,emodin undergoes a stereoselective dimerization reaction to generate skyrin under the catalysis of cytochrome P450 monooxygenase SkyG.The sixth part is to discuss the experimental results of this subjectThe seventh part is a summary and prospect of this research.The elucidation of the biosynthetic mechanism of skyrin will lay the foundation for the use of combined biosynthesis and synthetic biology to synthesize medicinal anthraquinone polymers,and it will also help to understand how microorganisms use simple natural product monomers to produce complex structures of polymers.This will enrich and develop related theories about the formation mechanism of natural product polymers.