Biosynthesis Of Diphenyl Ethers In Fungi

The natural fungus products with complex chemical structure and diversified bioactivity are the important sources of potential new drugs.For example,mycophenolic acid,an immunosuppressor,lovastatin,a drug for cholesterol lowering,and griseofulvin,an antifungal drug,etc.,have been used in clinical practices.The natural diphenyl ethers product from the plants and microorganisms have the same diphenyl oxide core framework,but the differences of the substituent groups on the benzene ring give them wide and good bioactivities,such as antibiosis,antivirus,anti-inflammation and antitumor,etc.Therefore,natural diphenyl ethers are one of the most important frameworks for new drug development.Fungus is one of the main sources of natural diphenyl ethers.The natural diphenyl ethers can be divided into hydroxy diphenyl ethers,polybrominated diphenyl ethers and chlorinated diphenyl ethers,etc.in accordance with the differences of the substituent groups on the benzene ring.The early study states the biosynthetic pathway of pescthic acid,the first natural diphenyl ethers coming from the fungus;there are 12 steps in total.Wherein,there is a polyketone backbone rearrangement reaction catalyzed by Cu oxidase.In addition,there is no other research report from the biosynthetic pathway of fungus natural diphenyl ethers.In the present article,we pay attention to diorcinol,a diphenyl ether with the simplest structure in fungus while with the most extensive distribution,and its derivatives.Diorcinol?3,3-dyhydroxy-5,5dimethyl diphenyl ether?has a symmetry chemical structure and it is with a good bioactivity.It does not only have bacteriostatic actions to Staphylococcus aureus,M.tuberculosis and Candida albicans,but also has antineoplastic activity and hemolytic activity.The further research indicates that,diorcinol is also able to restrain the gathering of amyloid?-peptides 42(A?₄₂).So it can be used as the candidate compound for the treatment of alzheimer's disease.Therefore,the clarification of the sample biosynthetic pathway of diorcinol can not only enrich the biosynthesis mechanism of fungus diphenyl ethers,but also provide the basis for later obtaining the diorcinol derivatives with more complex structure and more significant activity by the methods of synthetic biology and metabolic engineering.Clay.C.C.Wang research group has authenticated the relevant gene?ors gene cluster?of diorcinol biosynthesis in Aspergillus nidulans FGSC A4 by in vivo gene knockout experiment.The strains without gene AN7909?orsA?did not have the production capacity of orsellinic acid and diorcinol;the strains without AN7911?orsB?accumulated compounds gerfelin and C10-deoxy gerfelin;the missing of AN7912?orsC?,AN7913?orsD?and AN7914?orsE?did not affect the biosynthesis of diorcinol.This result indicates that,AN7909 and AN7911 are directly related to the generation of diorcinol,while other genes do not participate in the synthesis.However,the function of AN7910 in ors gene cluster and its correlation with diorcinol biosynthesis were not involved in the in vivo knockout experiment.The bioinformatics analysis indicates,AN7909 is non-reducing fungus polyketide synthase,containing SAT-KS-PT-ACP₁-ACP₂-TE structural domain.It is interred that it is able to catalyze 1 molecule acetyl coenzyme A and 3 molecules Malonyl-CoA to generate orsellinic acid;AN7911 is an amidohydrolase superfamily protein;while AN7910 is a hypothetical protein,it is a nuclear transcription factor 2?NTF2?superfamily protein.The domain analysis showed that it contains the SnoaL₄ domain.However,the specific catalytic activity of AN7909,AN7910 and AN7911,and their functions in diorcinol biosynthesis have not been verified yet.Therefore,this subject tries to verify the functions of AN7909,AN7910 and AN7911 to explore the biosynthetic pathway of diorcinol by yeast system heterologous expression system,enzymology in vitro biochemical characterization and other experiments.The results obtained by this subject are shown as follows:?1?AN7909,AN7910 and AN7911 genes are successfully co-expressed via yeast system heterologous expression platform to obtain diorcinol;?2?AN7909 is a special non-reducing polyketide synthase.The engineered strains with high expression in yeast and original manuscript aspergillus nidulans mainly generate diphenyl ether compound diorcinolic acid,other than orsellinic acid;?3?The point mutation experiment verifies that.ACP₁ and TE structural domain in the structural domain of AN7909 protein directly related to the generation of diorcinolic acid;?4?In vitro biological function verifies that,AN7910 is a decarboxylase family protein relied by new non-cofactors,it is able to exclusively catalyze diorcinolic acid C2 decarboxylation to generate C10-deoxy gerfelin,wherein,His68 and Arg16 are the active amino acid locus;?5?In vitro biological function verifies that,AN7911 is an amidohydrolase superfamily decarboxylase relied by nonmetallic ion,it is able to catalyze C10-deoxy gerfelin C4 decarboxylation to generate diorcinol.In conclusion,we have revealed the new sample biosynthetic pathway of fungus diphenyl ether natural product,which provides new enzymatic component for the reorganization of follow-up diphenyl ether natural products.