The Research Of Side Chain Biosynthesis Of Fungal Immunosuppressive Agent Mycophenolic Acid

Background:Filamentous fungi are one main source of pharmaceuticals with widely biological activities,such as antibiotic penicillin,cholesterol-lowering agent lovastatin and antifungal griseofulvin.Fungal natural products with structural diversity are classified into polyketides,terpenoids,polyketide-terpenoids,peptides,alkaloids based on biosynthetic pathway and structural frameworks.The polyketide-terpenoids consists of polyketide and isopentenyl units,the representative of which including immunosuppressive agent mycophenolic acid(MPA),anti-angiogenic drug fumacillin and the anti-proliferative drugs aspernidine A.Mycophenolic acid can block deoxyribonucleic acid(DNA)synthesis by inhibiting the rate-limiting enzyme inosine monophosphate dehydrogenase(IMPDH)in the guanine nucleotide biosynthetic pathway efficiently.Thus,MPA possesses a wide spectrum of bioactivities based on unique mechanism of action of it,such as antiviral,antifungal,antibacterial,antitumor,antipsoriasis and immunosuppressive activities.However,to a certain extent,there are various adverse effects during immunotherapy limit the application of MPA to a certain extent,such as diarrhea,leukopenia,sepsis and vomiting,etc.At present,the MPA derivative with higher activity and lower adverse reaction is the key to the transformation of its chemical structure based on modification of isopentenyl side chain of MPA.Up to now,the MPA gene cluster was cloned and identified in Penicillium brevicompactum IBT23078,P.brevicompactum NRRL 864,P.roqueforti CECT and P.roqueforti FM164.Mpa gene cluster contained seven putative open reading frames(ORFs)MpaA-H,including prenyltransferase(MpaA),nonreducing polyketide synthase(MpaC),cytochrome P450 oxidase-hydrolase fusion protein(MpaDE),inosine monophosphate dehydrogenase(MpaF),O-methyltransferase(MpaG),?-? hydrolase(MpaH)and unknown functional protein(MpaB).1)MpaF was heterologous expressed in Aspergillus nidulans that did not produce MPA.The results indicated that the resistance of A.nidulans to MPA was significantly increased,which demonstrated that MpaF is the target of MPA.2)The genetics in vivo and enzyme biochemical study showed that MpaC catalyze the production of 5-methylorsellinic acid(5-MOA)from one molecule of acetyl-CoA,three molecules of malonyl-CoA and one molecule of S-adenosylmethionine(SAM);the fusion protein MpaDE catalyzes a two-step sequential reaction to convert 5-MOA to 5,7-dihydroxy-4-methyl phthalide(DHMP);MpaG catalyzes the methylation of the C-5 position of the polyketide backbone DHMP in demethylmycophenolic(DMMPA)to form MPA.3)In addition to,MpaA,MpaH and MpaB may be involved in the uploading and cleavage of isoprenyl side chain of MPA,but the relevant functional studies of which have not been reported.Therefore,this study choose MpaA/B/H as the research object and attempts to elucidate the unique biosynthesis mechanism of isoprenyl side chain of MPA by comprehensive research strategies and methods,such as gene knock-out experiments in vivo,isolation and identification of relevant intermediates,chemical complementation and enzyme biochemical in vitro.The complete MPA biosynthetic pathway was clarified will set a solid theoretical foundation for the subsequent synthesis biology transformation of MPA based on biological enzyme engineering and metabolic engineering methods.Obejective:1.Indetifying MPA biosynthetic gene clusters in P.griseofulvum.2.Constructing MpaA/B/C/H knockout mutants,their corresponding accumulated products were detected.3.Elucidating the function of MpaA/B/H in biosynthesis of isopentenyl side chain of MPA through biochemical study of its in vitro,and then we discuss enzymatic reaction mechanism of its.4.Exploreing the significance of MpaA/B for the biological modification of isopentenyl side chain of MPA based on the above results of biochemical studies in vitro.Methods:1.Whole genome sequencing of P.griseofulvum and finding MPA gene cluster(mpa)in P.griseofulvum through local blast,and then 2ndFind,Pfam,NCBI and other software were used to analyze the gene cluster to get the coding sequences of all functional genes in the cluster.2.Snapgene software was used to design primers,DNAMAN for protein homology analysis,Phery2 for protein homology modeling,Clustalx 2.1 and MEGA 6.0 for phylogenetic tree analysis,TMHMM Server v.2.0 for predicting protein trans-membrane region.3.MpaA/B/C/H gene mutant strains were achieved by constructing split-marker knock-out plasmid and protoplast transformation method.High performance liquid chromatography(HPLC)was used to analyze the retention time and UV absorption of the intermediate product.The molecular weight of the corresponding compounds was identified by liquid chromatograph-mass spectrometer(LC-MS).The compounds were isolated by semi-prepared liquid phase.The structures of the compounds were identified by NMR.4.The yeast expression vector of MpaA/MpaB/MpaH/MpaG were constructed by enzyme digestion or yeast homologous recombination.The function of MpaA/B/H/G were initially verify via compound feeding experiments,which elucidate possible biosynthetic pathways of isopentenyl side chain of MPA.5.The corresponding protein was obtained by affinity chromatography(MpaB)or extraction of microsomes(MpaA).The biochemical reaction mechanism of the enzyme was studied and the function of MpaA/B in the synthesis of the isopentenyl side chain of MPA was elucidated in detail.Results:1.A 25.5-kb mpa gene cluster was identified in the genome of P.griseofulvum.The sequences of seven coding genes were highly consistent with those of mpa gene cluster from P.brevicompactum.2.The phylogenetic tree analysis of MpaA indicated that it has a similarity to Coq2,UbiA and LePGT1,all of which belong to the family of membrane-bound prenyltransferases.MpaA knockout strain lost its ability to produce MPA and compound 94 was accumulated,the structure of 94 was identified as DHMP.The results of MpaC knockout mutant strain fed DHMP in vivo showed that it is intermediates of MPA biosynthetic pathway.3.After in Saccharomyces cerevisiae FY::MpaA with DHMP,compound 95 was produced and its structure was identified as 6-farnesyl-5,7-dihydroxy-4-methyl phthalide(FDHMP).The results show that MpaA is the C6 position of DHMP farnesyl diphosphate(FPP)transferase.Further biochemical in vitro results showed that MpaA has a broad substrate,which can not only select FPP,but also can catalyze the formation of compound 101 by combining geranyl diphosphate(GPP)and DHMP.4.MpaH knockout strains did't lost its ability to produce MPA(MpaH may not be involved in the biosynthesis of MPA),but compounds 102 and 103 were accumulated.The structure of 102 and 103 were identified as the terminal carboxyl C2 side chain and carbonyl C6 side chain of MPA derivative,respectively.The results of MpaC knockout strain fed the compounds in vivo showed that neither 102 nor 103 cannot recovery the production of MPA,indicating that the two compounds are by-products of the MPA biosynthesis pathway.5.The two intermediates FDHMP and 104 were accumulated in MpaB knockout mutant strain,the structure of 104 was identified as the hydroxymethylation product at the C-3 position of benzene ring of FDHMP.The results of MpaC knockout strains fed FDHMP and 104 in vivo proved that they are intermediates of MPA biosynthetic pathway.6.After fed FDHMP and 104 in S.cerevisiae BJ5464::MpaB,new compounds 105,106 and 107,108 were produced,respectively.The structural characterization of 105 and 106 are that terminal carboxyl group and terminal hydroxyl group,respectively.Time course experiments analysis of MpaB showed that it catalyzes the production of compounds 105 and 106 at the same time,but 105 is the major product compared to 106.MpaC knockout strain fed 105 and 106 confirmed that they are intermediates of MPA biosynthetic pathway.7.Subsequently,the substrate diversity catalytic experiment of MpaB revealed that it failed to catalyze the reaction with MXH-12 and MXH-14,but it can convert ZGJ-7,ZGJ-8 and ZGJ-9 to 109',110' and 111',respectively.Conclusion:The biosynthetic mechanism of isopentenyl side chain of MPA was preliminarily elucidated based on the identification of MPA biosynthetic gene cluster mpa in P.griseofulvum,as well as the function of the three genes and proteins of MpaA/B/H was investigated in vivo and in vitro.1)MpaA,an membrane-dependent prenyltransferase,catalyzes the farnesyl pyrophosphorylation(FPP)of DHMP to form FDHMP(95);2)The unknown functional protein MpaB is responsible for catalyzing the carbon-carbon bond cleavage of the end of the FPP side chain of FDHMP(95)and 104 to forms carboxyl and hydroxyl products;3)The previously predicted MpaH gene that catalyzes the carbon-carbon bond cleavage,although it may not involve in the final formation of MPA based on gene knockout in vivo.The biochemical function of MpaA was confirmed in vitro,which not only enrich the function of membrane-dependent prenyltransferase family from fungal,but also provide a tool for synthesis or discovery of new mycophenolic acid derivatives in the future.The discovery of biochemical function of the unknown functional protein MpaB in vitro not only reveals the cleavage process of the isopentenyl side chain hidden in MPA biosynthesis pathway,but also broadens the biological enzyme resources that catalyze the carbon-carbon bond cleavage in nature.More importantly,MpaB catalyzes the cleavage of the isoprenyl side chain C15 of various polyketone-sesquiterpene hybrid compounds,which making it possible to apply it to the transformation of other natural products.However,it make us confused how the cleavage product of MpaB catalyze the substrate to form DMMPA or MPA.We will clarify the complete biosynthetic pathway of MPA by further explore.