Biosynthetic Study Of Fusidane-type Antibiotics

The fusidane-type antibiotics,a type of non-lanostane fungi-derived triterpene,which are formed through removal of C-4?methyl group,oxidation of C-20 methyl group to carboxylic acid,attachment of acetoxyl group at C-16?position and other modifications on the basis of protostadienol.Helvolic acid,fusidic acid,and cephalosporin P₁ are the most representative fusidane-type antibiotics,all of which exhibit potent activity against grampositive bacteria.Amongst them,fusidic acid has been used to treat staphylococcal skin infections,and it exerts antibacterial effects by inhibiting the synthesis of bacterial proteins by inhibiting the elongation factor EF-G.Due to the special antibacterial mechanism of fusidic acid,it has no obvious cross-resistance with commonly used antibiotics.With the increasingly serious problem of antibiotic resistance in the world,it is important to find a new type of fusidane derivative.So far,researchers have been focused on modifications of fusidane skeleton by means of chemical derivatization and biotransformation.However,none of these analogs were found to be more active than fusidic acid.Combinatorial biosynthesis has been an effective means of modifying the structural diversity of natural products.However,a prerequisite for its successful is need to reveal the biosynthetic pathway of the target compounds.In 2009,with the accessibility of Aspergillus fumigatus Af293 genome sequences,a gene cluster consisting of nine genes was proposed for helvolic acid biosynthesis.Preliminary heterologous expression studies in Saccharomyces cerevisiae revealed that HelA?oxidosqualene cyclase,OSC?,functioned to convert2,3?S?-oxidosqualene into Protostadienol,which underwent dehydrogenation to form3-keto by HelC?short-chain dehydrogenase/reductase,SDR?,or oxidation to generate4?-carboxylic acid by HelB1?cytochrome P450,P450?.To reveal the complete biosynthetic pathway of helvolic acid,Dr.Jian-Ming Lv introduced the nine genes into the heterologous expression system of Aspergillus oryzae by one-time and step-by-step method,confirming that the putative gene cluster is sufficient for helvolic acid biosynthesis,and constructed a complete biosynthetic pathway for helvolic acid.However,the mechanism of biosynthesis of fusidic acid and cephalosporin P₁ is not clear.Therefore,the subject will reveal the biosynthetic pathway of fusidic acid and cephalosporin P₁ on the basis of previous studies.In this study,we first searched for the homologues of HelA from the genome of Acremonium fusidioides ATCC 14700 and Acremonium chrysogenum ATCC 15500,which produce fusidic acid and cephalosporin P1 respectively.Finally,we found the potential biosynthetic gene clusters of fusidic acid and cephalosporin P₁.By homology analysis with the helvolic acid gene cluster,we found that all three gene clusters contain six conserved genes?helA,helB1,helB2,helD2,helB4,helC?.The six genes engage in the early stages of biosynthesis of helvolic acid.Based on the above results,we concluded that the early stages of biosynthesis of fusidane-type antibiotics was catalyzed by the conserved 6 gene,subsequent branching routes produce different fusidane antibiotics under the action of their respective modified enzymes.To verify the above hypothesis,we introduced two post-modification genes?fusC1 and fusB1?in the fusidic acid gene cluster and three post-modification genes?cepC2,cepB4 and cepD2?in the cephalosporin P₁ gene cluster into the previously constructed six genes expression strains respectively,confirmed that the putative gene cluster is sufficient for fusidic acid and cephalosporin P₁.Subsequently,we confirmed the function of modified gene and elucidated the biosynthetic pathway of fusidic acid and cephalosporin P₁ by heterologous expression,in vitro reaction and substrate feeding methods.In the biosynthetic pathway of fusidic acid,we confirmed that the function of FusC1 is to reduce the C-3 carbonyl to C-3?hydroxyl group,while FusB1 is responsible for the oxidation of C-11.Notably,we found two SDR enzymes?FusC1and FusC2?,which exhibit opposite stereoselectivity in 3-ketoreduction.Next,to further characterize the enzyme reaction,we measured the catalytic activity of FusC1and FusC2.We measured the optimal reaction cofactor of FusC1 was NADPH,pH was 7 and the temperature was 5 ^oC.We measured the optimal reaction cofactor of FusC2 was NADH,pH was 7 and the temperature was 37 ^oC.In the biosynthetic pathway of cephalosporin P₁,we confirmed that the function of CepC2 is to reduce the C-3 carbonyl to to C-3?hydroxyl group,while CepB4 is responsible for the double oxidation of C-6 and C-7.In addition,CepD2 is responsible for acetylation in the C-6?hydroxyl.Notably,we have demonstrated that the CepB4 of P450 monooxygenase is a multifunctional oxidase by in vitro reaction of microsomal.It can have multiple oxidation forms in C-6 and C-7.The CepB4 not only simultaneously oxidizes C-6 and C-7,but also oxidize C-6 to form C-6?hydroxyl groups and further oxidation to carbonyl groups,and also includes oxidize C-7 to form C-7?hydroxyl groups.By the means of heterologous expression in A.oryzae NSAR1,we obtained as much as 15 compounds,including 5 new compounds.We tested inhibitory effects of compounds against Staphylococcus aureus 209P by the 2-fold dilution method.The results revealed that most compounds showed activities against S.aureus 209P.By further structure-activity relationship analysis,we firstly found that C-6?monohydroxylation was beneficial to activity.In conclusion,this study systematically revealed the biosynthetic pathways of fusidic acid and cephalosporin P₁ on the basis of six genes expression strains of helvolic acid,which will open the door for yielding artificial fusidane-type antibiotics via combinational biosynthesis.