Biosynthetic Study Of A Fusidane-type Antibiotic Helvolic Acid

Fusidane-type antibiotics,a small group of fungi-derived triterpenes,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?1?,fusidic acid and cephalosporin P₁ are the most representative fusidane-type antibiotics,all of which exhibit potent activities against gram-positive bacteria.Amongst them,fusidic acid is far more active and has been clinically used to treat staphylococcal skin infections.The remarkable bioactivity and successful clinical usage of fusidic acid have inspired many attempts to search more active congeners.Great efforts 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.Biosynthetic engineering is a feasible option to expand chemical diversity and generate compounds featuring novel structures,which however requires a thorough understanding of biosynthetic pathways of target compounds.So far,the biosynthetic mechanisms of fusidane-type antibiotics remain unclear.In 2009,with the accessibility of Aspergillus fumigatus Af293 genome sequences,a gene cluster consisting of nine genes was proposed for helvolic acid biosynthesis,and the cluster encodes one oxidosqualene cyclase HelA,four cytochrome P450s HelB1-4,one short-chain dehydrogenase/reductase?SDR?HelC,two acyltransferases HelD1-2 and one ketosteroid-dehydrogenase HelE.Preliminary heterologous expression studies in yeast revealed that oxidosqualene cyclase HelA functioned to convert 2,3-?S?-oxidosqualene into the protostadienol tetracyclic backbone,which was then oxidized to generate 4?-carboxylic acid by the cytochrome P450 HelB1,or underwent dehydrogenation to form 3-keto catalyzed by the short-chain dehydrogenase/reductase HelC.However,the biosynthesis of helvolic acid remains unclear.Firstly,it hasn't been determined that whether the proposed gene cluster is responsible for the biosynthesis of helvolic acid.Secondly,if the proposed gene cluster leads to the biosynthesis of helvolic acid,it is unrevealed that how the left 6out of 9 genes in the cluster exert the tailoring.Thirdly,it is unknown that whether there are compounds exhibiting stronger activity than helvolic acid in the biosynthesis of helvolic acid.and was beyond the unraveling of helvolic acid biosynthesis.Thus,exploring helvolic acid biosynthesis could provide an opportunity to thoroughly decipher the biosynthesis of fusidane-type antibiotics and yield novel derivatives by virtue of combinational biosynthesis.In this work,we introduced all the nine genes in the cluster into the heterologous expression system quadruple auxotrophic Aspergillus oryzae NSAR1 and obtained helvolic acid,demonstrating that the proposed gene cluster is indeed dedicated to the biosynthesis of helvolic acid?1?.Then we introduced the genes into A.oryzae NSAR1in a stepwise manner and systematically investigated the biosynthetic pathway of helvolicacid?Fig.1?.TheoxidosqualenecyclaseHelAconverts2,3-?S?-oxidosqualeneintoprotostadienol?2?.Thentheshortchain dehydrogenase/reductase HelC converts the C-3?hydroxyl group to C-3 carbonyl group,and the cytochrome P450 HelB1 oxidizes the C-4?methyl group to C-4?carboxyl group.Collaboratively,HelC together with Hel B1 could trigger the demethylation to form 5.Following that,the cytochrom P450 HelB2 is responsible for the oxidation of C-16 to generate the C-16?hydroxyl group,and the acyltransfetrase HelD2 could attach an acetyl group at the C-16?position.After the cytochrome P450HelB4 oxidizes the C-20 methyl group to carboxylic acid,the cytochrome P450HelB3 functions to form C-6?hydroxyl group and C-7 carbonyl group.Then the acyltransferase HelD1 introduces the acetyl group at the C-6?position.Finally,the3-ketosteroid-?¹-dehydrogenase HelE forms the double bond between C-1 and C-2positions via dehydrogenation.By heterologous expression in A.oryzae NSAR1,we identified all the six uncharacterized genes involved in helvolic acid biosynthesis.According to the heterologous expression in A.oryzae NSAR1,it seemed that HelC could work with broad substrate specificity.Thus,we heterologously expressed his₆-tagged HelC in E.coli BL21?DE3?and obtained the purified homogeneity via the affinity chromatography column.We incubated HelC and 4 in the presence of cofactor NAD⁺,and detected the oxidative decarboxylation product 5.In the same manner,HelC could also accept 6,10 and 17 as substrates to generate 7,13 and 16,respectively.And HelC could also convert the three products 15,18 and 20 only containing C-3?hydroxyl group to 16,19 and 21.Impressively,in vitro enzymatic reactions showed that HelC could act on 16,19 and 21 to yield the corresponding reduced forms in the presence of NADH.To the best of our knowledge,HelC is the first reported SDR catalyzing the reversible reaction between 3?-hydroxyl and oxo groups in triterpenoids and steroids biosynthesis.Nowthat Compounds 5,7 and 16could be generated via oxidative decarboxylation catalyzed by HelC,and it was supposed that they couldn't be derived from the other biosynthetic pathway.Then we introduced cytochrome P450s HelB1,Hel B2 and HelB4 into A.oryzae NSAR1,respectively,constructing differnet trnasformant strains,which were fed on 3,5 and13,respectively.The results indicated that the three cytochrome P450s couldn't tailor the substrates 3,5 and 13 bearing 3-carbonyl group?Fig.1?.By the means of heterologous expression in A.oryzae NSAR1,we obtained as much as 25 compounds,including 16 new compounds.We tested inhibitory effects of compounds 1-25 against Staphylococcus aureus 209P by the 2-fold dilution method.The results revealed that most compounds showed activities against S.aureus 209P,among which 19,22 and 23 exhibited stronger activities than helvolic acid?1?,and especially Compound 23 showed the strongest antibacterial activity with minimum inhibition concentration?MIC?of 0.5?g/mL.By further structure-activity relationship analysis,we found Compound 16 showed stronger antibacterial activity than 13,indicating that oxidation of C-20 methyl group to carboxyl group could dramatically enhance the antibacterial activity,which was in accordance with that reported.The fact that 16,19 and 21 exhibited higher activity than the respective 15,18 and 20 suggested that 3-keto was more beneficial for activity than 3?-hydroxyl group,which was also consistent with that reported.In addition,we firstly found that acetylation at C-6 position was detrimental to the activity,as 19 and 23 exhibited stronger activities than their corresponding acetylated products at the C-6?position,21 and the end product helvolic acid?1?.In summary,heterologous reconstitution in A.oryzae NSAR1 enabled us to establish the complete biosynthetic pathway of helvolic acid,opening a door for yielding artificial fusidane-type antibiotics via combinational biosynthesis.In vitro assays revealed the short chain dehydrogenase/reductase HelC was featured with broad substrate specificity and multifunction,serving as a potential enzymatic tool for biotransformation of natural productsthat.The discovery of fusidane-type antibiotics with higher anti-Staphylococcus aureus activity than helvolic acid provided an opportunity for rational design of fusidane-type analogues.