Abstract
Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. However, their biosynthesis is poorly understood. Here, we perform a stepwise introduction of the nine genes from the proposed gene cluster for helvolic acid into Aspergillus oryzae NSAR1, which enables us to isolate helvolic acid (~20 mg L−1) and its 21 derivatives. Anti-Staphylococcus aureus assay reveals that the antibacterial activity of three intermediates is even stronger than that of helvolic acid. Notably, we observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis. These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics.
Highlights
Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics
In order to determine whether the proposed gene cluster is sufficient to produce helvolic acid, we constructed an A. oryzae NSAR1 transformant strain AO21 co-expressing all the nine genes
Following the cyclization to the tetracyclic protosta-17(20)Z,24-dien-3β-ol (2) by HelA, HelB1mediated and HelB2-mediated oxidation at C-4 and C-16, HelD2-dependent acetylation of 16-OH, oxidation of C-21 by HelB4, and HelC-dependent oxidative decarboxylation yield the fusidane skeleton 14, which is further modified in three additional steps mediated by HelB3, HelD1, and HelE to give helvolic acid (1) (Fig. 4)
Summary
Fusidane-type antibiotics represented by helvolic acid, fusidic acid and cephalosporin P1 are a class of bacteriostatic agents, which have drawn renewed attention because they have no cross-resistance to commonly used antibiotics. We observe an unusual C-4 demethylation process mediated by a promiscuous short-chain dehydrogenase/reductase (HelC) and a cytochrome P450 enzyme (HelB1), which is distinct from the common sterol biosynthesis These studies have set the stage for using biosynthetic approaches to expand chemical diversity of fusidane-type antibiotics. Preliminary heterologous expression studies in Saccharomyces cerevisiae revealed that HelA (oxidosqualene cyclase, OSC), functioned to convert (3S)-2,3-oxidosqualene into protosta-17 (20)Z,24-dien-3β-ol (2), which underwent dehydrogenation to form 3-keto (3) by HelC (short-chain dehydrogenase/reductase, SDR), or oxidation to generate 4β-carboxylic acid (4) by HelB1 (cytochrome P450, P450)[18] It is still not clear whether the proposed nine-gene cluster is enough for helvolic acid biosynthesis, and if so, what the functions and reaction order of the six other genes are, and whether biosynthetic intermediates with more potent antibacterial activity than the end product helvolic acid exist. These findings have provided a basis for constructing fusidane-type derivatives using biosynthetic approaches
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