Abstract
Terreic acid is a potential anticancer drug as it inhibits Bruton’s tyrosine kinase; however, its biosynthetic molecular steps remain unclear. In this work, the individual reactions of terreic acid biosynthesis were determined by stepwise pathway assembly in a heterologous host, Pichia pastoris, on the basis of previous knockout studies in a native host, Aspergillus terreus. Polyketide synthase AtX was found to catalyze the formation of partially reduced polyketide 6-methylsalicylic acid, followed by 3-methylcatechol synthesis by salicylate 1-monooxygenase AtA-mediated decarboxylative hydroxylation of 6-methylsalicylic acid. Our results show that cytochrome P450 monooxygenase AtE hydroxylates 3-methylcatechol, thus producing the next product, 3-methyl-1,2,4-benzenetriol. A smaller putative cytochrome P450 monooxygenase, AtG, assists with this step. Then, AtD causes epoxidation and hydroxyl oxidation of 3-methyl-1,2,4-benzenetriol and produces a compound terremutin, via which the previously unknown function of AtD was identified as cyclooxygenation. The final step involves an oxidation reaction of a hydroxyl group by a glucose-methanol-choline oxidoreductase, AtC, which leads to the final product: terreic acid. Functions of AtD and AtG were determined for the first time. All the genes were reanalyzed and all intermediates and final products were isolated and identified. Our model fully defines the molecular steps and corrects previous results from the literature.
Highlights
Fungal secondary metabolites are well known for their wide-ranging biological activities
The high activity of 6-MSA synthase (6-MSAS) in P. pastoris indicates that this host may be a suitable chassis organism for proteins from A. terreus, and it was chosen for heterologous expression of the terreic acid (TA) pathway in the present study
The product of 3-methylcatechol was identified by liquid chromatography with mass spectrometry (LC-MS) and 1H nuclear magnetic resonance (NMR) analysis (Supplementary Fig. 2) in a comparison with other experimental results[11]. These findings proved that AtA but not AtE catalyzes the decarboxylative hydroxylation after AtX in TA biosynthesis, in agreement with gene knockout results in a native strain[7]
Summary
Fungal secondary metabolites are well known for their wide-ranging biological activities. The atA-encoded 6-MSA decarboxylase catalyzes decarboxylation and hydroxylation reactions to form a predicted compound: 3-methylcatechol (compound 5, Fig. 1), followed by a hydroxylation reaction catalyzed by the atE-encoded cytochrome P450 monooxygenase to produce a predicted compound, 3-methyl-1,2,4-benzenetriol (compound 6, Fig. 1) This reaction could be catalyzed by a catechol 1,2-dioxygenase encoded by a gene outside the at cluster resulting in formation of a nonaromatic compound: (2E,4Z)-2-methyl-2,4-hexadienedioic acid www.nature.com/scientificreports/. Deficiency in atD and atG blocks TA synthesis but no intermediates or shunt products have been identified, leaving a gap in functional characterization of both enzymes[7]. The high activity of 6-MSAS in P. pastoris indicates that this host may be a suitable chassis organism for proteins from A. terreus, and it was chosen for heterologous expression of the TA pathway in the present study. The heterologous biosynthesis of TA and of the intermediates was realized via combinatorial expression of various functional genes, and the functions of the biosynthetic genes were confirmed and redefined, thereby correcting previous results from the literature and describing all the reactions of the TA biosynthesis pathway
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