Combinatorial Enzymatic Synthesis of Unnatural Long-Chain β-Branch Pyrones by a Highly Promiscuous Enzyme

[Image: see text] In this study, we described in detail a combinatorial enzymatic synthesis approach to produce a series of unnatural long-chain β-branch pyrones. We attempted to investigate the catalytic potential of a highly promiscuous enzyme type III PKS to catalyze the non-decarboxylative condensation reaction by two molecules of fatty acyl diketide-N-acetylcysteines (diketide-NACs) units. Two non-natural long-chain (C(16), C(18)) fatty acyl diketide-NACs were prepared successfully for testing the ability of non-decarboxylative condensation. In vitro, 12 novel naturally unavailable long-chain β-branch pyrones were generated by one-pot formation and characterized by ultraviolet–visible spectroscopy and high-resolution liquid chromatography–mass spectrometry. Interestingly, enzymatic kinetics result displays that this enzyme exhibits the remarkable compatibility to various non-natural long-chain substrates. These results would be useful to deeply understand the catalytic mechanism of this enzyme and further extend the application of enzymatic synthesis of non-natural products.