De novo biosynthesis of 8-prenylnaringenin in Saccharomyces cerevisiae improved by screening and engineering of prenyltransferases and precursor pathway

Abstract

8-Prenylnaringenin (8-PN) is a valuable medical phytoestrogen, which is a precursor to many prenylated flavonoids. However, the availability of 8-PN is limited by inefficient prenyltransferases (PTs) and inadequate substrate precursor levels in microbial chassis. First, six PTs from different sources and their truncated cognates were expressed in a (2S)-naringenin producing strain. Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate, tSfN8DT-1, could synthesize 8-PN. Second, tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1\(^{Q12E}\) with greater catalytic activity was obtained. Third, key genes, tHMGR and IDI1, of the mevalonate (MVA) pathway were overexpressed using a copy number combinatorial strategy, which greatly improved 8-PN titer by 368.75%. Fourth, a predicted structure of tSfN8DT-1\(^{Q12E}\) was used for molecular docking and virtual saturation mutagenesis. Two key residues, P229 and N305, were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M. The best-performing mutant, tSfN8DT-1\(^{Q12E N305M}\), produced 49.35 \(\pm {}\) 0.05 mg/L (5.57 \(\pm {}\) 0.01 mg/g DCW) 8-PN in a shaking flask. Finally, 101.40 \(\pm {}\) 2.55 mg/L of 8-PN was obtained in a 5-L bioreactor, which is the greatest titer reported to date for 8-PN. This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1. The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods, which may provide a feasible strategy for the biosynthesis of prenylated flavonoids.