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

8-Prenylnaringenin(8-PN)is a valuable medical phytoestrogen,which is a precursor to many prenylated flavonoids.How-ever,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 synthe-size 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^(Q12EN305M),produced 49.35±0.05 mg/L(5.57±0.01 mg/g DCW)8-PN in a shaking flask.Finally,101.40±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.