N-terminal truncation of prenyltransferase enhances the biosynthesis of prenylnaringenin

Abstract:

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values.It also serves as an important precursor for many prenylated flavonoids.Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors.In this work,a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation.The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops,along with a long side chain formed by nearly 120 amino acids.SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae.A mutant expressing the truncated enzyme at K62 site,designated as SfND8T-1-t62,produced the highest 8-PN titer.Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue.Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates,which also indicated K185 was a potentially critical residue.Besides,the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation,while the saturated mutation of K185 showed a great decrease in 8-PN production,indicating K185 is vital for the activity of SfN8DT-1.Subsequently,overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L,which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis.Finally,44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h,which is the highest 8-PN titer reported to date.