Expanding the pathway to synthesize isopiperitenol in Escherichia coli

Abstract:

As a plant-derived aromatic compound, isopiperitenol has a high economic value. Little is known about the production of isopiperitenol in Escherichia coli engineering strains. [Objective] To construct the engineering strains of E. coli for the biosynthesis of natural isopiperitenol. [Methods] We modified the limonene-3-hydroxylase (LIM3H) gene PM2 of cytochrome P450 family from Mentha×piperita by truncating the N-terminal hydrophobic region or adding 17α short peptide or 2B1 short peptide of LIM3H to increase its hydrophilicity. The activity of LIM3H was detected by CO differential spectroscopy. The NADPH-cytochrome P450 reductase gene with truncated N-terminal hydrophobic region (trATR) from Arabidopsis thaliana was fused with the modified LIM3H (Modi-LIM3H) gene and the fusion gene was expressed in E. coli. The obtained fusion protein was used to catalyze the production of isopiperitenol from limonene. [Results] LIM3H expression and isopiperitenol production were detected in all the strains with the LIM3H N-terminus modified in three different ways. The LIM3H modified by addition of 17α short peptide showed the highest protein level, and the strain carrying this modified gene had the highest yield (1.94 mg/L) of isopiperitenol.[Conclusion] In this study, exogenous LIM3H and ATR were introduced into E. coli and the fusion protein catalyzed the production of isopiperitenol from limonene, which provided a new method for the production of natural isopiperitenol.