Metabolic Engineering of Escherichia coli for Borneol Production Using an Artificial Pathway

Abstract:

Objective This study aims to construct an artificial concise isopentenol utilization pathway (IUP) with isoprenol as the substrate, thus addressing the limitations (such as excessive pathway length and high cofactor demand) of the conventional MEP/MVA pathways and enhancing the biosynthesis efficiency of borneol.Method A two-step enzymatic catalysis pathway with isoprenol as the substrate was designed for the synthesis of precursor IPP/DMAPP in Escherichia coli (E.coli） BL21 to synthesize borneol. Subsequently, metabolic engineering strategies were employed to further improve the synthesis of borneol, such as improving the soluble expression level of the bornyl diphosphate synthase CbTPS1, screening different sources of prenyltransferases to enhance the metabolic flux of IPP/DMAPP to GPP, optimizing the expression levels of key enzymes in the borneol biosynthesis pathway, and screening highly active phosphatases. Finally, the effects of different metal ions on the biosynthesis of bornol were explored.Result The artificial IUP increased the borneol production efficiency by 12.70% compared with the MEP pathway. Through enzyme and pathway engineering, the titer of borneol reached 2 642.8 μg/L. Further supplementation with Mg²⁺ increased the borneol titer to 5 912.4 μg/L.Conclusion The biosynthesis of borneol was achieved via the artificial IUP, which can provide theoretical and methodological references for the biosynthesis of other terpenoids.