Microbial synthesis of plant-based (-)-menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (-)-limonene and (+)-cis-isopulegone. Herein, the first artificial and effective system was developed for (+)-cis-isopulegone biosynthesis from (-)-limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene-3-hydroxylase, and isopiperitenol dehydrogenase. A cofactor self-regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)-cis-isopulegone (up to 281.2 mg L-1 ) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (-)-menthol.
Keywords: biocatalysis; biosynthesis; enzyme catalysis; microbial cell factory; oxidoreductases.
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