Enzymatic Menthol Production: One-Pot Approach Using Engineered Escherichia coli

Area of Science:

• Biotechnology
• Synthetic Biology
• Biocatalysis

Background:

• Menthol isomers are valuable monoterpenoids naturally produced by mint plants.
• Current production relies on extraction, with biosynthetic routes offering cleaner alternatives.
• Metabolic engineering of host cells is a common but limited strategy for terpenoid production.

Purpose of the Study:

• To develop and optimize a cell-free, one-pot biotransformation system for menthol and neomenthol synthesis.
• To circumvent limitations associated with host cell toxicity in metabolic engineering.
• To apply pathway assembly and biocatalysis for efficient production of mint compounds.

Main Methods:

• Engineered cell-free one-pot biotransformation using recombinant Escherichia coli extracts.
• Coexpression of specific enzymes: NtDBR (ene-reductase), MMR, and MNMR (menthone dehydrogenases).
• Modular engineering strategy to optimize individual enzymatic steps.

Main Results:

• Successful one-pot biosynthesis of (1R,2S,5R)-(-)-menthol and (1S,2S,5R)-(+)-neomenthol from pulegone.
• Achieved moderate to high purity: 79.1% menthol and 89.9% neomenthol.
• Demonstrated improved production levels through modular optimization.

Conclusions:

• The developed cell-free system offers an efficient and optimizable approach for producing menthol and neomenthol.
• This strategy overcomes host cell toxicity issues, enabling easier optimization and modularity.
• It provides a valuable biocatalysis strategy for synthesizing valuable and potentially toxic compounds.