Abstract
Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants, Mentha spp. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell. We have developed a one-pot (bio)synthesis of (1R,2S,5R)-(-)-menthol and (1S,2S,5R)-(+)-neomenthol from pulegone, using recombinant Escherichia coli extracts containing the biosynthetic genes for an "ene"-reductase (NtDBR from Nicotiana tabacum) and two menthone dehydrogenases (MMR and MNMR from Mentha piperita). Our modular engineering strategy allowed each step to be optimized to improve the final production level. Moderate to highly pure menthol (79.1%) and neomenthol (89.9%) were obtained when E. coli strains coexpressed NtDBR with only MMR or MNMR, respectively. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds.
Highlights
Menthone reduction resulted in a 2:1 ratio of menthol and neomenthol
Products formed during biotransformations of DMN cell extracts in strains A) 6 and B) 7 at different IPTG
Product yields were determined by GC analysis using a DB-WAX column
Summary
Epimerisation of menthone with sodium hydroxide yielded a mixture of menthone and isomenthone (70/30), separable by column chromatography.[1] These compounds were stored at reduced temperature to retard the re-equilibration process. Menthone and isomenthone were treated non-selectively with sodium borohydride to reduce the ketones to the respective secondary alcohols (SI Table 1).[2] These products were separable by column chromatography, including minor products derived from the contaminating isomer present in the starting material. Product distributions for sodium borohydride reduction of menthone and isomenthone. Subsequent basic ester hydrolysis yielded the remaining compound isomenthol (SI Table 1). Product yields were determined by GC analysis using a DB-WAX column. Data point colours: menthone = dark blue; isomenthone = red; menthol = green; neoisomenthol = purple; neomenthol = light blue; isomenthol = orange.
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