Abstract
(−)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (−)-carvone is desirable. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli. Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (−)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (−)-carvone production from 100 mg/L (−)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.
Highlights
(−)-Carvone is a monoterpenoid with a spearmint flavor
The P450 spearmint (Mentha spicata) gene, CYP71D18, was codon-optimized for E. coli, and the cytochrome P450 reductase (CPR) gene ATR2 of Arabidopsis thaliana was used as the native sequence
When (−)-carveol and (−)-carvone were incubated with the wild type E. coli BL21(DE3) strain, dihydrocarvone formation was observed. These results indicated that the exogenous peppermint enzyme carveol dehydrogenase (CDH) (ISPD) generated dihydrocarveol, and the endogenous E. coli enzyme generated dihydrocarvone as by-products
Summary
(−)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (−)-carvone is desirable. Kinetic parameter information of these two enzymes was limited, but it appeared that the P450 reaction was rate-limiting in spearmint p lants[9] Based on these prior studies, we hypothesized that the expression levels of P450 and CDH are quite different, leading to an imbalance in the carvone biosynthesis pathway in E. coli, resulting in a low conversion rate from (−)-limonene to (−)-carvone. To investigate this hypothesis, a protein quantification method with high sensitivity was required to conduct a comparative study among various strains by the abundance ratio of pathway enzymes. DNA synthesis has become extremely accessible, such that the QconCAT method has the potential to become more popular in synthetic biology
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