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Background
Natural mint flavor is produced by extraction from mint, which is not efficient enough to make it environment friendly process. (−)-Carvone is the monoterpenoid with key flavor of spearmint, and there has been an attempt to produce (−)-carvone by recombinant Escherichia coli. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in E. coli independently, the yield of (−)-carvone was low in the previous study.
Results
We have found a by-product formation when cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in single cell. These by-products were determined as dihydrocarveol and dihydrocarvone. We hypothesized that the enzymatic kinetics and the expression levels of P450 and CDH are quite different in E. coli. Therefore, two strains independently expressing P450/CPR and CDH were mixed with different mixing ratio, confirming increase in carvone production and decrease in by-product formation when CDH input was reduced. To determine the optimum balance of enzyme expressions, proteome analysis quantification concatemer (QconCAT) method to quantify P450, CPR, and CDH was developed. Using the QconCAT standard protein that was artificially created by concatenating the tryptic peptides, the ratio between P450 and CDH was calculated, and their optimum ratio to maximize (−)-carvone production was shown. Then, a single strain expressing both P450/CPR and CDH was constructed to imitate the superior expression ratio. The upgraded strain showed 15-fold improvement compared to the initial strain, showing 44 ± 6.3 mg/L of (−)-carvone production from 100 mg/L (−)-limonene as a starting substrate.
Conclusions
Improved expression balance of P450 and CDH in recombinant E. coli increased the (−)-carvone production using (−)-limonene as the direct substrates by the whole-cell biocatalysis, showing approximately 150 times higher titer than previous report. Our study showed the usefulness of proteome analysis QconCAT method in the strain development for industrial biotechnology field.
Keywords
Carvone, Proteomics, QconCAT, Bioconversion, Escherichia coli, Whole-cell biocatalysis
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 06 Jan, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Natural mint flavor is produced by extraction from mint, which is not efficient enough to make it environment friendly process. (−)-Carvone is the monoterpenoid with key flavor of spearmint, and there has been an attempt to produce (−)-carvone by recombinant Escherichia coli. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in E. coli independently, the yield of (−)-carvone was low in the previous study.
Results
We have found a by-product formation when cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in single cell. These by-products were determined as dihydrocarveol and dihydrocarvone. We hypothesized that the enzymatic kinetics and the expression levels of P450 and CDH are quite different in E. coli. Therefore, two strains independently expressing P450/CPR and CDH were mixed with different mixing ratio, confirming increase in carvone production and decrease in by-product formation when CDH input was reduced. To determine the optimum balance of enzyme expressions, proteome analysis quantification concatemer (QconCAT) method to quantify P450, CPR, and CDH was developed. Using the QconCAT standard protein that was artificially created by concatenating the tryptic peptides, the ratio between P450 and CDH was calculated, and their optimum ratio to maximize (−)-carvone production was shown. Then, a single strain expressing both P450/CPR and CDH was constructed to imitate the superior expression ratio. The upgraded strain showed 15-fold improvement compared to the initial strain, showing 44 ± 6.3 mg/L of (−)-carvone production from 100 mg/L (−)-limonene as a starting substrate.
Conclusions
Improved expression balance of P450 and CDH in recombinant E. coli increased the (−)-carvone production using (−)-limonene as the direct substrates by the whole-cell biocatalysis, showing approximately 150 times higher titer than previous report. Our study showed the usefulness of proteome analysis QconCAT method in the strain development for industrial biotechnology field.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
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