Dataset Information

Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis

ABSTRACT: The cytochrome P450 limonene-6-hydroxylase (P450), cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in Escherichia coli for (−)-carvone production from (−)-limonene. The optimum ratio of enzyme expression to maximize (−)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method.

ORGANISM(S): Escherichia Coli

SUBMITTER: Fumio Matsuda

PROVIDER: PXD028077 | JPOST Repository | Wed Aug 24 00:00:00 BST 2022

REPOSITORIES: jPOST

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Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis.

Yoshida Erika E Kojima Motoki M Suzuki Munenori M Matsuda Fumio F Shimbo Kazutaka K Onuki Akiko A Nishio Yousuke Y Usuda Yoshihiro Y Kondo Akihiko A Ishii Jun J

Scientific reports 20211111 1

(-)-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) w ...[more]

PMID: 34764337

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Project description:BackgroundN-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell.ResultsThe host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis.ConclusionsThese results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

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