Bacterial Production of Pinene by a Laboratory-Evolved Pinene-Synthase

Miki Tashiro, Hiroshi Kiyota, Shigeko Kawai-Noma, Kyoichi Saito, Masahiko Ikeuchi, Yoko Iijima, Daisuke Umeno

Research output: Contribution to journalArticlepeer-review

41 Citations (Scopus)

Abstract

Successful feeding of the substrate geranylpyrophosphate (GPP) to monoterpene synthase is critical to the efficient microbial production of monoterpenes. Overexpression of GPP synthases, metabolic channeling from GPP synthase to terpene synthases, and down-tuning of endogenous competitors have been successfully used to increase the production of monoterpene. Nevertheless, the production of monoterpenes has remained considerably lower than that of hemi-/sesqui-terpenoids. We tested whether it is effective to improve the cellular activity of monoterpene synthases. To this end, we developed a high-throughput screening system to monitor for elevated GPP consumption. Through a single round of mutagenesis and screening, we isolated a pinene synthase variant that outperformed the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibited drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenylpyrophosphate isomerase, and GPP synthase yielded 140 mg/L pinene in a flask culture.

Original languageEnglish
Pages (from-to)1011-1020
Number of pages10
JournalACS Synthetic Biology
Volume5
Issue number9
DOIs
Publication statusPublished - 2016 Sep 16
Externally publishedYes

Keywords

  • carotenoid
  • cyanobacteria
  • directed evolution
  • monoterpene bioproduction
  • terpene synthase

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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