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*

*この研究の対応する著者

研究成果: Article査読

55 被引用数 (Scopus)

抄録

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.

本文言語English
ページ(範囲)1011-1020
ページ数10
ジャーナルACS Synthetic Biology
5
9
DOI
出版ステータスPublished - 2016 9月 16
外部発表はい

ASJC Scopus subject areas

  • 生体医工学
  • 生化学、遺伝学、分子生物学(その他)

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