Journal
ACS SYNTHETIC BIOLOGY
Volume 5, Issue 9, Pages 1011-1020Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00140
Keywords
terpene synthase; monoterpene bioproduction; carotenoid; directed evolution; cyanobacteria
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Funding
- PRESTO (JST)
- MEXT/JSPS [23108507]
- JSPS
- Hamaguchi Foundation for the Advancement of Biochemistry
- Mishima-Kaiun Memorial Foundation
- Futaba Electronics Memorial Foundation
- Shorai Foundation for Science and Technology
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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.
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