Development of Corynebacterium glutamicum as a monoterpene production platform

Luckie, B. A.; Kashyap, M.; Pearson, A. N.; Chen, Y.; Liu, Y.; Valencia, L. E.; Romero, A. C.; Hudson, G. A.; Tao, X. B.; Wu, B.; Petzold, C. J.; Keasling, J. D.

doi:10.1101/2023.10.31.565027

bioRxiv · 10.1101/2023.10.31.565027

Development of Corynebacterium glutamicum as a monoterpene production platform

Luckie, B. A.·Kashyap, M.·Pearson, A. N.·Chen, Y.·Liu, Y.·Valencia, L. E.·Romero, A. C.·Hudson, G. A.·Tao, X. B.·Wu, B.·Petzold, C. J.·Keasling, J. D.

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Abstract

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at nearly 1500 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes. HighlightsO_LIDesign of a mevalonate-based monoterpene production platform in C. glutamicum C_LIO_LIHighest production titers of geranoids, eucalyptol, and linalool reported in C. glutamicum to date C_LIO_LIIdentification of citronellal as an intermediate in the reduction of geraniol to citronellol by C. glutamicum C_LI

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Luckie, B. A., Kashyap, M., Pearson, A. N., Chen, Y., Liu, Y., Valencia, L. E., Romero, A. C., Hudson, G. A., Tao, X. B., Wu, B., Petzold, C. J., Keasling, J. D.. 2023-11-02. Development of Corynebacterium glutamicum as a monoterpene production platform. https://doi.org/10.1101/2023.10.31.565027

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