Development of Corynebacterium glutamicum as a monoterpene production platform

Bridget A. Luckie, Meera Kashyap, Allison N. Pearson, Yan Chen, Yuzhong Liu, Luis E. Valencia, Alexander Carrillo Romero, Graham A. Hudson, Xavier B. Tao, Bryan Wu, Christopher J. Petzold, Jay D. Keasling*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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 1504.6 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.
Original languageEnglish
JournalMetabolic Engineering
Volume81
Pages (from-to)110-122
ISSN1096-7176
DOIs
Publication statusPublished - 2024

Keywords

  • Monoterpene biosynthetic production
  • Geraniol
  • Citronellol
  • Eucalyptol
  • Linalool
  • Citral
  • Citronellic acid
  • Corynebacterium glutamicum

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