Development of Corynebacterium glutamicum as a monoterpene production platform

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 initial plasmid design iterations, we demonstrated an increased flux through the mevalonate-based bypass pathway to increase isoprenol production to the highest reported titers to date at nearly 1.5 g/L. These designs also evaluated the effects of backbone, promoter, and GPP synthase homolog source on 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 486.3 mg/L (± 54.6 mg/L) of geranoids, 557.3 mg/L of 1,8-cineole (± 12.3 mg/L), and 209.7 mg/L of linalool (±23.9 mg/L). Furthermore, we determined that C. glutamicum oxidizes geraniol through an aldehyde intermediate before asymmetrically reducing geraniol to citronellol.