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 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.

Improved polyketide production in C. glutamicum by preventing propionate-induced growth inhibition.

Corynebacterium glutamicum is a promising host for production of valuable polyketides. Propionate addition, a strategy known to increase polyketide production by increasing intracellular methylmalonyl-CoA availability, causes growth inhibition in C. glutamicum. The mechanism of this inhibition was unclear before our work. Here we provide evidence that accumulation of propionyl-CoA and methylmalonyl-CoA induces growth inhibition in C. glutamicum. We then show that growth inhibition can be relieved by introducing methylmalonyl-CoA-dependent polyketide synthases. With germicidin as an example, we used adaptive laboratory evolution to leverage the fitness advantage of polyketide production in the presence of propionate to evolve improved germicidin production. Whole-genome sequencing revealed mutations in germicidin synthase, which improved germicidin titer, as well as mutations in citrate synthase, which effectively evolved the native glyoxylate pathway to a new methylcitrate pathway. Together, our results show that C. glutamicum is a capable host for polyketide production and we can take advantage of propionate growth inhibition to drive titers higher using laboratory evolution or to screen for production of polyketides.

Vaccination with Recombinant Cryptococcus Proteins in Glucan Particles Protects Mice against Cryptococcosis in a Manner Dependent upon Mouse Strain and Cryptococcal Species.

Development of a vaccine to protect against cryptococcosis is a priority given the enormous global burden of disease in at-risk individuals. Using glucan particles (GPs) as a delivery system, we previously demonstrated that mice vaccinated with crude Cryptococcus-derived alkaline extracts were protected against lethal challenge with Cryptococcus neoformans and Cryptococcus gattii The goal of the present study was to identify protective protein antigens that could be used in a subunit vaccine. Using biased and unbiased approaches, six candidate antigens (Cda1, Cda2, Cda3, Fpd1, MP88, and Sod1) were selected, recombinantly expressed in Escherichia coli, purified, and loaded into GPs. Three mouse strains (C57BL/6, BALB/c, and DR4) were then vaccinated with the antigen-laden GPs, following which they received a pulmonary challenge with virulent C. neoformans and C. gattii strains. Four candidate vaccines (GP-Cda1, GP-Cda2, GP-Cda3, and GP-Sod1) afforded a significant survival advantage in at least one mouse model; some vaccine combinations provided added protection over that seen with either antigen alone. Vaccine-mediated protection against C. neoformans did not necessarily predict protection against C. gattii Vaccinated mice developed pulmonary inflammatory responses that effectively contained the infection; many surviving mice developed sterilizing immunity. Predicted T helper cell epitopes differed between mouse strains and in the degree to which they matched epitopes predicted in humans. Thus, we have discovered cryptococcal proteins that make promising candidate vaccine antigens. Protection varied depending on the mouse strain and cryptococcal species, suggesting that a successful human subunit vaccine will need to contain multiple antigens, including ones that are species specific.IMPORTANCE The encapsulated fungi Cryptococcus neoformans and Cryptococcus gattii are responsible for nearly 200,000 deaths annually, mostly in immunocompromised individuals. An effective vaccine could substantially reduce the burden of cryptococcosis. However, a major gap in cryptococcal vaccine development has been the discovery of protective antigens to use in vaccines. Here, six cryptococcal proteins with potential as vaccine antigens were expressed recombinantly and purified. Mice were then vaccinated with glucan particle preparations containing each antigen. Of the six candidate vaccines, four protected mice from a lethal cryptococcal challenge. However, the degree of protection varied as a function of mouse strain and cryptococcal species. These preclinical studies identify cryptococcal proteins that could serve as candidate vaccine antigens and provide a proof of principle regarding the feasibility of protein antigen-based vaccines to protect against cryptococcosis.