Azotobacter vinelandii as a Nitrogen-Negative Chassis for Bio-Oil and Bio-Wax Production of Heterologous and Native Lipids

The biosynthetic production of energy-dense petrochemical substitutes is an important goal to address sustainability. The diazotrophic soil microbe Azotobacter vinelandii is a model microbe for the study of biological nitrogen fixation. In addition to capturing atmospheric nitrogen and converting it into usable nitrogen compounds, it is also regarded for the ability to accumulate the bioplastic poly-β-hydroxybutyrate and the extracellular polysaccharide alginate. Here, we demonstrate the potential to broaden the chemical products repertoire of A. vinelandii by demonstrating the accumulation of several classes of biological lipids and waxes. These products include the expanded accumulation of wax esters and fatty alcohols through heterologous expression of foreign genes and pathways, and increased production of the native lipid alkylresorcinol, accomplished by deregulating specific internal pathways and removing competitive pathways for alternative products. As a result, we demonstrate a sevenfold increase in the accumulation of alkylresorcinol, manifesting as intracellular inclusions that are easily extracted with simple solvents and account for nearly 20% of the cellular biomass. By selecting a diazotrophic microbe as a chassis for lipid accumulation, we produced these lipids without any requirement for industrial nitrogen sources in the growth medium, resulting in a net positive nitrogen process as well.