(188bc) Photocatalytic Production of the Jet Fuel Limonene in Synechococcus Sp. PCC 7002

In response to increasing global temperatures and rising levels of greenhouse gasses in the atmosphere, metabolic engineering efforts to develop sustainable fuel and chemical sources have increased, bringing with them a particular interest for cyanobacterial platforms. These photosynthetic bacteria are particularly promising because they are capable of directly producing high-value chemical products from naturally abundant CO₂ and do not require an expensive carbohydrate feedstock for large-scale production.

Terpenoids are a class of chemicals that have been identified as promising for the use of renewable drop-in biofuels, due to their high energy density and structural diversity that allows them to mimic various classes of gasoline, diesel, and jet fuels. The properties of the monocyclic terpenoid limonene (C₁₀H₁₆) make it suitable as a precursor for next generation jet fuels, as well as a fuel additive to enhance cold-weather performance. Limonene is currently extracted from the peels of citrus fruit as a byproduct of juice processing, a production method that is energy-intensive and subject to volatile pricing fluctuations from seasonal farming yields.

In particular, the cyanobacterium Synechococcus sp. PCC 7002 is considered a model organism for such biotechnological applications due to its rapid growth and ability to survive in salt water, as well as varying temperature, nutrient and light irradiation conditions. We have therefore engineered Synechococcus for the photosynthetic production of limonene from atmospherically-harmful CO₂. Heterologous expression of the L-limonene synthase gene from the Mentha spicata plant (commonly, Spearmint) has imbued our base strain with the ability to produce 4 mg limonene per L cell culture, the highest reported limonene yield among photosynthetic engineered organisms with comparable genetic manipulation. To address the goal of logically engineering a commercially competitive strain, it is imperative to first identify metabolic bottlenecks and competing side reactions that limit limonene production.

We will discuss our rationale engineering strategy to further increase limonene titers, specifically focusing on results from isotope assisted metabolic flux analysis (¹³C-MFA) used to measure intracellular carbon fluxes. In addition, we will examine how metabolic modeling provides useful tools for predicting the most impactful target pathways for genetic engineering. Finally, we will assess the methylerythritol phosphate (MEP) pathway and its unique features in cyanobacteria.

(This material is supported by the National Science Foundation Energy for Sustainability Program. Grant #1604691.)