Product specificity is a key advantage of biochemical fuel production, enabling synthesis of bio-advantaged fuels and blendstocks with highly tunable production and combustion characteristics via precise manipulation of chemical structure. Historically, production of such bio-advantaged fuels was constrained by the limited number of tractable, high-flux metabolic pathways to reduced fuel molecules. Recent advances in synthetic biology have greatly increased the accessibility of new pathways to metabolic engineering, enabling production of an array of new structures, previously unreachable via chemical synthesis pathways. To illustrate the potential of this bio-advantaged approach, we demonstrate production and characterization of fuel molecules across two key metabolic classes: isoprenoid-derived products and polyketide synthase (PKS)-derived products. Unsaturated C5 isoprenoid alcohols have potential as high-octane blendstocks, but are challenged by production characteristics including product toxicity, volatility, and high solubility in aqueous media. We have overcome these limitations via manipulation of production conditions and product targets, resulting in production and in situ recovery of C5 alcohols at high titer, rate and yield. Canonical isoprenoid-derived molecules are synthesized in C5 units, restricting metabolic flexibility - moving forward, utilization of promiscuous enzymes for production of non-canonical C6 and C7 isoprenoid units enables synthesis of previously uncharacterized molecules with improved energy density and water solubility, including C6 and C7 isoprenoid alcohols. Biological production of diesel-range fuels has traditionally relied on straight-chain, fatty-acid derived fuels, resulting in undesirably high melting points. Utilization of PKS pathways eliminates this challenge by allowing highly controlled branching and functionalization. To demonstrate the viability of this approach, we have developed a system for conversion of biofuels to branched, C5-C7 ketones at titers exceeding 1 g/L. Building on this platform, engineering iterative PKS modules enables production of highly branched fuels with cetane exceeding 40 and predicted melting points below -40°C, as well as poly-cycloproponated molecules featuring up to 8 iterative cyclopropane units, with predicted energy densities above 47 MJ/L.

Towards bio-advantaged fuels: Leveraging new biosynthetic pathways for design of next-generation fuels and blendstocks

ACS Fall 2020 Virtual Meeting

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