Engineering an Escherichia coli platform to synthesize designer biodiesels

Abstract

Biodiesels, fatty acid esters (FAEs), can be synthesized by condensation of fatty acid acyl CoAs and alcohols via a wax ester synthase in living cells. Biodiesels have advantageous characteristics over petrodiesels such as biodegradability, a higher flash point, and less emission. Controlling fatty acid and alcohol moieties are critical to produce designer biodiesels with desirable physiochemical properties (e.g., high cetane number, low kinematic viscosity, high oxidative stability, and low cloud point). Here, we developed a flexible framework to engineer Escherichia coli cell factories to synthesize designer biodiesels directly from fermentable sugars. In this framework, we designed each FAE pathway as a biodiesel exchangeable production module consisting of acyl CoA, alcohol, and wax ester synthase submodules. By inserting the FAE modules in an engineered E. coli modular chassis cell, we generated E. coli cell factories to produce targeted biodiesels (e.g., fatty acid ethyl (FAEE) and isobutyl (FAIbE) esters) with tunable and controllable short-chain alcohol moieties. The engineered E. coli chassis carrying the FAIbE production module produced 54mg/L FAIbEs with high specificity, accounting for>90% of the total synthesized FAEs and ∼4.7 fold increase in FAIbE production compared to the wildtype. Fed-batch cultures further improved FAIbE production up to 165mg/L. By mixing ethanol and isobutanol submodules, we demonstrated controllable production of mixed FAEEs and FAIbEs. We envision the developed framework offers a flexible, alternative route to engineer designer biodiesels with tunable and controllable properties using biomass-derived fermentable sugars.

A Prototype for Modular Cell Engineering

“…Overall, these results validated the designed property that the modular cell is auxotrophic due to imbalance of redox and precursor metabolites and requires strong coupling with a production module for growth and efficient production of target chemicals. This strategy of strong coupling has been previously shown for production of butanol 42, 43 , isobutanol 44 , succinate 45 , short-chain esters 3, 4, 34, 35 , isopentenol 46 , itaconic acid 47 from glucose and ethanol from glycerol 48 . Since synthesis and regulation of redox and precursor metabolites are linked within cellular metabolism, any perturbation will affect redox state and precursor requirement for cell growth and maintenance 49 .…”

Harnessing a Novel P450 Fatty Acid Decarboxylase fromMacrococcus caseolyticusfor Microbial Biosynthesis of Odd Chain Terminal Alkenes

“…For FAs analysis, sample preparation and GC/MS methods were described previously (40). For terminal alkene analysis, 500 μL of samples (cells plus supernatants) were transferred to a 2 ml polypropylene microcentrifuge tube with a screw cap containing 100-200 mg of glass beads (0.25-0.30 mm in diameter), 60 μL of 6 N HCl, and 500 μL of ethyl acetate solution containing 1 mg/L of ethyl pentadecanoate as an internal standard.…”

Comparison of Multi-Objective Evolutionary Algorithms to Solve the Modular Cell Design Problem for Novel Biocatalysis

“…This modular cell design approach, known as ModCell, uses multi-objective optimization to account for the competing cellular objectives when cellular metabolism is (re)designed in a modular fashion to produce a diverse class of target chemicals. ModCell has been experimentally demonstrated for biosynthesis of alcohols [7,11,12] and esters [13][14][15][16][17] in Escherichia coli.…”