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.

1,3-Propanediol (1,3-PDO) is a bifunctional molecule, and used in applications similar to those of ethylene glycol, propylene glycol, 1,3-butanediol and 1,4-butanediol. The use of glycerol as a feedstock is an alternative to reduce production costs for both 1,3-PDO and biodiesel, since biodiesel glycerol can be used for the production of 1,3-PDO by bacteria. Also, using metabolic engineering, it is possible to manipulate the metabolic routes and obtain high value products, reduce or eliminate the formation of undesirable byproducts. The aim of the study was to produce 1,3-propanediol in E. coli cloned with dha genes from Klebsiella pneumoniae GLC29. Six genes responsible for 1,3-PDO production in Klebsiella pneumoniae GLC29 were cloned. These genes were assembled in pSB1C3 as an expression vector: Genes dhaB1, dhaB2, dhaB3 and dhaT (pSB1C3dhaB123T), and another vector with genes dhaF and dhaG (pSB1C3dhaB123TFG) were derived using Gibson's Assembly technique. Escherichia coli TCS099 and SZ63 stains were used as hosts for 1,3-PDO production, and kept at -80°C for long-term storage. Glycerol was used as the sole or main carbon source in all experiments. Fermentations were performed in flasks in aerobic and anaerobic conditions using minimal media. Also, two stage fermentation (aerobic-anaerobic) was performed for 1,3-propanediol production. Only pSB1C3dhaB123TFG was able to produce high amounts of 1,3-PDO in shake flasks experiments, producing 2.5 g/L in micro-aerobic conditions, using E. coli TCS099 as host. Besides, E. coli SZ63 hosting pSB1C3dhaB123TFG was able to produce high amounts of 1,3-PDO, corresponding to 11.3 g/L of 1,3-PDO using a two-stage fermentation process using low concentration of vitamin B12 (1 mg/L). Plasmid pSB1C3dhaB123TFG shows potential for producing high amounts of 1,3-PDO, specially because of dhaF and dhaG, reaffirming the importance of this genes on 1,3-PDO production, especially with the addition of low amounts of vitamin B12, which is an expensive compound.