Engineered respiro-fermentative metabolism for the production of biofuels and biochemicals from fatty acid-rich feedstocks

Abstract

Although lignocellulosic sugars have been proposed as the primary feedstock for the biological production of renewable fuels and chemicals, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. In addition to their abundance, the metabolism of FAs is very efficient and could support product yields significantly higher than those obtained from lignocellulosic sugars. However, FAs are metabolized only under respiratory conditions, a metabolic mode that does not support the synthesis of fermentation products. In the work reported here we engineered several native and heterologous fermentative pathways to function in Escherichia coli under aerobic conditions, thus creating a respiro-fermentative metabolic mode that enables the efficient synthesis of fuels and chemicals from FAs. Representative biofuels (ethanol and butanol) and biochemicals (acetate, acetone, isopropanol, succinate, and propionate) were chosen as target products to illustrate the feasibility of the proposed platform. The yields of ethanol, acetate, and acetone in the engineered strains exceeded those reported in the literature for their production from sugars, and in the cases of ethanol and acetate they also surpassed the maximum theoretical values that can be achieved from lignocellulosic sugars. Butanol was produced at yields and titers that were between 2- and 3-fold higher than those reported for its production from sugars in previously engineered microorganisms. Moreover, our work demonstrates production of propionate, a compound previously thought to be synthesized only by propionibacteria, in E. coli. Finally, the synthesis of isopropanol and succinate was also demonstrated. The work reported here represents the first effort toward engineering microorganisms for the conversion of FAs to the aforementioned products.

FIG. 1.

Pathways engineered in E. coli for the conversion of fatty acids to fuels (red) and chemicals (green). Also shown is the catabolism of fatty acids via the β-oxidation pathway (orange) and of glucose through the Embden-Meyerhof-Parnas pathway (blue). Relevant reactions are represented by the names of the genes coding for the enzymes (E. coli genes unless otherwise specified in parentheses as follows: C. acetobutylicum, ca; C. beijerinckii, cb): aceA, isocitrate lyase; aceB, malate synthase A; adc, acetoacetate decarboxylase (ca); ackA, acetate kinase; adh, secondary alcohol dehydrogenase (cb); adhE, acetaldehyde/alcohol dehydrogenase; adhE2, secondary alcohol dehydrogenase (ca); atoA and atoD, acetyl-CoA:acetoacetyl-CoA transferase; atoB, acetyl-CoA acetyltransferase; bcd, butyryl-CoA dehydrogenase (ca); crt, crotonase (ca); etfAB, electron transfer flavoprotein (ca); fadA, 3-ketoacyl-CoA thiolase; fadB, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; fadD, acyl-CoA synthetase; fadE, acyl-CoA dehydrogenase; hbd, β-hydroxybutyryl-CoA dehydrogenase (ca); icd, isocitrate dehydrogenase; pta, phosphate acetyltransferase; sdhABCD, succinate dehydrogenase; scpA, methylmalonyl-CoA mutase; scpB, methylmalonyl-CoA decarboxylase; scpC, propionyl-CoA:succinate CoA transferase; sucA, 2-oxoglutarate dehydrogenase; sucB, dihydrolipoyltranssuccinylase; and sucCD, succinyl-CoA synthetase. Abbreviations: 2[H] = NADH = FADH₂ = QH₂ = H₂; P/O, amount of ATP produced per oxygen consumed in the oxidative phosphorylation.

FIG. 2.

Engineering E. coli for the production of ethanol (a and b) and butanol (c and d) from fatty acids. Values represent the means and standard deviations for triplicate cultures. Gene overexpression and deletion are indicated by + and Δ, respectively, next to the corresponding gene or operon. Details about the pathways can be found in Fig. 1. (a) Ethanol concentration (line) and ethanol and caproic acid yields (bars) for 72-hour cultures of wild-type MG1655 and strains containing engineered pathways for ethanol production (oxygen-tolerant acetaldehyde/alcohol dehydrogenase, adhE*) and efficient catabolism of FAs [fadR* atoC(Con)]. k_La, volumetric oxygen transfer coefficient (h⁻¹). (b) Fermentation profile for strain MG1655 ΔadhE fadR*atoC(Con) [adhE*+] in minimal medium with 5 g/liter palmitic acid (C_16:0) and using a k_La of 6.7 h⁻¹. Additions of palmitic acid (5 g/liter each) were made every 24 h. ΔCDW, increase in cell dry weight with respect to the initial value. (c) Butanol concentration (line) and butanol, ethanol, and caproic acid yields (bars) for 72-hour cultures of strains engineered by (i) expression of the C. acetobutylicum pathway for the synthesis of butanol from acetoacetyl-CoA (hbd, crt, bcd, etfAB, and adhE2), (ii) overexpression of the E. coli acetyl-CoA acetyltransferase (atoB) for the conversion of acetyl-CoA to acetoacetyl-CoA, (iii) elimination of the E. coli native ethanol pathway (adhE), and (iv) engineering the β-oxidation pathway for efficient catabolism of FAs [fadR*atoC(Con)]. A k_La of 6.7 h⁻¹ was used. (d) Fermentation profile for strain MG1655 ΔadhE fadR*atoC(Con) [atoB+ hbd+ crt+ bcd+ etfAB+ adhE2+] in minimal medium with 5 g/liter palmitic acid (C_16:0) and a k_La of 6.7 h⁻¹. Two additions of palmitic acid (5 g/liter each) were made at 24 and 72 h. ΔCDW, increase in cell dry weight with respect to initial value.

FIG. 3.

Production of acetate (a and b), acetone (c), isopropanol (d), succinate (e), and propionate (f) from FAs in engineered E. coli strains. Details about the engineered pathways are shown in Fig. 1. Gene overexpression and deletion are indicated by + and Δ, respectively, next to the corresponding gene or operon. A k_La of 14.5 h⁻¹ was used in all experiments. Values represent the means and standard deviations for 72-hour triplicate cultures. (a) Acetate concentration (line) and acetate and caproic acid yields (bars) in wild-type MG1655 and recombinant strains constructed by overexpressing the native phopshoacetyltransferase (pta)-acetate kinase (ackA) pathway for conversion of acetyl-CoA to acetate and modifying the β-oxidation pathway for efficient catabolism of FAs [fadR*atoC(Con)]. (b) Fermentation profile for strain MG1655 fadR*atoC(Con) [ackA+ pta+] in minimal medium with 5 g/liter palmitic acid (C_16:0). Additions of palmitic acid (5 g/liter each) were made every 12 h. ΔCDW, increase in cell dry weight with respect to initial value. (c) Acetone and caproic acid yields in strains constructed by overexpressing E. coli acetyl-CoA and acetoacetyl-CoA transferases (atoDAB) and C. acetobutylicum acetoacetate decarboxylase (adc) along with amplification of the native acetate pathway (ackA-pta). These modifications were implemented in wild-type MG1655 and the fadR*atoC(Con) derivative. (d) Isopropanol and caproic acid yields in strains engineered for acetone production and overexpressing a secondary alcohol dehydrogenase from C. butyricum (adh), which converts acetone to isopropanol. (e) Effects of fadR*atoC(Con) genotype and deletions of genes encoding TCA cycle enzymes on succinate and caproic acid yields. (f) Propionate and caproic acid yields in strains overexpressing a metabolic cycle that catalyzes the decarboxylation of succinate to propionate (scpA, scpB, scpC). The effect of amplification of this cycle, along with the deletion of succinate dehydrogenase (ΔsdhB), was evaluated in wild-type MG1655 and the fadR*atoC(Con) derivative.