The path to next generation biofuels: successes and challenges in the era of synthetic biology

Abstract

Volatility of oil prices along with major concerns about climate change, oil supply security and depleting reserves have sparked renewed interest in the production of fuels from renewable resources. Recent advances in synthetic biology provide new tools for metabolic engineers to direct their strategies and construct optimal biocatalysts for the sustainable production of biofuels. Metabolic engineering and synthetic biology efforts entailing the engineering of native and de novo pathways for conversion of biomass constituents to short-chain alcohols and advanced biofuels are herewith reviewed. In the foreseeable future, formal integration of functional genomics and systems biology with synthetic biology and metabolic engineering will undoubtedly support the discovery, characterization, and engineering of new metabolic routes and more efficient microbial systems for the production of biofuels.

Figure 1

General bioprocess scheme for the production of fuels from renewable feedstocks. Different feedstocks are listed according to their environmental and economical sustainability. Feedstock deconstruction releases elementary building blocks such as pentoses, hexoses, polyols, fatty acids, etc. that are then microbially converted to biofuels.

Figure 2

Engineered pathways for microbial production of ethanol from carbohydrates. Orange, red and green boxes indicate pathways for pentose and hexose sugars utilization, and ethanol synthesis respectively. The dashed lines indicate multiple steps. Abbreviations: ADH, alcohol dehydrogenase; AR, aldose reductase; ARAA, L-arabinose isomerase; ARAB, L-ribulokinase; ARAD, L-ribulosephosphate 4-epimerase; FDH, formate dehydrogenase; FHL, formate hydrogen lyase; LAD, L-arabitol 4-dehydrogenase; LXR1, L-xylulose reductase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate lyase; XDH, xylitol dehydrogenase; XR, xylose reductase; XYLA, xylose isomerase; XYLB, xylulokinase.

Figure 3

Current metabolic engineering efforts for 1-butanol production. Heterologous expression of clostridial genes in S. cerevisiae and E. coli. Gene names in red indicate steps engineered for butanol biosynthesis. Source of the genes is reported in parenthesis. c.a., Clostridium acetobutylicum; c.b., Clostridium beijerinckii; e.c., Escherichia coli; s.c., Saccharomyces cerevisiae; r.e., Ralstonia eutropha; s.c., Streptomyces collinus. || indicates gene knock-outs. Gene/enzyme names: adh, adhE1, adhE2, alcohol dehydrogenase;bcd-etfAB, butyryl-CoA dehydrogenase;ccr, butyryl-CoA dehydrogenase;crt, crotonase; erg10, thiolase; fdh1, formate dehydrogenase; phaA, thiolase; phaB, acetoacetyl-Coa reductase; thl, thiolase.

Figure 4

Synthetic pathways for advanced biofuels. Fuels generated by the keto-acid mediated pathway are shown in red. Isoprenoids biosynthesis including the mevalonate and methylerythritol pathways is shown in blue. Fatty acid pathway for fatty acids ethyl esters (FAEE), fatty alcohols and long-chain alkanes/alkenes is reported in green. The dashed lines indicate multiple steps. Fuel products are shown in boxes. Abbreviations: ACP, acyl carrier protein; CoA, Coenzyme A; DMAPP, dimethylallyl pyrophosphate; FAEE, fatty acids ethyl esters; FPP, farnesyl diphosphate; Gly-3P, glyceraldehydes-3Phosphate; GPP, geranyl pyrophosphate; GGPP, geranyl-geranyl pyrophosphate; IPP, isopentenyl pyrophosphate; PEP, posphoenolpyruvate.

Figure 5

Engineered pathways for microbial production of biofuels from bio-oil constituents glycerol and fatty acids. Glycerol dissimilation pathway is shown in red. Fatty acids catabolism is shown in blue. The dashed line indicates multiple steps. || indicates gene knock-outs. Fuels are reported in red boxes. Genes in parenthesis indicate clostridial synthetic steps engineered in E. coli for butanol production. 2 [H] = NAD(P)H = FADH₂ = H₂. Gene/enzyme names: adhE, adhE1, adhE2, alcohol dehydrogenase; atoB, thiolase;dhaKLM, dihydroxyacetone kinase; bcd-etfAB, butyryl-CoA dehydrogenase; fadA, 3-ketoacyl-CoA thiolase; fadB, enoyl-CoA hydratase, 3-hydroxybutyryl-CoA epimerase; fadD, fatty acyl-CoA synthetase; fadE, acyl-CoA dehydrogenase; fdhF, formate dehydrogenase; frdABCD, fumarate reductase; gldA, glycerol dehydrogenase; hbd, β-hydroxybutyryl-CoA dehydrogenase; hycB-I, hydrogenase; ldhA, lactate dehydrogenase; lpdA-aceEF, pyruvate dehydrogenase; pflB, pyruvate formate lyase; pykF, pyruvate kinase; thil, thiolase. Abbreviations: DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; PEP, posphoenol pyruvate.