Metabolic engineering of microorganisms for the production of higher alcohols

Abstract

Due to the increasing concerns about limited fossil resources and environmental problems, there has been much interest in developing biofuels from renewable biomass. Ethanol is currently used as a major biofuel, as it can be easily produced by existing fermentation technology, but it is not the best biofuel due to its low energy density, high vapor pressure, hygroscopy, and incompatibility with current infrastructure. Higher alcohols, including 1-propanol, 1-butanol, isobutanol, 2-methyl-1-butanol, and 3-methyl-1-butanol, which possess fuel properties more similar to those of petroleum-based fuel, have attracted particular interest as alternatives to ethanol. Since microorganisms isolated from nature do not allow production of these alcohols at high enough efficiencies, metabolic engineering has been employed to enhance their production. Here, we review recent advances in metabolic engineering of microorganisms for the production of higher alcohols.

FIG 1

Strategies for the production of linear, primary alcohols. (A) Production of 1-butanol and 1-hexanol through the native or reconstructed clostridial pathway. The dotted arrow adjacent to Bcd-EtfAB indicates the weak activity of the Bcd enzyme in microbes other than clostridia. (B) Production of long-chain primary alcohols. In contrast to short-chain alcohols, long-chain fatty alcohols can be produced via various routes. The blue box in the reverse β-oxidation indicates the essential genetic manipulation to activate this pathway in the presence of glucose. The crp* gene encodes the mutant catabolite repressor protein for catabolite derepression. Points to be considered for further engineering in enzymatic and cellular levels are indicated in cyan and green boxes, respectively. For each reaction, the names of the corresponding enzymes used in the metabolic engineering studies are shown. The source of the enzyme was noted together with the enzyme, except for E. coli. The abbreviations of the species are as follows: Ac, Acinetobacter calcoaceticus; Aca, Aeromonas caviae; Ca, Clostridium acetobutylicum; Ch, Cuphea hookeriana; Ma, Marinobacter aqualeolei; Mm, Mus musculus; Mma, Mycobacterium marinum; Re, Ralstonia eutropha; Se, S. elongatus; Sp, Synechocystis sp. PCC 6803; Td, Treponema denticola; Uc, Umbellularia californica. See the main text for the abbreviations of the enzymes.

FIG 2

Production of branched-chain and secondary alcohols. Higher alcohols are shown in the red boxes, and the 2-ketoacid precursors are indicated in red text. The reactions in the isopropanol production pathway are shown with orange arrows. As in Fig. 1, points to be considered are indicated in cyan and green boxes. The source of the enzyme is noted together with the enzyme except E. coli, and follows that in Fig. 1. Additional abbreviations of the species are Ll, Lactococcus lactis; Sc, Saccharomyces cerevisiae. The enzymes shown are as follows: AckA, acetate kinase A and propionate kinase II; AdhE^mut, aerobically functional alcohol dehydrogenase; IlvA, threonine dehydratase; IlvC, ketol-acid reductoisomerase; IlvD, dihydroxyacid dehydratase; IlvE, branched-chain amino-acid aminotransferase; IlvIH, acetolactate synthase I; IlvBN, acetolactate synthase III complex; KivD, 2-ketoacid decarboxylase; LeuA, 2-isopropylmalate synthase; LeuB, 3-isopropylmalate dehydrogenase; LeuCD, 3-isopropylmalate isomerase complex; YqhD, NADPH-dependent aldehyde reductase; AtoB and Thl, acetyl-CoA acetyltransferase; AtoDA, acetyl-CoA:acetoacetyl-CoA synthase; CtfAB, CoA transferase; Adc, acetoacetate decarboxylase; Adh_B-593, primary/secondary alcohol dehydrogenase from C. beijerinckii B-593.