Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli

Abstract

1-Butanol, an important chemical feedstock and advanced biofuel, is produced by Clostridium species. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway in Escherichia coli to provide an irreversible reaction catalyzed by trans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

Fig. 1.

Synthetic build-up of NADH and acetyl-CoA (shown on the left) in the 1-butanol production system. Shown on the right is the 1-butanol production pathway engineered in E. coli from C. acetobutylicum (boxed). Acetyl-CoA initiates the NADH-consuming reactions. A total of four NADH molecules is needed to make one 1-butanol molecule. Enzymes that utilize NADH directly as the electron donor (Ter) are coupled more tightly to the NADH driving force than the enzymes that require additional electron transfer mediators (Bcd). Elimination of Pta, involved in acetate production, not only increased the pools of available acetyl-CoA but also reduced ATP synthesis, both of which can be used as driving forces to increase 1-butanol production. SUC, succinate; PEP, phosphoenolpyruvate; LAC, lactate; ETF, electron transfer flavoprotein; Fd, ferrodoxin. CB, C. boidinii; CA, C. acetobutylicum; EC, E. coli.

Fig. 2.

(a) Comparison of anaerobic 1-butanol production using different Ter homologues and mutants. Production level with C. acetobutylicum Bcd-EtfAB is also shown. Successful Ter mutants (dashed box) as a result of first-round growth selection and their corresponding 1-butanol titers are shown, with point mutations specified in the table at the bottom. Host strain JCL166 carrying plasmid pEL11 harboring the artificial operon atoB-adhE2-crt-hbd, along with a second plasmid containing Bcd-EtfAB (pIM11) or various Ter homologous and mutants, was used in all production assays. The extent of anaerobic growth rescue of each strain is indicated in the top panel, where one “+” refers to an OD₆₀₀ of around 0.2 to 0.3 in liquid cultures. It is important to note that anaerobic growth rescue and 1-butanol production procedures were performed in separate experiments under different culturing conditions (Materials and Methods). CA, Clostridium acetobutylicum; TD, Treponema denticola; TV, Treponema vincentii; FJ, Flavobacterium johnsoniae; FS, Fibrobacter succinogenes; NA, not applicable. JCL166, BW ΔldhA ΔadhE ΔfrdBC. (b) Effect of aeration level on 1-butanol production. Fermentations of strain JCL166 harboring plasmids pEL11 and pIM8 were performed under two different oxygen conditions, as indicated. Detailed procedures for each condition are described in Materials and Methods. Samples were taken after 24 h. Cell densities are listed on the right y axis. JCL166, BW ΔldhA ΔadhE ΔfrdBC. (c) Effect of pH adjustment on anaerobic 1-butanol production. Fermentations of strain JCL166 harboring plasmids pEL11 and pIM8 were performed with or without pH adjustments. “Time” indicates time since inoculation. JCL166, BW ΔldhA ΔadhE ΔfrdBC. Error bars show standard deviations. L, liter.

Fig. 3.

(a) Effects of Fdh overexpression and Pta deletion on anaerobic 1-butanol production. Time courses of alcohol production, cell growth, and glucose consumption are shown. A much higher yield and productivity of 1-butanol was achieved in JCL299 than in JCL166. It is important to note that other components present in the TB medium (such as yeast extracts) also contributed slightly to the 1-butanol titer, therefore affecting the yield. Solid black lines (Δldh Δadh Δfrd Δpta/Fdh⁺) refer to JCL299 transformed with plasmids pEL11, pIM8, and pCS138. Solid gray lines (Δldh Δadh Δfrd/Fdh⁺) represent JCL166 transformed with plasmids pEL11, pIM8, and pCS138. Dashed gray lines (Δldh Δadh Δfrd/Fdh⁻) refer to JCL166 transformed with plasmids pEL11 and pIM8. “Time” indicates time since inoculation. JCL166, BW ΔldhA ΔadhE ΔfrdBC; JCL299, BW ΔldhA ΔadhE ΔfrdBC Δpta. (b) Comparison of intracellular NADH levels and anaerobic 1-butanol production titers in the wild type and the engineered strains. All strains contained plasmids pEL11 and pIM8. Strains indicated as “Fdh over-expressed” also carry plasmid pCS138. A concentration of 20 mM formate was fed to the culture at the time of anaerobic switch where noted. The intracellular NADH level was measured using crude extracts prepared from the production culture after 24 h of fermentation. JCL16, wild type; JCL166, BW ΔldhA ΔadhE ΔfrdBC; JCL299, BW ΔldhA ΔadhE ΔfrdBC Δpta. (c) Medium analysis for anaerobic 1-butanol production. Fermentations of strain JCL299 harboring plasmids pEL11, pIM8, and pCS138 were performed in different medium compositions as indicated on the x axis (“−” indicates the absence of the particular component). The contribution of every element in the TB medium to 1-butanol production was analyzed by the subtraction of each component one by one (Glc, glucose; YE, yeast extract). Samples were taken after 30 h of fermentation. Cell densities are listed on the right y axis. Error bars show standard deviations. L, liter.

Fig. 4.

Stoichiometric balances for 1-butanol synthesis.

Fig. 5.

(a) Multiple sequence alignments of Ter homologues from various organisms using ClustalW. The M11K amino acid substitution found in the F. succinogenes Ter mutants is shaded. Fully conserved residues are noted with asterisks. TD, T. denticola; TV, T. vincentii; FS, F. succinogenes; FJ, F. johnsoniae. (b) Anaerobic 1-butanol production in the pH-controlled fed batch fermentor with gas-stripping using either the T. denticola (Tde) Ter or the F. succinogenes (Fsu) Ter M11K mutant. Strain JCL299 was transformed with plasmids pEL11 and pCS138, in addition to plasmid pIM8 carrying the T. denticola Ter or plasmid pHJ6 carrying the F. succinogenes Ter mutant. Similar performances were observed in both cases in terms of 1-butanol titer (30 g/liter), productivity (0.18 g/liter/h), and cumulative yield (around 70% of the theoretical). “Time” indicates time since inoculation. JCL299, BW ΔldhA ΔadhE ΔfrdBC Δpta. L, liter.