Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli

Abstract

Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we took advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from Methanococcus jannaschii. This new pathway, which directly converts pyruvate to 2-ketobutyrate, bypasses threonine biosynthesis and represents the shortest keto acid-mediated pathway for producing 1-propanol and 1-butanol from glucose. Directed evolution of CimA enhanced the specific activity over a wide temperature range (30 to 70 degrees C). The best CimA variant was found to be insensitive to feedback inhibition by isoleucine in addition to the improved activity. This CimA variant enabled 9- and 22-fold higher production levels of 1-propanol and 1-butanol, respectively, compared to the strain expressing the wild-type CimA. This work demonstrates (i) the first production of 1-propanol and 1-butanol using the citramalate pathway and (ii) the benefit of the 2-keto acid pathway that enables a growth-based evolutionary strategy to improve the production of non-growth-related products.

FIG. 1.

Schematic representation of the pathway for 1-propanol and 1-butanol production. The engineered citramalate pathway consists of four enzymatic steps from pyruvate to 2-ketobutyrate.

FIG. 2.

Transfer of the citramalate pathway to E. coli. (A) Schematic representation of the synthetic operons. (B) Time courses for the growth of E. coli strain SA405 (ΔilvA ΔtdcB) and JCL16. OD₆₀₀, optical density at 600 nm. Cells were incubated in M9 medium containing glucose at 30°C. Diamonds, JCL16; circles, squares, and triangles, SA405 with pSA63 (circles), pCS27 (without cimA-leuABCD) (squares), or l-isoleucine (39.5 μg/ml) (triangles).

FIG. 3.

Progress of the evolution of CimA. (A) Amino acid mutations are shown in the schematic representation of CimA. The gray bar indicates the putative regulator domain. (B) Time courses for the growth of an E. coli strain (SA408 [ΔilvA ΔtdcB ΔilvI]) containing the derivative of pSA63 (cimA-leuABCD). Cells were incubated in M9 medium containing glucose at 30°C. Circles, wild-type (WT) CimA; triangles, CimA2; diamonds, CimA3.7; and squares, control (blank plasmid).

FIG. 4.

Enzyme assays. (A) Specific activities (M CoA produced/min/M protein) of the wild type (WT) (squares) and CimA3.7 (circles) at various temperatures. (B) Specific activities of the wild type and CimA3.7 at 30°C in the presence of various concentrations of l-isoleucine.

FIG. 5.

1-Propanol and 1-butanol production with the citramalate pathway. (Left panel) 1-Propanol production. (Right panel) 1-Butanol production in the same strain. The host is KS145, and overexpressed genes are indicated below the axis. Cultures were grown at 30°C in M9 medium containing 5 g/liter yeast extract with or without 72 g/liter glucose for 40 h.

FIG. 6.

1-Propanol and 1-butanol production with CimA3.7. Time profiles of cell growth with IPTG (squares) and without IPTG (open circles) (A); 1-propanol (B), 1-butanol (C), and ethanol (D) production; glucose consumption (E); and organic acid production (acetate [diamonds], lactate [circles], formate [triangles]) (F) from KS145/pSA55/pSA142 (containing cimA3.7). Cultures were grown at 30°C in M9 medium containing 72 g/liter glucose and 5 g/liter yeast extract. OD600, optical density at 600 nm.

FIG. 7.

Sequence analysis of CimA3.7. (A) Structure of Mycobacterium tuberculosis LeuA. The residues in the active site and the bound 2-ketoisovalerate are colored blue and orange, respectively. The image on the left contains the regulator domain, while the image on the right does not. The residues corresponding to mutations in CimA3.7 are colored red. (B) Amino acid sequence alignment of CimA (M. jannaschii), LeuA (E. coli), and LeuA (M. tuberculosis). Multiple sequence alignment was carried out using ClustalW (17). Fully conserved residues are shaded. The residues in the active site are shown with asterisks. Residue mutations in CimA3.7 are labeled in red. Gaps in the sequence are shown with dashes.