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Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain.

ABSTRACT: Fermentation of glucose to D-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growing on M9 minimal medium. After the ECOM3 strain was subjected to adaptive evolution on glucose M9 medium for 60 days, a growth rate equivalent to that of anaerobic wild-type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 populations acquired different phenotypes: one produced lactate as a sole fermentation product, while the other two strains exhibited a mixed-acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed a D-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analyses were employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN and sodAB explains the remaining oxygen uptake that was observed in evolved ECOM3 strains. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.

SUBMITTER: Portnoy VA

PROVIDER: S-EPMC2607145 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain.

Portnoy Vasiliy A VA Herrgård Markus J MJ Palsson Bernhard Ø BØ

Applied and environmental microbiology 20081024 24

Fermentation of glucose to D-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growi ...[more]

PMID: 18952873

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Project description:The R481 residue of cytochrome bo(3) ubiquinol oxidase from E. coli is highly conserved in the heme-copper oxidase superfamily. It has been postulated to serve as part of a proton loading site that regulates proton translocation across the protein matrix of the enzyme. Along these lines, proton pumping efficiency has been demonstrated to be abolished in many R481 mutants. However, R481Q in bo(3) from E. coli has been shown to be fully functional, implying that the positive charge of the arginine is not required for proton translocation [ Puustinen , A. and Wikstrom , M. ( 1999 ) Proc. Natl. Acad. Sci. U.S.A. 96 , 35 - 37 ]. In an effort to delineate the structural role of R481 in the bo(3) oxidase, we used resonance Raman spectroscopy to compare the nonfunctional R481L mutant and the functional R481Q mutant, to the wild type protein. Resonance Raman data of the oxidized and reduced forms of the R481L mutant indicate that the mutation introduces changes to the heme o(3) coordination state, reflecting a change in position and/or coordination of the Cu(B) located on the distal side of heme o(3), although it is approximately 10 A away from R481. In the reduced-CO adduct of R481L, the frequencies of the Fe-CO and C-O stretching modes indicate that, unlike the wild type protein, the Cu(B) is no longer close to the heme-bound CO. In contrast, resonance Raman data obtained from the various oxidation and ligation states of the R481Q mutant are similar to those of the wild type protein, except that the mutation causes an enhancement of the relative intensity of the beta conformer of the CO-adduct, indicating a shift in the equilibrium between the alpha and beta conformers. The current findings, together with crystallographic structural data of heme-copper oxidases, indicate that R481 plays a keystone role in stabilizing the functional structure of the Cu(B) site through a hydrogen bonding network involving ordered water molecules. The implications of these data on the proton translocation mechanism are considered.

Dataset Information

Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain.

Publications

Aerobic fermentation of D-glucose by an evolved cytochrome oxidase-deficient Escherichia coli strain.

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