RESUMO
The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.
Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b , Citocromos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Hemoglobinas/metabolismo , Oxirredutases/metabolismo , Aerobiose , Proteínas de Bactérias/genética , Reatores Biológicos , Respiração Celular , Células Cultivadas , Clonagem Molecular , Grupo dos Citocromos d/genética , Grupo dos Citocromos d/metabolismo , Citocromos/genética , Metabolismo Energético , Escherichia coli/classificação , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Hemoglobinas/genética , Oxirredução , Oxigênio/metabolismo , Consumo de Oxigênio , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Especificidade da Espécie , Hemoglobinas TruncadasRESUMO
The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counter-balancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration.