title: Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrations.
abstract: The effect of dissolved carbon dioxide (dCO(2)) concentration on the stoichiometric and kinetic constants and by-product accumulation was determined for Escherichia coli cells producing recombinant green fluorescent protein (GFP). Constant dCO(2), in the range of 20-300 mbar, was maintained during batch cultures by manipulating the inlet gas composition. As dCO(2) increased, specific growth rate (micro) decreased, and acetate accumulation and the time for onset of GFP production increased. Maximum biomass yield on glucose and GFP concentration were affected for dCO(2) above 70 and 150 mbar, respectively. Expression analysis of 16 representative genes showed that E. coli can respond at the transcriptional level upon exposure to increasing dCO(2), and revealed possible mechanisms responsible for the detrimental effects of high dCO(2). Genes studied included those involved in decarboxylation reactions (aceF, icdA, lpdA, sucA, sucB), genes from pathways of production and consumption of acetate (ackA, poxB, acs, aceA, fadR), genes from gluconeogenic and anaplerotic metabolism (pckA, ppc), genes from the acid resistance (AR) systems (adiA, gadA, gadC), and the heterologous gene (gfp). The transcription levels of tricarboxylic acid (TCA) cycle genes (icdA, sucA, sucB) and glyoxylate shunt (aceA) decreased as dCO(2) increased, whereas fadR (that codes for a negative regulator of the glyoxylate operon) and poxB (that codes for PoxB which is involved in acetate production from pyruvate) were up-regulated as dCO(2) increased up to 150 mbar. Furthermore, transcription levels of genes from the AR systems increased as dCO(2) increased up to 150 mbar, indicating that elevated dCO(2) triggers an acid stress response in E. coli cells. Altogether, such results suggest that the increased acetate accumulation and reduction in mu, biomass yield and maximum GFP concentration under high dCO(2) resulted from a lower carbon flux to TCA cycle, the concomitant accumulation of acetyl-CoA or pyruvate, and the acidification of the cytoplasm.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, México.
authors.forename: Antonino
authors.initials: A
authors.lastname: Baez
authors.forename: Noemí
authors.initials: N
authors.lastname: Flores
authors.forename: Francisco
authors.initials: F
authors.lastname: Bolívar
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
mesh_terms: D000085:Acetates / Q000032:analysis; D018533:Biomass; D002245:Carbon Dioxide / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013312:Stress, Physiological
chemical_list: D000085:Acetates; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D049452:Green Fluorescent Proteins