Premium
Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrations
Author(s) -
Baez Antonino,
Flores Noemí,
Bolívar Francisco,
Ramírez Octavio T.
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22379
Subject(s) - glyoxylate cycle , escherichia coli , citric acid cycle , chemistry , biochemistry , biology , microbiology and biotechnology , decarboxylation , gene , metabolism , catalysis
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 ( µ ) 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 µ , 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. Biotechnol. Bioeng. 2009; 104: 102–110 © 2009 Wiley Periodicals, Inc.