Transcriptional and metabolic response of recombinant Escherichia coli to spatial dissolved oxygen tension gradients simulated in a scale-down system.
Escherichia coli, expressing recombinant green fluorescent protein (GFP), was subjected to dissolved oxygen tension (DOT) oscillations in a two-compartment system for simulating gradients that can occur in large-scale bioreactors. Cells were continuously circulated between the anaerobic (0% DOT) and aerobic (10% DOT) vessels of the scale-down system to mimic an overall circulation time of 50 s, and a mean residence time in the anaerobic and aerobic compartments of 33 and 17 s, respectively. Transcription levels of mixed acid fermentation genes (ldhA, poxB, frdD, ackA, adhE, pflD, and fdhF), measured by quantitative RT-PCR, increased between 1.5- to over 6-fold under oscillatory DOT compared to aerobic cultures (constant 10% DOT). In addition, the transcription level of fumB increased whereas it decreased for sucA and sucB, suggesting that the tricarboxylic acid cycle was functioning as two open branches. Gene transcription levels revealed that cytrochrome bd, which has higher affinity to oxygen but lower energy efficiency, was preferred over cytochrome bO3 in oscillatory DOT cultures. Post-transcriptional processing limited heterologous protein production in the scale-down system, as inferred from similar gfp transcription but 19% lower GFP concentration compared to aerobic cultures. Simulated DOT gradients also affected the transcription of genes of the glyoxylate shunt (aceA), of global regulators of aerobic and anaerobic metabolism (fnr, arcA, and arcB), and other relevant genes (luxS, sodA, fumA, and sdhB). Transcriptional changes explained the observed alterations in overall stoichiometric and kinetic parameters, and production of ethanol and organic acids. Differences in transcription levels between aerobic and anaerobic compartments were also observed, indicating that E. coli can respond very fast to intermittent DOT conditions. The transcriptional responses of E. coli to DOT gradients reported here are useful for establishing rational scale-up criteria and strain design strategies for improved culture performance at large scales.
Lara
Alvaro R
AR
Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico (UNAM), Mexico City, Mexico.
Leal
Lidia
L
Flores
Noemí
N
Gosset
Guillermo
G
Bolívar
Francisco
F
Ramírez
Octavio T
OT
D000332:Aerobiosis; D000693:Anaerobiosis; D019149:Bioreactors / Q000382:microbiology*; D002952:Citric Acid Cycle / Q000235:genetics; D003580:Cytochromes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005285:Fermentation; D006038:Glyoxylates / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D010100:Oxygen / Q000378:metabolism*; D014158:Transcription, Genetic
D003580:Cytochromes; D029968:Escherichia coli Proteins; D006038:Glyoxylates; D049452:Green Fluorescent Proteins; C031150:glyoxylic acid; D010100:Oxygen