title: Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli.
abstract: Guanosine-3',5'-tetraphosphate (ppGpp) and sigmaS, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.
authors.affiliation: Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Halle, Germany.
authors.forename: Hongying
authors.initials: H
authors.lastname: Lin
authors.forename: Frank
authors.initials: F
authors.lastname: Hoffmann
authors.forename: Aleksei
authors.initials: A
authors.lastname: Rozkov
authors.forename: Sven-Olof
authors.initials: SO
authors.lastname: Enfors
authors.forename: Ursula
authors.initials: U
authors.lastname: Rinas
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
mesh_terms: D018929:Cell Culture Techniques; D015169:Colony Count, Microbial; D003470:Culture Media / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development* / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D013997:Time Factors
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000242:Cyclic AMP