Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations.
A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fed-batch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.
Prytz
Ingela
I
The Swedish Centre for Bioprocess Technology, Stockholm Centre for Physics, Astronomy and Biotechnology, SE-106 91 Stockholm, Sweden.
Sandén
Anna Maria
AM
Nyström
Thomas
T
Farewell
Anne
A
Wahlström
Asa
A
Förberg
Cecilia
C
Pragai
Zoltan
Z
Barer
Mike
M
Harwood
Colin
C
Larsson
Gen
G
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C116144:UspB protein, E coli; C078564:universal stress protein A, Bacteria; D001616:beta-Galactosidase; D005947:Glucose