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.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Bacterial Proteins

genetics

metabolism

Bioreactors

microbiology

Cell Culture Techniques

methods

Escherichia coli

cytology

enzymology

genetics

growth & development

Escherichia coli Proteins

genetics

metabolism

Gene Expression Regulation

Bacterial

physiology

Gene Expression Regulation

Enzymologic

physiology

Glucose

metabolism

Heat-Shock Proteins

genetics

metabolism

Membrane Proteins

genetics

metabolism

Multienzyme Complexes

genetics

metabolism

Promoter Regions

Genetic

Protein Engineering

methods

Recombinant Proteins

biosynthesis

beta-Galactosidase

biosynthesis

genetics

stress promoters

Biochemistry

Publikations- och innehållstyp

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art (ämneskategori)