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] super(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.