Fed‐batch production of recombinant β‐galactosidase using the universal stress promoters <i>uspA</i> and <i>uspB</i> in high cell density cultivations

Abstract

Abstract 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, β‐galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the usp A than uspB vectors where uspA showed considerably higher basal level. The data indicate that the σ 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 β‐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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 595–603, 2003.