Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene

Abstract

The glyceraldehyde-3-phosphate dehydrogenase promoter, P-GAP, was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P-GAP was constructed and the elemental parameters affecting the expression of HSA from P-GAP were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P-GAP. Glycerol supported a higher level of HSA expression from P-GAP along with a higher cell density than either glucose or methanol. The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth. A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol. The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P-GAP can be a practical alternative of the MOX promoter in the large-scale production of HSA from H. polymorpha. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.