Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin inKluyveromyces lactis

ABSTRACT KlADH4 is a gene of Kluyveromyces lactisencoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host toSaccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression inK. lactis of the bacterial β-glucuronidase and of the human serum albumin (HSA) genes under the control of theKlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation ofKlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction ofKlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source.

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Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4359-4363.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4359-4363 ◽

Cited By ~ 29

Author(s):

Tiziana Lodi ◽

Barbara Neglia ◽

Claudia Donnini

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Protein Conformation ◽

Protein Production ◽

Heterologous Protein ◽

Kluyveromyces Lactis ◽

Heterologous Proteins ◽

Disulfide Bridges ◽

Genes Encoding

ABSTRACT The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

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