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 1␤, 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.In eukaryotes, the specific folding of proteins targeted to the secretory pathway or to extracellular space occurs in the endoplasmic reticulum (ER). For many secretory proteins, the proper folding requires the formation of intra-and intermolecular disulfide bonds (for reviews, see references 15, 21, and 39). The pathway of oxidative protein folding has been extensively studied in Saccharomyces cerevisiae. Genes have been identified that are involved in redox homeostasis within the ER. The protein folding process requires numerous chaperones and enzymes. The core pathway contains two conserved proteins: Pdi1p and Ero1p. Protein disulfide isomerase (PDI) catalyzes formation, isomerization, and reduction of disulfide bonds of substrate proteins (18,22,23,33). The ER membrane-associated protein Ero1p (ER oxidoreduction) introduces oxidizing equivalents through a flavin-dependent mechanism, engaging thiol-disulfide exchange with Pdi1p (17,38). Mutations in ERO1 and PDI1 result in cells that are sensitive to the reducing agent dithiothreitol (DTT) and that accumulate proteins that normally contain disulfide bonds in reduced form in the ER. The accumulation of reduced proteins induces the unfolded protein response (16,19,29,30). Overexpression of ERO1 results in cells resistant to DTT (16). A few other proteins functionally related to Pdi1p or to Ero1p have also been identified in S. cerevisiae. Overproduction of Mpd1, Mpd2, Eug1, and Eps1 can partially complement the loss of Pdi1p, and overproduction of Erv2 partially complements the loss of Ero1. Unlike ERO1 a...

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1␤ compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position ؉527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N-and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.Yeasts are very useful hosts for the production of heterologous proteins. The yeast Kluyveromyces lactis presents several advantages over other yeast species. It is positive for lactose fermentation, is able to grow on cheap substrates such as residual whey from dairy industries, and has competitive secretory properties, excellent large-scale fermentation characteristics, and food grade status; also, both episomal and integrative expression vectors are available for it (for reviews, see references 20, 40, and 50). Its ability to secrete heterologous proteins into the medium at a concentration higher than that secreted by Saccharomyces cerevisiae was demonstrated previously (50), although the secretory and glycosylation processes and their regulation are still poorly understood for K. lactis (1,42,43).For K. lactis, the regulation of primary carbon metabolism differs markedly from that for S. cerevisiae and reflects the dominance of respiration over fermentation that is typical for the majority of yeast species (7). In K. lactis, respiration is not repressed by glucose, and fermentative and oxidative metabolism can take place simultaneously. Glucose repression, however, does exist: several enzymes that are required for alternate carbohydrate metabolism have been shown to be subject to glucose repression (6,13,17,25,30). The K. lactis genes involved in glucose repression include RAG1, encoding a lowaffinity glucose permease (23, 48); DGR151 (or RAG5), encoding the single hexokinase of this yea...