Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

Abstract

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...

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

“…In particular, in Kluyveromyces lactis, a yeast used as a host for secreted production of mammalian proteins (11,13,14,40), duplication of KlPDI1 increases the amount of human serum albumin (HSA), a protein rich in disulfide bonds (4), that is secreted.…”

Improved secretory production of calf prochymosin by codon optimization and disruption of PMR1 in Kluyveromyces lactis GG799

“…Over expression of GDP-mannose pyrophosphorylase, KlPSa1p gene of K. lactis has also been found to enhance the heterologous protein secretion (Uccelletti et al, 2005). Glucose repression defective mutant of K. lactis is also found to enhance the production of heterologous proteins (Donnini et al, 2004). The feasibility of improving chymosin secretion in K. lactis GG799 depends on whether the several steps of protein processing are close to their limits.…”

How to achieve high-level expression of microbial enzymes

“…129,[153][154][155] A glucose repression-defective mutant of K. lactis was used to improve the production yield of glucoamylase. 156 The dimorphic species A. adeninivorans uses adenine, xanthine, uric acid, putrescine, glucose and n-alkylamines. Among the few recombinant enzymes that have been expressed in A. adeninivorans, a tannase has been successfully over-produced using various plasmids carrying one or two expression modules.…”