RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.     

Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1 . Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C₁₈ fatty acid. The KlLAC1 protein preferred long-chain (C₁₈) fatty acids to very-long-chain (C₂₆) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide.     

Induction by glucose of an antimycin-insensitive,azide-sensitive respiration in the yeast Kluyveromyces lactis

Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (Q_{O 2} of 80 l O₂·h^-1·mg^-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 l O₂·h^-1·mg^-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive Q_{O 2} is necessary for cellular growth when the normal respiratory pathway is not functional.The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 m azide or 1mm cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.     

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis

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-1beta 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.     

Transformation of Kluyveromyces lactis by Electroporation

The physical and biological parameters involved in efficient transformation of Kluyveromyces lactis by electroporation have been analyzed. By using an optimum voltage and a constant volume of cell suspension in a cuvette, the efficiency of transformation increased with increases in cell numbers and plasmid concentration. However, the most important parameter was the time of the pulse. Changes of 1 ms decreased the efficiency of transformation more than 70 to 80%. Under our best conditions, between 10⁶ and 10⁷ transformants per μg of plasmid DNA could be obtained. Under certain conditions, the size of the plasmid also affected electroporation efficiency. In any case, we did not obtain integrative transformation with an autonomously replicating plasmid.     

Evidence for an inducible glucose transport system in Kluyveromyces lactis.

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly. In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably beacuse of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.     

Expression of the Arxula adeninivorans glucoamylase gene in Kluyveromyces lactis

 The glucoamylase gene of the yeast Arxula adeninivorans was expressed in Kluyveromyces lactis by using the GAP promoter from Saccharomyces cerevisiae and a multicopy plasmid vector. The transformants secreted 90.1% of the synthesized glucoamylase into the culture medium. The secreted glucoamylase activities are about 20 times higher in comparison to those of Saccharomyces cerevisiae transformants using the same promoter. Secreted glucoamylase possesses identical N-terminal amino acid sequences to those secreted by A. adeninivorans showing that cleavage of the N-terminal signal peptide takes place at the same site. Biochemical characteristics of glucoamylase expressed by K. lactis and A. adeninivorans are very similar. Received: 12 June 1995/Received revision: 17 July 1995/Accepted: 26 July 1995