Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

Genetic manipulation of yeast linear DNA plasmids, particularly of k1 and k2 from the non-conventional dairy yeast Kluyveromyces lactis, has been advanced by the recent establishment of DNA transformation-mediated one-step gene disruption and allele replacement techniques. These methods provide the basis for a strategy for the functional analysis of plasmid genes and DNA elements. By use of double selection regimens, these single-gene procedures have been extended to effect disruption of individual genes on plasmid k2 and transplacement of a functional copy onto plasmid k1, resulting in the production of yeast strains with an altered plasmid composition. This cytoplasmic gene shuffle system facilitates the introduction of specifically modified alleles into k1 or k2 in order to study the function, expression (from UCS promoters) and regulation of cytoplasmic linear plasmid genes. Additionally, identification, characterization and localization of plasmid gene products of interest are made possible by shuffling GFP-, epitope- or affinity purification-tagged alleles between k2 and k1. The gene shuffle approach can also be used for vector development and heterologous protein expression in order to exploit the biotechnical potential of the K. lactis k1/k2 system in yeast cell factory research.     

Evolution of linear plasmids

Summary Linear plasmids are genetic elements commonly found in yeast, filamentous fungi, and higher plants. In contrast to all other plasmids they possess terminal inverted repeats and terminal bound proteins and encode their own DNA and RNA polymerases. Here we present alignments of conserved amino acid sequences of both the DNA and RNA polymerases encoded by those linear plasmids for which DNA sequence data are available. Additionally these sequences are compared to a number of polymerases encoded by related viral and cellular entities. Phylogenetic trees have been established for both types of polymerases. These trees appear to exhibit very similar subgroupings, proving the accuracy of the method employed.Abbreviations TIR terminal inverted repeats - mt mitochondrial - ORF open reading frame Offprint requests to: F. Kempken     

Intraspecific gene expression variability in the yeast Kluyveromyces lactis revealed by micro-array analysis

Using the Genolevures sequencing data, we developed an expression micro-array for the yeast Kluyveromyces lactis consisting of 482 genes, mainly involved in central metabolism, compound transport facilitators and stress response. The array was validated using the LAC/GAL system. By comparing gene expression in the laboratory reference strain CBS2359 and in an industrial strain B1, we demonstrated the influence of two carbon sources, glucose and lactose, on the expression of genes involved in the respiratory and in the fermentative metabolic pathways. We also showed that the two strains, although both originating from dairies, display unexpected differences in gene expression on each type of carbon source.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

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.     

An approach to the hypoxic and oxidative stress responses in Kluyveromyces lactis by analysis of mRNA levels

Genome duplication, after the divergence of Saccharomyces cerevisiae from Kluyveromyces lactis along evolution, has been proposed as a mechanism of yeast evolution from strict aerobics, such as Candida albicans, to facultatives/fermentatives, such as S. cerevisiae. This feature, together with the preponderance of respiration and the use of the pentose phosphate pathway in glucose utilization, makes K. lactis a model yeast for studies related to carbon and oxygen metabolism. In this work, and based on the knowledge of the sequence of the genome of K. lactis, obtained by the Génolevures project, we have constructed DNA arrays from K. lactis including a limited amount of selected probes. They are related to the aerobiosis-hypoxia adaptation and to the oxidative stress response, and have been used to test changes in mRNA levels in response to hypoxia and oxidative stress generated by H(2)O(2). The study was carried out in both wild-type and rag2 mutant K. lactis strains in which glycolysis is blocked at the phosphoglucose isomerase step. This approach is the first analysis carried out in K. lactis for the majority of the genes selected.     

Molecular cloning of the neutral trehalase gene from Kluyveromyces lactis and the distinction between neutral and acid trehalases

We cloned the Kluyveromyces lactis KlNTH1 gene, which encodes neutral trehalase. It showed 65.2% and 68.5% identity at nucleotide and amino acid sequence level, respectively, with the Saccharomyces cerevisiae NTH1 gene. Multiple alignment of the predicted trehalase protein sequences from yeasts, bacteria, insects, and mammals revealed two major domains of conservation. Only the yeast trehalases displayed in an N-terminal extension two consensus sites for cAMP-dependent protein phosphorylation and a putative Ca²⁺-binding sequence. Gene disruption of the KlNTH1 gene abolished neutral trehalase activity and clearly revealed a trehalase activity with an acid pH optimum. It also resulted in a high constitutive trehalose level. Expression of the KlNTH1 gene in an S. cerevisiae nth1Δ mutant resulted in rapid activation of the heterologous trehalase upon addition of glucose to cells growing on a nonfermentable carbon source and upon addition of a nitrogen source to cells starved for nitrogen in a glucose-containing medium. In K. lactis, the same responses were observed except that rapid activation by glucose was observed only in early-exponential-phase cells. Inactivation of K. lactis neutral trehalase by alkaline phosphatase and activation by cAMP in cell extracts are consistent with control of the enzyme by cAMP-dependent protein phosphorylation. Received: 19 March 1996 / Accepted: 15 October 1996     

Heterologous complementation of the Klaac null mutation of Kluyveromyces lactis by the Saccharomyces cerevisiae AAC3 gene encoding the ADP/ATP carrier

The KlAAC gene, encoding the ADP/ATP carrier, has been assumed to be a single gene in Kluyveromyces lactis, an aerobic, petite-negative yeast species. The Klaac null mutation, which causes a respiratory-deficient phenotype, was fully complemented by AAC2, the Saccharomyces cerevisiae major gene for the ADP/ATP carrier and also by AAC1, a gene that is poorly expressed in S. cerevisiae. In this study, we demonstrate that the Klaac null mutation is partially complemented by the ScAAC3 gene, encoding the hypoxic ADP/ATP carrier isoform, whose expression in S. cerevisiae is prevented by oxygen. Once introduced into K. lactis, the AAC3 gene was expressed both under aerobic and under partial anaerobic conditions but did not support the growth of K. lactis under strict anaerobic conditions.     

Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1

Summary The yeast Kluyveromyces lactis haboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes. Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation. pK192S was always accompanied by pK192L in subclones of KUV192. Both plasmids were derived from pGKL1 by deletion of the large right part of it. pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence. Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L. The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1S in addition to pGKL2 and competed with pGKL1 or pGKL1S for their maintenance. Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.     

Evaluation of the tetracycline promoter system for regulated gene expression in Kluyveromyces marxianus

A tetracycline repressible promoter system designed for Saccharomyces cerevisiae was evaluated for use in Kluyveromyces marxianus. A plasmid was constructed containing the Escherichia coli beta-glucuronidase (gus) gene cloned downstream of the yeast tet-off promoter, the tetR-VP16 activator protein gene, and the URA3 gene for selection. The tet-off promoter-gus construct was integrated into the chromosomal DNA and tested under varying growth conditions in complex medium. The repressors tetracycline and doxycycline were both found to be effective for inhibiting gene expression. Doxycycline levels of 0.5 microg/mL or greater were sufficient to nearly completely suppress Gus synthesis. For most transformants, the induction ratio was approximately 2,000-fold. The tet-off promoter was effective at 30, 37, and 42 degrees C, although the overall Gus activity was highest at 37 degrees C. During exponential growth, little product was formed; expression increased dramatically in late exponential and early stationary phase. The promoter thus shows promise for protein synthesis following cell growth. No inducer is required and the repressor is only needed to prevent expression during the seed culture.