Michele M. Bianchi

The ‘petite-negative’ yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity

We cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcAΔ strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low‐affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.

Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene

ABSTRACT A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using aKluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene (KlPDC1) and transformed with the heterologous lactate dehydrogenase gene (LDH). The LDH gene was placed under the control theKlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g−1, suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we usedK. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g−1 (maximum theoretical yield, 1 g g−1), and with high LDH activity.

Regulation of the Expression of the Kluyveromyces lactis PDC1 Gene: Carbon Source-Responsive Elements and Autoregulation

The yeast Kluyveromyces lactis has a single structural gene coding for pyruvate decarboxylase (KlPDC1). In order to study the regulation of the expression of KlPDC1, we have sequenced (EMBL Accession No. Y15435) its promoter and have fused the promoter to the reporter gene lacZ from E. coli. Transcription analysis in a Klpdc1Δ strain showed that KlPDC1 expression is subject to autoregulation. The PDC1 gene from Saccharomyces cerevisiae was able to complement the Rag− phenotype of the Klpdc1Δ mutant strain and it could also repress transcription of the KlPDC1–lacZ fusion on glucose. A deletion analysis of the promoter region was performed to study carbon source‐dependent regulation and revealed that at least two cis‐acting regions are necessary for full induction of gene expression on glucose. Other cis‐elements mediate repression on ethanol. Copyright

Large‐scale phenotypic analysis reveals identical contributions to cell functions of known and unknown yeast genes

Sequencing of the yeast genome has shown that about one‐third of the yeast ORFs code for unknown proteins. Many other have similarity to known genes, but still the cellular functions of the gene products are unknown. The aim of the B1 Consortium of the EUROFAN project was to perform a qualitative phenotypic analysis on yeast strains deleted for functionally orphan genes. To this end we set up a simple approach to detect growth defects of a relatively large number of strains in the presence of osmolytes, ethanol, high temperature, inhibitory compounds or drugs affecting protein biosynthesis, phosphorylation level or nucleic acids biosynthesis. We have now developed this procedure to a semi‐quantitative level, we have included new inhibitors, such as hygromycin B, benomyl, metals and additional drugs interfering with synthesis of nucleic acids, and we have performed phenotypic analysis on the deleted strains of 564 genes poorly characterized in respect to their cellular functions. About 30% of the deleted strains showed at least one phenotype: many of them were pleiotropic. For many gene deletions, the linkage between the deletion marker and the observed phenotype(s) was studied by tetrad analysis and their co‐segregation was demonstrated. Co‐segregation was found in about two‐thirds of the analysed strains showing phenotype(s). Copyright

HOW TO BRING ORPHAN GENES INTO FUNCTIONAL FAMILIES

In the framework of the B1 Consortium of the EUROFAN‐1 project, we set up a series of simple phenotypic tests that can be performed on a large number of strains at a time. This methodological approach was intended to help assign functions of putative genes coding for unknown proteins to several specific aspects of cell biology. The tests were chosen to study phenotypes which should be affected by numerous genes. In this report, we examined the sensitivity/resistance or the adaptation of the cell to physical or chemical stresses (thermotolerance, osmotolerance and ethanol sensitivity), the effects of the alteration of the level of protein phosphorylation (sensitivity or resistance to compounds affecting the activity of protein kinases or phosphatases) and the effects of compounds interfering with synthesis of nucleic acids or proteins. Deletions in 66 genes of unknown function have been tested in 21 different conditions. In many deletant strains, phenotypes were observed and, for the most promising candidates, tetrad analysis was performed in order to verify co‐segregation of the deletion marker with the phenotype. Copyright

Gene expression waves: Cell cycle independent collective dynamics in cultured cells

The ergodic hypothesis, which assumes the independence of each cell of the ensemble from all the others, is a necessary prerequisite to attach single cell based explanations to the grand averages taken from population data. This was the prevailing view about the interpretation of cellular biology experiments that typically are performed on colonies of billions of cells. By analysing gene expression data of different cells going from yeast to mammalian cell cultures, we demonstrate that cell cultures display a sort of ‘ecology‐in‐a‐plate’ giving rise to a rich dynamics of gene expression that are independent from reproductive cycles, hence contradicting simple ergodic assumptions The aspecific character of the observed coordinated gene expression activity inhibits any simple mechanistic hypothesis and highlights the need to consider population effects in the interpretation of data coming from cell cultures.

Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

ABSTRACT The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

Plasmid functions involved in the stable propagation of the pKD1 circular plasmid in kluyveromyces lactis

SummaryPlasmid factors involved in the stable propagation of pKD1-derived vectors in Kluyveromyces lactis transformants have been identified. Three genes (A, B and C) have been found to be present in pKD1: the interruption of the B and C genes led to high plasmid instability. Stability could be restored in trans when host cells contained pKD1 as the resident plasmid (pKD1+ strains). The A gene, which codes for a site-specific recombinase, did not affect plasmid partitioning. Vectors bearing only the pKD1 replication origin (or a chromosomal ARS), and no other pKD1 sequence, were very unstable both in the presence and absence of the resident plasmid in host cells. These vectors could be stabilized in pKD1+ strains, but not in pKD1o strains, by the insertion of a 200 pb-long pKD1 sequence. This sequence, called the cis-acting stability locus (CSL), together with the products of the B and C genes, ensured plasmid partitioning at cell divison. Possible hairpin structures and direct repeats were regularly spaced within the CSL. This region, and the corresponding cis-acting stabilizing elements of other yeast plasmids, did not have sequence homology but shared some structural regularities.

Optimization of recombinant fungal laccase production with strains of the yeast Kluyveromyces lactis from the pyruvate decarboxylase promoter

Laccases are multicopper oxidases of wide specificity that catalyze the oxidation of phenolic and related compounds using molecular oxygen as the electron acceptor. Here, we report the production of the Lcc1 laccase of the fungus Trametes trogii in strains of the yeast Kluyveromyces lactis, using the pyruvate decarboxylase promoter (KlPDC1) as an expression system. We assayed laccase production in various strains, with replicative and integrative transformants and with different cultivation parameters. A comparison with Lcc1 enzymes from other yeasts and from the original organism was also performed. The best production conditions were obtained with integrative transformants of an individual strain, whereas cultivation conditions were less stringent than the use of the regulated KlPDC1 promoter could anticipate. The secreted recombinant laccase showed better enzyme properties than the native enzyme or recombinant enzyme from other yeasts. We conclude that selected K. lactis strains, with opportune physiological properties and transcription regulation of the heterologous gene, could be optimal hosts for laccase isoenzyme production.

A dual signalling pathway for the hypoxic expression of lipid genes, dependent on the glucose sensor Rag4, is revealed by the analysis of the KlMGA2 gene in Kluyveromyces lactis.

In the respiratory yeast Kluyveromyces lactis, little is known about the factors regulating the metabolic response to oxygen shortage. After searching for homologues of characterized Saccharomyces cerevisiae regulators of the hypoxic response, we identified a gene that we named KlMGA2, which is homologous to MGA2. The deletion of KlMGA2 strongly reduced both the fermentative and respiratory growth rate and altered fatty acid composition and the unsaturation index of membranes. The reciprocal heterologous expression of MGA2 and KlMGA2 in the corresponding deletion mutant strains suggested that Mga2 and KlMga2 are functional homologues. KlMGA2 transcription was induced by hypoxia and the glucose sensor Rag4 mediated the hypoxic induction of KlMGA2. Transcription of lipid biosynthetic genes KlOLE1, KlERG1, KlFAS1 and KlATF1 was induced by hypoxia and was dependent on KlMga2, except for KlOLE1. Rag4 was required for hypoxic induction of transcription for both KlMga2-dependent (KlERG1) and KlMga2-independent (KlOLE1) structural genes.