High levels of inducible expression of cloned β-galactosidase ofKluyveromyces lactis inSaccharomyces cerevisiae

Summary The LAC4 gene ofKluyveromyces lactis CBS2360 coding for -galactosidase was isolated from aK. lactis gene bank. The gene was when cloned in a yeast expression vector pBCL26, derived from pLG2 (Guarente 1983), under the control of the inducible GAL1-10 USA/CYC1 yeast hybrid promoter. Two constructions were obtained, pBCLG2 and pBCLG4, that bear the LAC4 gene in the two opposite orientations and we have studied the expression ofK. lactis -galactosidase in yeast cells transformed with these two plasmids and under different growth conditions. High levels of expression induced by galactose were observed with pBCLG2, which bears the LAC4 gene in the correct orientation, while a low constitutive level of expression was observed in pBCLG4 transformants both in glucose and in galactose media. The expression of the heterologous protein, under induced conditions, appears to be strongly influenced by the growth phase of the culture, with a sharp increase of the specific activity of the enzyme and of its level, calculated as percent of total protein, at the beginning of the stationary phase of growth, during which time the -galactosidase reaches a level of 15% of total cellular protein.     

Efficient secretion of human lysozyme from the yeast, <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, 100 g lysozyme secretion ml ^–1 culture: this level was 10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.     

The consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae

Summary The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5-ATCACGTGA-3) flanked by an AT-rich (±90%) stretch of ± 160 by followed by another conserved box (5-TNNTTTATGTTTCCGAAAATTAATAT-3).These three elements were named K1CDEI, K1CDEII, and K1CDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 by AT-rich (±90%) element, named K1CDE0, was found ± 150 by upstream of K1CDEI. The sequences of both K1CDEI and K1CDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161–164 by in K. lactis versus 78–86 by in S. cerevisiae and the presence in K. lactis of K1CDEO, which is not found in S. cerevisiae.     

A vital function for mitochondrial DNA in the petite-negative yeastKluyveromyces lactis

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeastKluyveromyces lactis, specific mutations in the nuclear genesMGI2 andMGI5 encoding the- and-subunits of the mitochondrial F₁-ATPase, allow mtDNA to be lost. In this study we show that wild-typeK. lactis does not survive in the absence of its mitochondrial genome and that the function ofmgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing amgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned theK. lactis homologue of theSaccharomyces cerevisiae mitochondrial genome maintenance geneMGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption ofMGM101 is not lethal in a haploidmgi strain with a specific mutation in a subunit of the mitochondrial F₁-ATPase. These observations suggest that mtDNA inK. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of F₀.     

Optimization of an electroporation procedure for Kluyveromyces lactis transformation

Summary An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into Kluyveromyces lactis yeasts. The plasmid pCR1, derived from a native Kluyveromyces plasmid, was used to transform K. lactis. This plasmid produces a wheat -amylase and contains both the biosynthetic marker URAA and G418 resistance genes. Transformation was optimal at 4500 V/cm, 25 F, and with 0.2 g plasmid DNA. Transformation efficiencies in the range 10⁴–10⁵ transformants/10⁷ cells/g DNA were obtained.     

Some properties ofSaccharomyces kluyveri

Four isolates of aSaccharomyces species which differed fromS. kluyveri by their ability to use cellobiose were analyzed genetically in relation to the latter species. Isolated single spores had low viability. Spore tetrads segregated mating types 2 2, with sexual agglutination occurring between complementary mating types. All single-spore isolates assimilated cellobiose indicating that these isolates were not naturally occurring hybrids betweenS. kluyveri and a cellobiose assimilatingSaccharomyces species.Two cell types were exhibited by single-spore cultures ofS. kluyveri, one granulated (G-type) and one vacuolated (g-type). G-type cultures formed fertile hybrids with complementary mating types of both G- and g-type cultures. Hybrids between two g-type cultures were sterile. They would, however, give fertile hybrids when mixed with G-type cultures.Sporulating hybrids betweenSaccharomyces sp. andS. kluyveri were produced. However the percentage spore germination was low. Single-spore cultures examined had cell types atypical of either parent. The ability to assimilate cellobiose was dominant and appeared to segregate with mating type and cell type.Weak mating reactions occurred when the (+) and (-) mating types ofSaccharomyces sp. were mixed with (a) and () mating types ofS. cerevisiae, respectively.The species ofSaccharomyces isolated from the Pacific Coast are designated as strains ofS. kluyveri.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the - and -subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the -subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the -subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the -subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.Communicated by C. P. Hollenberg     

Secretion of β-lactamase from K. lactis using pEPS1, a convenient episomal vector designed for the secretion of foreign proteins

Summary A convenient shuttle vector that enables high level secretion of proteins from Kluyveromyces lactis has been developed. The vector, pEPS1, contains a unique cloning site that allows the construction, in a single ligation step, of episomal plasmids capable of directing secretion of foreign gene products from K. lactis. As an example we demonstrate the production of -lactamase and determine optimal conditions for its secretion into the culture media.     

Development of a bisphenol A-adsorbing yeast by surface display of the<Emphasis Type="Italic"> Kluyveromyces</Emphasis> yellow enzyme on<Emphasis Type="Italic"> Pichia pastoris</Emphasis>

A novel surface-engineered strain of yeast Pichia pastoris was constructed that displays at its surface Kluyveromyces lactis Yellow Enzyme (KYE) fused to the C-terminal half of Saccharomyces cerevisiae -agglutinin. The expression of the fusion protein was controlled by the AOX1-promoter. The new strain showed an increased sorption of the xenoestrogen Bisphenol A (BPA). It was shown that sorption of BPA depended on the presence of methanol in the growth medium and on the pH of the binding assays. The binding kinetics were typical for binding at a surface. The present results demonstrate that the -agglutinin surface display system can be used in the yeast P. pastoris.     

Partial characterization of anrpoD-like gene oflactoccocus lactis subsp.lactis ML3 with a polymerase chain reaction-based approach

With degenerated oligonucleotide primers for conserved regions of bacterial sigma factor proteins, a 117-bp internal DNA fragment of anrpoD-like gene ofLactoccocus lactis subsp.lactis ML3 was amplified by the polymerase chain reaction (PCR). The DNA sequence of this PCR product was determined by cycle sequencing, and the deduced amino acid sequence of this internal fragment showed an extensive homology with the known sigma factor sequences from six other microorganisms and present a 13-amino acid region corresponding to the typical RpoD box of primary sigma factors. This PCR product was used as a probe to specifically detect sigma homologs inPediococcus acidilactici, Leuconostoc lactis, Lactobacillus helveticus, Lactobacillus acidophilus, Enterococcus faecalis, Streptococcus thermophilus, andLactococcus lactis subsp.cremoris. These data are consistent with the existence of a high similarity between the primary sigma factors from diverse Gram-positive microorganisms.     

Genetics of resistance toPuccinia graminis tritici andPuccinia recondite tritici in four South African wheats

Summary Genes for resistance toPuccinia graminis tritici andPuccinia recondita tritici identified in four South African wheats were:Sr6,Sr8a,Sr9e, andLr13 in W3762;Sr5,Sr8a,Sr9b,Sr12,Sr24,Lr13, andLr24 in W3760;Sr2,Sr24,SrC,Lr13, andLr24 in W3751; andSr7a,Sr23,Sr36, andLr16 in W3755. GenesSr2,Sr9e, andSr24 also conferred adult plant resistance to the predominant pathotypes ofP. graminis tritici. GenesSr7a,Sr23, andSrC, when present alone, did not confer acceptable adult plant resistance, even though low seedling reactions were associated with them when tested with the same pathotypes. Genetic recombination betweenLr13 andSr9e was estimated at 12.5%±2.3%.     

Analysis of coenzyme Q systems,monosaccharide patterns of purified cell walls,and RAPD-PCR patterns in the genus Kluyveromyces

Analysis of the coenzyme Q system and the monosaccharide pattern of purified cell walls were used for species characterization in the genus Kluyveromyces. All the type strains of the genus possess coenzyme Q-6 and the mannose-glucose (Saccharomyces type) cell wall sugar pattern. With the help of Random Amplified Polymorphic DNA-Polymerase Chain Reaction analysis 17 species were separated: K. aestuarii, K. africanus, K. bacillisporus, K. blattae, K. delphensis, K. dobzhanski, K. lactis (anamorph Candida sphaerica), K. lodderae, K. marxianus (syn. K. fragilis, K. bulgaricus, K. cicerisporus anamorphs Candida macedoniensis, C. pseudotropicalis, C. kefyr), K. phaffii, K. piceae, K. polysporus, K. sinensis, K. thermotolerans (syn. K. veronae, anamorph Candida dattila), K. waltii, K. wickerhamii, K. yarrowii (anamorph Candida tannotolerans). A strain of K. drosophilarum showed with the type strain of K. lactis only 63% similarity. The strain originally described as the type strain of K. cellobiovorus nom. nud. was excluded from the genus (Q-9), and found to be conspecific with the type strain of Candida intermedia.Abbreviations nDNA nuclear DNA - RAPD-PCR Random Amplified Polymorphic DNA-Polymerase Chain Reaction - T type strain of the epithet     

Biochemical and molecular-genetic properties of a cytochrome-c-deficient mutant ofKluyveromyces lactis

We have isolated a respiration-deficient nuclear mutant of the yeastKluyveromyces lactis that exhibited diminished levels of all cytochromes and did not grow on glycerol and other nonfermentable carbon sources. The mutant namedcyc1 was transformed with aK. lactis genomic library and the DNA fragment conferring its wild-type properties was isolated and sequenced. The sequence of the isolated gene showed extensive homology with other eukaryotic cytochrome-c genes. The highest level of homology, based on the deduced amino acid sequences, was observed between the gene products ofK. lactis andHansenula anomala.     

Heat shock-induced protein synthesis inLactococcus lactis subsp.lactis

Heat shock inLactococcus lactis subsp.lactis may induce as many as 16 proteins after a temperature shift from 30° to 40°C. Five induced proteins were found to be immunologically related to theEscherichia coli GroEL, DnaK, DnaJ, and GrpE proteins, and to theBacillus subtilis ⁴³ factor. From these initial studies we conclude that, inL. lactis subsp.lactis, a heat shock response similar to that known to occur in other prokaryotes might exist.     

The yeastKluyveromyces lactis as an efficient host for heterologous gene expression

Several different yeast species have been developed into systems for efficient heterologous gene expression. In this paper we review foreign gene expression in the dairy yeastKluyveromyces lactis. This yeast presents several advantageous properties in comparison to other yeast species. These include its impressive secretory capacities, its excellent fermentation characteristics on large scale, its food grade status and the availability of both episomal and integrative expression vectors. Moreover, in contrast to the methylotrophic yeasts that are frequently used for the expression of foreign genes,K. lactis does not require explosion-proof fermentation equipment. Here, we present an overview of the available tools for heterologous gene expression inK. lactis (available promoters, vector systems, etc). Also, the production of prochymosin, human serum albumin and pancreatic phospholipase byK. lactis is discussed in more detail, and used to rate the achievements ofK. lactis with respect to other micro-organisms in which these proteins have been produced.     

Phylohydrax (Rubiaceae-Spermacoceae)—a new genus to accommodate the African and Madagascan“Hydrophylax” species

Hydrophylax, a genus of maritime strand or dune plants, was previously thought to comprise three species, one from India, Sri Lanka and Thailand (H. maritima), and two from Africa and Madagascar (H. carnosa and H. madagascariensis respectively). Evidence is presented that the African and Madagascan species are better placed into a separate genus (Phylohydrax). Differential characters betweenH. maritima andP. carnosa—P. madagascariensis, the relationships between the three species, and the affinities ofPhylohydrax andHydrophylax to otherSpermacoceae are discussed in detail.     

Generation of a stable non-reverting Leu− mutant of Kluyveromyces lactis by gene disruption

The LEU2 gene coding for -isopropylmalate dehydrogenase of the yeast Kluyveromyces lactis strain AWJ137 was disrupted. In the resulting Leu^– strain a 0.57 × 10³-base pairs PstI/BglII fragment of the LEU2 coding region was replaced by the TRP1 gene of Saccharomyces cerevisiae. The mutant strain was characterized by stability tests and a physical map of the disrupted region was established by restriction-enzyme analysis combined with hybridization experiments. The usefulness of the mutant strain as a recipient was shown by transformation experiments.