T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used. Received: 11 May 1998 / Accepted: 16 October 1998     

Kinetics of lactose hydrolysis by beta-galactosidase of Kluyveromyces lactis immobilized on cotton fabric

A mathematic model for describing the Michaelis-Menten-type reaction kinetics with product competitive inhibition and side-reaction is proposed. A multiresponse nonlinear simulation program was employed to determine the coefficients of a four-parameter rate expression. The rate expression was compared with the conventional Michaelis-Menten reaction rate models with and without product inhibition. Experimental data were obtained using beta-galactosidase of Kluyveromyces lactis immobilized on cotton fabric in a batch system at a temperature of 37 degrees C and at various initial concentrations of dissolved lactose ranging from 3-12.5% (w/v). The reaction is followed by concentration changes with time in the tank. Samples were obtained after the outlet stream of the packed bed reactor is mixed in a well-stirred tank. High-performance liquid chromatography (HPLC) was applied to monitor the concentrations of all the sugars (reactants as well as products). The four-parameter rate model is featured with a term to describe the formation of trisaccharides, a side-reaction of the enzymatic hydrolysis. The proposed model simulates the process of lactose hydrolysis and the formation of glucose and galactose, giving better accuracy compared with the previous models.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized beta-galactosidase from Kluyveromyces lactis

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free -galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the -galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized -glucosidase and with added co-solvents such as hexanol.     

Phylogenetic circumscription of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora

Genera currently assigned to the Saccharomycetaceae have been defined from phenotype, but this classification does not fully correspond with species groupings determined from phylogenetic analysis of gene sequences. The multigene sequence analysis of Kurtzman and Robnett [FEMS Yeast Res. 3 (2003) 417-432] resolved the family Saccharomycetaceae into 11 well-supported clades. In the present study, the taxonomy of the Saccharomyctaceae is evaluated from the perspective of the multigene sequence analysis, which has resulted in reassignment of some species among currently accepted genera, and the proposal of the following five new genera: Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora.     

The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane. Received: 22 July 1997 / Accepted: 27 October 1997     

Comparison of Cytokine Induction by Lipopolysaccharide of Bacteroides fragilis with Salmonella typhimurium in Mice

Comparison of cytokine stimulation by lipopolysaccharide (LPS) of Bacteroides fragilis and Salmonella typhimurium was done to study the early events occurring in vivo. Mice injected intraperitoneally with either LPS demonstrated endogenous production of all the cytokines studied (tumor necrosis factor-alpha, interferon-gamma and interleukin-6) within 6 hr in the bloodstream. However induction of all the cytokines by B. fragilis LPS (50 μg/mouse) was much weaker compared with S. typhimurium LPS (50 μg/mouse). Even a dose of S. typhimurium LPS 40 times smaller (1.2 μg/mouse) induced cytokines more strongly compared with B. fragilis LPS. Thus, a weak biological response to B. fragilis LPS as evidenced by chick embryo lethality, limulus lysate gelation, LD₅₀ for mice and rabbit pyrogenicity could be due to weak induction of bioactive mediators by LPS.     

The Protonema of Chara fragilis Desv.: Regenerative Formation,Photomorphogenesis, and Gravitropism

When exposed to constant white light for four weeks, isolated nodes of Chara fragilis Desv. regenerated side branches, rhizoids, and multicellular protonemata, the latter being similar to those germinated from oospores. When kept in darkness the nodes developed protonemata exclusively. These were single-celled, colourless, and tip-growing and, with the light microscope, they looked like rhizoids. Upon exposure to blue light, but not to red or far-red, the growth rates of the protonemata rapidly declined, the cell apices swelled, and the nucleus migrated acropetally. Within 24 h the cells went through the first of a series of divisions resulting in the formation of multicellular protonemata. When returned to darkness after a blue light pulse of 5 h the cell divisions proceeded normally, but the protonemata showed etiolated growth. While growth of the internode was drastically promoted, the development of the multicellular apex and the lateral initial were suppressed. Both uni- and multicellular etiolating protonemata showed negative gravitropism but were phototropically insensitive. It is argued that the single-celled protonema is an organ specialized for the penetration of mud covering the nodes or oospores of Chara and thus serves to search for light, comparable to etiolated hypocotyls and stems in seedlings of higher plants.