Induction of β galactosidase in the yeast Kluyveromyces lactis

Summary The inductive effect of lactose, -methyl-thio-D-galactopyranoside, (TMG) and glucose on galactosidase synthesis in Kluyveromyces lactis has been studied. Whereas TMG gave a five fold stimulation of the rate of -galactosidase synthesis, lactose only gave a small stimulation. Glucose caused represssion at levels above 10^-3M but stimulated -galactosidase synthesis when added at lower concentrations.     

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 basis of specificity in tetrameric Kluyveromyces lactis β-galactosidase

β-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli β-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a β-galactosidase used in food processing.     

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.     

Effects of cultivation conditions on the production of heterologous α-galactosidase by Kluyveromyces lactis

Growth conditions relevant for the large-scale production of heterologous proteins with yeasts were studied on a laboratory scale. A strain of Kluyveromyces lactis, containing 15 copies of an expression cassette encoding guar -galactosidase integrated into its ribosomal DNA, was used as a model. By using urea as a nitrogen source, it was possible to produce active extracellular -galactosidase in shake-flask cultures grown on a defined mineral medium. Inclusion of urea instead of ammonium sulphate prevented unwanted acidification of cultures. With urea-containing mineral medium, enzyme production in shake flasks was comparable to that in complex media containing peptone. In contrast, the presence of peptone was required to achieve high productivity in chemostat cultures. The low productivity in chemostat cultures growing on mineral media was not due to loss oft the expression cassette, since addition of peptone to such cultures resulted in an immediate high rate of -galactosidase production. The discrepancy between the behaviour of shake-flask and chemostat cultures during growth on mineral medium illustrates the necessity of physiological studies for the scalling-up of heterologous protein production from laboratory to production scale.     

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO₂. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol. Received 16 October 1997/ Accepted in revised form 17 April 1998     

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.     

Influence of culture conditions on the production of heterologous interleukin 1β by Kluyveromyces lactis

Summary Under the control of the repressible PHO5 promoter, the expression of gene encoding interleukin 1 (Il1) was derepressed when the medium was depleted of free inorganic phosphate (Pi). Maximum heterologous protein synthesis was obtained in the presence of 75 mg KH₂PO₄/1 (for 20 g glucose/l). The successful heterologous protein production greatly depends on nutritional culture conditions as Il1 production efficiency was increased by 83% through optimization of the growth medium. Comparison of different phosphate-limited cultivation strategies led to the development of a batch culture procedure with nutrient pulses to delay induced oxido-fermentative glucose metabolism and increase the Il1 production to 135 mg/l.     

Selection of potentially probiotic Kluyveromyces lactis for the fermentation of cheese whey–based beverage

This work aimed to assess the probiotic potential of different Kluyveromyces lactis strains isolated from Canastra cheese and to produce a fermented cheese whey beverage added to beetroot juice using the selected strain. Kluyveromyces lactis strains were tested for their resistance to the passage through the simulated gastrointestinal tract, adhesion properties, and functional effects such as inhibition of enteric pathogens, short-chain fatty acids (SCFA) production, and β-galactosidase activity. The selected strain was used to produce a fermented cheese whey beverage added to beetroot juice in different proportions. The produced beverages were characterized using HPLC for sugars, Folin-Ciocalteu for total phenolic content, DPPH for antioxidant activity, and GC-MS for volatiles compounds. Except B51, all strains showed viability above 75% after exposure to the simulated gastric and duodenal juices. The aggregation rates were above 84% in 24 h. Only B9 and C16 strains presented hydrophobicity above 60%. The highest B9 β-galactosidase activities were 2.17 U/g and 2.21 U/g for pH 7 and 9, respectively. The B9 SCFA profile was similar to that found for Saccharomyces bourllardi. The fermented cheese whey beverages presented phenolic content ranging from 102.75 to 291.61 μg EAG/mL and inhibition of DPPH ranging from 38.69 to 81.02% after 21 days of storage, besides being lactose free. Esters and acetates were the most abundant compounds. Kluyveromyces lactis B9 presented interesting results as a potential probiotic yeast. The produced beverages allowed the delivery of K. lactis B9 through innovative product with functional properties.     

Heterologous Kluyveromyces lactis β-galactosidase secretion by Saccharomyces cerevisiae super-secreting mutants

Several Saccharomyces cerevisiae strains with a super-secreting phenotype have been transformed using a secretion plasmid containing the LAC4 gene and have proven to be effective in the secretion of Kluyveromyces lactis -galactosidase. The strain CGY1585 (ssc1-1) showed the highest secretion (1.7 EU ml^–1) in the culture medium. As far as we know, Kluyveromyces lactis -galactosidase is the largest sized protein and the only intracellular one among those secreted by these mutants hitherto. The recombinant strains all grew in lactose media.     

猪胰岛素前体在酵母Kluyveromyces lactis中的分泌…

通过对包括猪胰岛素前体（ＰＩＰ）基因在内的表达框架克隆至质粒ｐＫＤ１衍生的两种载体上而在酵母Ｋｌｕｙｖｅｒｏｍｙｃｅｓ ｌａｃｔｉｓ中分泌表达猪胰岛素前体。根据放射免疫测定结果,猪胰岛素前体的表达水平为２０－３０ｍｇ／Ｌ,猪胰素胶体经过胰肽被转变基因工程人胰岛素,分析结果表明,来自Ｋ．ｌａｃｔｉｓ的人胰岛素,其氨基酸组成、晶体形状和生物活力天然胰岛素相同。     

The Kluyveromyces lactis CPY homologous genes — Cloning and characterization of the KlPCL1 gene

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.     

Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564

An investigation was conducted on the production of β-galactosidase (β-gal) by different strains of Kluyveromyces, using lactose as a carbon source. The maximum enzymatic activity of 3.8 ± 0.2 U/mL was achieved by using Kluyveromyces lactis strain NRRL Y1564 after 28 h of fermentation at 180 rpm and 30 °C. β-gal was then immobilized onto chitosan and characterized based on its optimal operation pH and temperature, its thermal stability and its kinetic parameters (K_m and V_max) using o-nitrophenyl β-d-galactopyranoside as substrate. The optimal pH for soluble β-gal activity was found to be 6.5 while the optimal pH for immobilized β-gal activity was found to be 7.0, while the optimal operating temperatures were 50 °C and 37 °C, respectively. At 50 °C, the immobilized enzyme showed an increased thermal stability, being 8 times more stable than the soluble enzyme. The immobilized enzyme was reused for 10 cycles, showing stability since it retained more than 70% of its initial activity. The immobilized enzyme retained 100% of its initial activity when it was stored at 4 °C and pH 7.0 for 93 days. The soluble β-gal lost 9.4% of its initial activity when it was stored at the same conditions.     

Immobilization of β-d-galactosidase from Kluyveromyces lactis on functionalized silicon dioxide nanoparticles: characterization and lactose hydrolysis

β-D-Galactosidase (BGAL) from Kluyveromyces lactis was covalently immobilized to functionalized silicon dioxide nanoparticles (10-20 nm). The binding of the enzyme to the nanoparticles was confirmed by Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Functionalized nanoparticles showed 87% immobilization yield. Soluble and immobilized enzyme preparation exhibited pH-optima at pH 6.5 and 7.0, respectively, with temperature optima at 35 and 40°C, respectively. Michaelis constant (K(m)) was 4.77 and 8.4mM for free and immobilized BGAL, respectively. V(max) for the soluble and immobilized enzyme was 12.25 and 13.51 U/ml, respectively. Nanoparticle immobilized BGAL demonstrated improved stability after favoring multipoint covalent attachment. Thermal stability of the immobilized enzyme was enhanced at 40, 50 and 65°C. Immobilized nanoparticle-enzyme conjugate retained more than 50% enzyme activity up to the eleventh cycle. Maximum lactose hydrolysis by immobilized BGAL was achieved at 8h.     

Simultaneous hydrolysis of cheese whey and lactulose production catalyzed by β-galactosidase from Kluyveromyces lactis NRRL Y1564

Bioprocess and Biosystems Engineering - β-Galactosidase was produced by the yeast Kluyveromyces lactis NRRL Y1564 in cheese whey supplemented with yeast extract under the optimal temperature...     

Suppression of ρ 0 lethality by mitochondrial ATP synthase F1 mutations in Kluyveromyces lactis occurs in the absence of F0

Specific mgi mutations in the α, β or γ subunits of the mitochondrial F₁-ATPase have previously been found to suppress ρ⁰ lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F₁ is dependent on the F₀ sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, ρ⁰ mutants can be isolated, upon treatment with ethidium bromide, that lack six major F₀ subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of ρ⁰ mutants indicates that an F₁-complex carrying a mgi mutation can assemble in the absence of F₀ subunits and that suppression of ρ⁰ lethality is an intrinsic property of the altered F₁ particle.     

Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21–KlMAL22 bi-directional promoter

We have identified the Kluyveromyces lactis maltase (KlMAL22) and maltose permease (KlMAL21) intergenic region as a candidate bi-directional promoter for heterologous gene expression. The expressions of cyan and yellow fluorescent proteins from, respectively, the KlMAL22 and KlMAL21 orientations of the promoter, were compared between two promoter variants during growth in media containing glucose, galactose or glycerol. Expression from both orientations of the native promoter was repressed during growth in glucose and galactose and was induced during growth in glycerol. Disruption of a putative Mig1p binding site caused some de-repression of the maltase orientation of the promoter by 48 h of growth in glucose. The KlMAL21–-KlMAL22 bi-directional promoter can be used to carry out regulated expression of heterologous gene products.     

The β Subunit of the Heterotrimeric G Protein Triggers the Kluyveromyces lactis Pheromone Response Pathway in the Absence of the γ Subunit

The Kluyveromyces lactis heterotrimeric G protein is a canonical Gαβγ complex; however, in contrast to Saccharomyces cerevisiae, where the Gγ subunit is essential for mating, disruption of the KlGγ gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Gγ, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and ΔGγ cells of K. lactis. In contrast to the moderate sterility shown by the single ΔKlGα, the double ΔKlGα ΔKlGγ mutant displayed full sterility. A partial sterile phenotype of the ΔKlGγ mutant was obtained in conditions where the KlGβ subunit interacted defectively with the Gα subunit. The addition of a CCAAX motif to the C-end of KlGβ, partially suppressed the lack of both KlGα and KlGγ subunits. In cells lacking KlGγ, the KlGβ subunit cofractionated with KlGα in the plasma membrane, but in the ΔKlGα ΔKlGγ strain was located in the cytosol. When the KlGβ-KlGα interaction was affected in the ΔKlGγ mutant, most KlGβ fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gβ subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Gγ subunit.