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.     

Production of secreted guar α-galactosidase by Lactococcus lactis

A plant -galactosidase gene was inserted in the expression vector pGKV259. The resulting plasmid pGAL2 consisted of the replication functions of the broad-host-range lactococcal plasmid pWV01, the lactococcal promoter P59, and the DNA sequences encoding the -amylase signal sequence from Bacillus amyloliquefaciens and the mature part of the -galactosidase from Cyamopsis tetragonoloba (guar). Lactococcus cells of strain MG1363 harbouring this vector produced the plant -galactosidase and secreted the enzyme efficiently as judged by Western blotting and activity assays. Expression levels of up to 4.3 mg extracellular -galactosidase g (dry weight) of biomass^–1 were achieved in standard laboratory batch cultures. The -galactosidase produced by Lactococcus was active on the chromogenic substrate 5-bromo-4-chloro-3-indolyl -d-galactopyranoside, the trisaccharide raffinose and on the galactomannan substrate, guar gum.     

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.     

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 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     

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.     

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.     

Quantification of extracellular α-acetolactate oxidative decarboxylation in diacetyl production by an α-acetolactate overproducing strain of Lactococcus lactis sp. lactis bv. diacetylactis

The quantification of -acetolactate (AAL) extracellular oxidative decarboxylation during an AAL overproducing strain culture shows that this reaction is at the origin of about 90% of the diacetyl production and that only a small proportion of extracellular AAL is readily transformed to diacetyl. These results, compared with previous ones obtained with a non AAL accumulating strain, allow research options to be put forward for the improvement of microbiological diacetyl production.     

Mg2+ ligands (Glu-384, Glu-429) of β-galactosidase from Lactococcus lactis ssp. lactis 7962

The secondary and the tertiary structures of -galactosidase from Lactococcus lactis ssp. lactis 7962 were designed by Nnpredict and Sybyl Version 6.3. Structural modeling of -galactosidase has shown that Glu-384 and Glu-429 are ligands for Mg²⁺ and Mg²⁺ is required for maximum activity. To confirm this prediction, we generated seven site specific mutants: Glu-384-Gln; Glu-384-Val; His-386-Phe; Asn-428-Asp; Glu-429-Gln; 384Gln-429Gln and 384Val-429Gln. The -galactosidases substituted at Glu-384 or Glu-429 had < 1% of the activity of the native enzyme with ONPG as substrate. The substitution of Glu-384 or Glu-429, which removed only one of the coordinating ligand for Mg²⁺, was still affected by Mg²⁺, but the mutants 384Gln-429Gln or 384Val-429Gln, which had been modified both Mg²⁺-binding sites, were not affected by Mg²⁺. Thus, Glu-384 and Glu-429 are probably ligands of Mg²⁺ and the three dimensional disposition of Mg²⁺ and its neighborhood interactions (Glu-384, Glu-429, Asp-428 or His-386) are important in the maintenance of –galactosidase activity.     

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.     

Decarboxylation of α-Keto Acids by Streptococcus lactis var. maltigenes

Decarboxylation rates for a series of C-3 to C-6 α-keto acids were determined in the presence of resting cells and cell-free extracts of Streptococcus lactis var. maltigenes. The C-5 and C-6 acids branched at the penultimate carbon atom were converted most rapidly to the respective aldehydes in the manner described for α-carboxylases. Pyruvate and α-ketobutyrate did not behave as α-carboxylase substrates, in that O₂ was absorbed when they were reacted with resting cells. The same effect with pyruvate was noted in a nonmalty S. lactis, accounting for CO₂ produced by some “homofermentative” streptococci. Mixed substrate reactions indicated that the same enzyme was responsible for decarboxylation of α-ketoisocaproate and α-ketoisovalerate, but it appeared unlikely that this enzyme was responsible for the decarboxylation of pyruvate. Ultrasonic disruption of cells of the malty culture resulted in an extract inactive for decarboxylation of pyruvate in the absence of ferricyanide. Dialyzed cell-free extracts were inactive against all keto acids and could not be reactivated.     

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.     

Increasing the stability of immobilizedLactococcus lactis cultures stored at 4 °C

Summary Immobilized cell technology was used to prepare concentrated cultures ofLactococcus lactis that lost only 22% of viability over a 30-day storage period at 4°C. Concentrated cultures ofL lactis CRA-1 were immobilized in calcium alginate beads and added to glycerol, NaCl or sucrose-NaCl solutions in order to obtain a_w readings ranging from 0.91 to 0.97. The suspensions were subsequently placed at 4°C and viability (CFU g^–1 of bead) was followed during storage. Viability losses were high at a_w readings of 0.95 and 0.97 and pH dropped significantly (up to one unit) in the unbuffered solutions. Addition of 1% soytone or glycerophosphate helphed stabilize pH, and a beneficial effect on viability during storage was observed in the glycerol-soytone mix when the beads were added to the conservation solutions immediately following immobilization. When beads were added to the conservation solution immediately following immobilization, a 70% drop in cell counts occurred during the first 5 days of incubation. Dipping theL lactis-carrying beads in milk for 2h before mixing with the glycerolsoytone 0.93 a_w solution reduced this initial 5-day viability loss. Cultures grown in the alginate beads also had good stability in the 0.93 a_w glycerol-soytone solution, where 78% of the population was viable after 30 days at 4°C. The process could be used to store immobilized cells at a processing plant, or by suppliers of lactic starters who wish to ship cultures without freezing or drying.     

Secretion of human interferon-β 1b by recombinant Lactococcus lactis

Interferon-beta has anti-viral, anti-proliferation and multifunctional immunomodulatory activities and shows promising clinical effects for treatment of inflammatory disorders. The recombinant human interferon-beta (huIFN-beta) 1b was expressed in the food-grade lactic acid bacterium, Lactococcus lactis, using a nisin-controlled gene expression system. huIFN-beta production from recombinant strains (with and without LEISSTCDA propeptide) was approximately 21 and 7 mug l(-1), respectively. Moreover, 95% (former strain) and 88% (latter strain) of total recombinant proteins were secreted into the culture medium. The biological activities of huIFN-beta from recombinant strains revealed similar antiviral activities of 10(7) I.U. mg(-1). These results demonstrate the potential application of recombinant strains as a food grade vehicle to deliver bioactive huIFN-beta in vivo.     

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     

Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.