# Patent

## Title
Kluyveromyces lactis yeast strain and methods for the production of sugars, ethanol, beta-galactosidase and biomass

## Abstract

A Kluyveromyces lactis yeast strain comprising the sequence identified by SEQ ID NO: 1, and methods for the production of sugars (glucose and galactose), ethanol, β-galactosidase and biomass is provided, in which the Kluyveromyces lactis yeast strain is cultured in the presence of a lactose-containing medium. The lactose-containing medium may be milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This Application is a 371 of PCT/ES2011/070550 filed on Jul. 27, 2011, which claims the priority of Spanish Patent Application No, 201131065 filed on Jun. 24, 2011, both applications are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The present invention is related to a Kluyveromyces lactis yeast strain modified by means of genomic integration capable of secreting β-galactosidase into the medium. Said yeast strain is used in methods for the production of sugars, biomass, ethanol and β-galactosidase in lactose-containing media, such as milk or whey.
BACKGROUND OF THE INVENTION
Whey is the liquid remaining after the precipitation and separation of casein from milk during cheese elaboration. This whey retains 55% of the nutrients in milk (it is 85-90% of the milk volume) and has high biological and chemical oxygen demand so it is considered a contaminating by-product, and an environmental problem important for dairies.
Until now no technology has been developed that has proven to be sufficiently profitable for processing large volumes of whey. The main drawback is the small number of microorganisms capable of growing in the milk whey. The wild strains of respiring yeasts, such as Kluyveromyces lactis, are unable to achieve alcohol concentrations sufficient to recoup the investment.
The problem posed by the art is to provide a Kluyveromyces lactis strain capable of natively and actively secreting β-galactosidase into the medium capable of achieving alcohol concentrations higher than the wild strain. The solution proposed by the present invention is a Kluyveromyces lactis yeast strain deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Culture Collection with deposit number DSM 24900, comprising the sequence identified by SEQ ID NO: 1.
DESCRIPTION OF THE INVENTION
The present invention is a Kluyveromyces lactis yeast strain deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Culture Collection with deposit number DSM 24900, comprising the sequence identified by SEQ ID NO: 1.
The Kluyveromyces lactis yeast strain of the invention, was deposited on Jun. 6, 2011, under the provisions of the Budapest Treaty, in the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) Culture Collection in Inhoffenstraβe 7 B, 38124 Braunschweig (Germany), by the depositor Queizuar, SL, A Silva-Bama, s/n, 15822 Touro, A Coruna (Spain).
The Kluyveromyces lactis yeast strain received the deposit number DSM 24900 after the Deposit International Authority found that the strain was viable. All restrictions to the deposited material will be irrevocably removed upon issuance of the patent application.
The strain of the invention comprises a DNA construct identified by SEQ ID NO: 1, comprising a signal sequence of the preprofactor α of Kluyveromyces lactis fused in the same reading frame with the mature form of the β-galactosidase of Kluyveromyces lactis. The signal sequence of the preprofactor α of Kluyveromyces lactis is able to promote the secretion of the β-galactosidase of the strain of the invention. It has been surprisingly found that, the yeast strain containing said construction, although it has levels of growth lower than those of the native strain, secrets β-galactosidase into the medium in its active form with yields up to 113% higher than the same K. lactis strain without modification. The strain can be used for the production of sugars, biomass, ethanol and β-galactosidase from a lactose-rich culture medium.
The construction identified by SEQ ID NO: 1 also comprises the coding sequence for the FLAG peptide, recognized by a monoclonal antibody and which can serve to recognize the resulting fusion protein by immunoaffinity chromatography. Said sequence can be substituted by sequences encoding tag peptides with an equivalent function such as c-myc, HA, E. It can also be substituted by peptide sequences which allow the isolation or purification of the peptide or fusion protein, for example, a polyhistidine sequence.
Preferably, the tag peptide is the FLAG epitope, and is linked to the C-terminus of the β-galactosidase.
One embodiment is a strain of the invention further comprising the sequences identified by SEQ ID NO: 3, 4 and 5.
This strain comprises a promoter for the expression of the protein. Said promoter is the promoter of the LAC4 gene, which is the sequence identified by SEQ ID NO: 3 and 4. Said strain also comprises a transcriptional terminator. Said transcriptional terminator is the terminator of the LAC4 gene, which is identified by SEQ ID NO: 5.
Another embodiment is a strain of the invention comprising a sequence having an identity of 95% with respect to SEQ ID NO: 1. And another embodiment is that said identity is 90%.
In the present application, said percentage of identity in a certain sequence is calculated taking into account, that an identity of 95% means that 95% of residues of the complete sequence of the DNA construction identified by the sequence SEQ ID NO: 1 are identical to the residues of that certain sequence.
A preferred embodiment is a protein obtained from the strain of the invention, the amino acid sequence of which is identified by SEQ ID NO: 2.
Another embodiment is a vector comprising the sequences identified by SEQ ID NO: 1, 3, 4 and 5.
Said vector is used to introduce said sequences into the strain of the invention. There are different methods suitable for introducing a DNA molecule in the strain of the invention:

    
    
        Spheroplast transformation, which involves the removal of the yeast cell wall and contacting the spheroplasts with the plasmid in the presence of PEG,
        Transformation with Li+, which involves treating the yeast cells with monovalent alkali cations (Na+, K+, Rb+, Cs+ and Li+) in combination with PEG to stimulate the uptake of DNA by the intact cells.
        Electroporation, which involves the administration of electrical pulses to yeast resulting in the opening of pores in the membrane of spheroplasts and intact yeast cells.
    
    


The strains of the invention are capable of growing in a lactose-rich medium, which is degraded forming glucose and galactose. Such that an embodiment of the invention is a method for the obtainment of sugars, wherein a Kluyveromyces lactis yeast strain comprising the sequence identified by SEQ ID NO: 1 is cultured in the presence of lactose-containing medium. A preferred embodiment is the method for the obtainment of sugar of the invention wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.
A preferred embodiment is a method for the obtainment of sugars of the invention, wherein said sugar is glucose and/or galactose.
Another preferred embodiment is a method for the obtainment of sugars of the invention, wherein said yeast strain comprises a sequence that has an identity of 95% with respect to SEQ ID NO: 1. In another more preferred embodiment, said identity is 90%.
In the method for the obtainment of sugars, the cell respiration of the strain of the invention is forced by means of high stirring and aeration, thereby avoiding yield losses by fermentation of the sugars to ethanol.
The lactose-containing media that can be used as carbon source in the context of the present invention include both synthetic media and natural products and derivatives thereof. Synthetic or semi-synthetic media that can be used in the context of the present invention include YPL, containing 1% yeast extract, 2% bactopeptone, and an amount of lactose that varies between 0.5% and 6%.
Lactose-rich natural products that can be used as a culture medium for the strain of the invention include milk and derivatives thereof, such as skim milk, the whey resulting from the preparation of butter (buttermilk), the whey resulting after casein precipitation or the permeate of a milk product which can be a milk permeate or a whey permeate. The present invention contemplates the use of milk of virtually any origin, including, without limitation, cow, human, goat, sheep, camel, and buffalo milk and the like. Preferably, the milk is subjected to treatment with rennet of animal origin (extract obtained from the abomasums of the stomach of ruminants), plant origin or recombinant at temperatures between 30 and 40° C., which results in the coagulation of milk casein, which carries most of the fat fraction of the same. After the elimination of the clot, the whey is obtained, which can be used as such (the so-called “sweet whey”) or it may be subjected to an additional deproteinization process, for example, by ultrafiltration or other separation techniques based on porous membranes with a separation limit of 17-20 kDa. Likewise, the invention contemplates the use of the so-called acid whey, resulting from the precipitation of milk proteins in acid medium.
The whey can be concentrated by aerosol spraying to result in fractions with a higher content of dry solid matter than the original whey, including a solid product called whey permeate. Typical contents in dry solid matter range from 5 to 6% in the whey, through values above 30%, particularly between 50 and 60% of the concentrates. The lactose concentration varies between 70 and 75% of total dry solid matter in the case of sweet whey and reaches values between 82 and 86% in the case of whey permeates.
Glucose and galactose which are formed as a result of the hydrolysis of lactose may be recovered from the culture medium using techniques well known by the person skilled in the art. Preferably, glucose and galactose are purified by adsorption with activated carbon preparations with different properties or by using ceramic membranes.
The lactose in the culture medium is digested by the strains of the invention to give glucose and galactose which, in turn, are suitable substrates for alcoholic fermentation producing alcohols. So that one embodiment of the invention is a method for the obtainment of ethanol wherein a Kluyveromyces lactis yeast strain comprising the sequence identified by SEQ ID NO: 1 is cultured in the presence of a lactose-containing medium.
A preferred embodiment is a method for the obtainment of ethanol of the invention, wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.
Another preferred embodiment is a method for the obtainment of ethanol of the invention, wherein said yeast strain comprises a sequence that has an identity of 95% with respect to SEQ ID NO: 1. In another more preferred embodiment, said identity is 90%.
Ethanol can be used as fuel, in beverages or at an industrial level. Normally, the alcoholic fermentation for producing ethanol is carried out for 30-60 hours, at a temperature around 32° C. The ethanol is recovered from the medium using conventional techniques, such as distillation.
The strain of the invention is able to produce and secrete β-galactosidase into the medium. So that a preferred embodiment of the invention is a method for the obtainment of the protein identified by SEQ ID NO: 2, wherein a Kluyveromyces lactis yeast strain comprising the sequence identified by SEQ ID NO: 1 is cultured in the presence of lactose-containing medium.
A preferred embodiment is a method for the obtainment of the protein identified by SEQ ID NO: 2 of the invention, wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.
Another preferred embodiment is a method for the obtainment of the protein identified by the sequence SEQ ID NO: 2 of the invention, wherein said yeast strain comprises a sequence that has an identity of 95% with respect to SEQ ID NO: 1. In another more preferred embodiment, said identity is 90%.
The β-galactosidase protein can be conveniently purified in a single step of affinity chromatography using substrate analogs, such as p-amino phenyl-β-D-thiogalactopyranoside.
The purification of β-galactosidase of K. lactis and its variants can be carried out using methods known in the art. The determination of the degree of purity of the β-galactosidase can be estimated by the value of the specific enzyme activity which is calculated by dividing the number of units of enzyme activity by the amount of mg of protein in a given volume. Preferably, the enzyme activity is determined by the method of Guarente (Guarente, L., 1983, Methods in Enzymology, 101: 181-191).
A preferred embodiment of the invention is a method for the obtainment of biomass, wherein a Kluyveromyces lactis yeast strain comprising the sequence identified by SEQ ID NO: 1 is cultured in the presence of a lactose-containing medium.
A preferred embodiment is a method for the obtainment of biomass of the invention, wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.
Another preferred embodiment is a method for the obtainment of biomass of the invention, wherein said yeast strain comprises a sequence that has an identity of 95% with respect to SEQ ID NO: 1. In another more preferred embodiment, said identity is 90%.
The biomass can be recovered from the culture medium by any technique known to those skilled in the art including, without limitation, centrifugation, deposition or filtration. Preferably, the technique used must significantly reduce the damage to the cells. In case that the same culture is used for preparation of ethanol and biomass, the separation of the biomass of the yeast cells must significantly reduce the loss of ethanol.
Normally, the yeast recovered from the culture medium is washed with an aqueous solution to remove unwanted materials that could be associated with the yeast. Preferably, the protein content in yeast is between 35 and 65%.
The yeast biomass recovered can be used as ingredient in food products without further processing. The recovered biomass can also be lysed and, optionally, the intact cells can be separated. Lysed yeast cells can be used in culture media as yeast extract or may be further processed to separate their various components, such as peptides, nucleotides, amino acids or components specific of the cell wall such as chitin, glucans, mannans and oligosaccharides.
Free Text of the Sequence Listing
In the following, the free text that appears in the sequence listing is shown.
SEQ ID NO: 1. Secretion domain of the Kluyveromyces lactis preprofactor α, Kluyveromyces lactis β-galactosidase and FLAG peptide.
SEQ ID NO: 3. Region 1 of the Kluyveromyces lactis β-galactosidase promoter.
SEQ ID NO: 4. Region 2 of the Kluyveromyces lactis β-galactosidase promoter.
SEQ ID NO: 5. Kluyveromyces lactis β-galactosidase terminator.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Growth and ethanol production of the Kluyveromyces lactis strain in whey. Upper panel, growth and yield in grams of ethanol per gram of cells of the Kluyveromyces lactis wild strain. Lower panel, growth and yield in grams of ethanol per gram of cells of the Kluyveromyces lactis strain of the invention.
FIG. 2: Growth, lactose consumption and ethanol production of the Kluyveromyces lactis strain of the invention in whey in a fermenter at 30° C.
FIG. 3: Growth, intracellular and extracellular β-galactosidase activity of the Kluyveromyces lactis strain of the invention growing in a lactose-rich medium in 5% YPL culture medium (1% yeast extract, 0.5% bactopeptone, and 5% lactose) in flasks at 30° C. and 150 rpm stirring.
FIG. 4. It shows a schematic representation of the structure of the vector comprising SEQ ID NO: 1.



PREFERRED EMBODIMENTS
EXAMPLE 1
Construction of the Vector and Transformation of the Strain of the Invention
From the LAC4 sequence encoding the K. lactis β-galactosidase, oligonucleotides primers were designed in order to amplify the gene by polymerase chain reaction (PCR) with the FLAG peptide and subsequently ligate it into a yeast expression vector to obtain the corresponding plasmid.
The vector contains the promoter of the K. lactis β-galactosidase which is induced in the presence of galactose and/or lactose, a sequence encoding the signal sequence of the yeast preprofactor α, and the terminator of the K. lactis β-galactosidase. The structure of the vector is represented in FIG. 4.
The gene is incorporated into the yeast in the LAC4 locus by homologous recombination. In the case of this strain, several tandem copies of the gene were integrated in the yeast genome.
The yeast strain was transformed with the resulting constructs using the lithium acetate method from Ito et al. (Ito et al., 3983, J. Bacteriol., 153: 163-168).
EXAMPLE 2
Media and Culture Conditions of the Strata of the Invention
With the recombinant strain obtained, cultures in media rich in galactose and/or lactose, as well as other carbon sources (glucose, glycerol, etc.) were carried out in flasks of different volumes (50, 100, 250, 500, 1000 and 2000 ml), trying to maintain a ratio of 1/2,5 with respect to the liquid in the flask volume. Cultures were also carried out in larger volumes (fermenters).
The cultures were carried out at the optimal temperature of the yeast, and with different degrees of stirring (50 rpm-500 rpm) to verify optimal production of biomass, protein or ethanol.
In the case of the use of whey, this was autoclaved and centrifuged to obtain a lactose-rich medium as clean as possible.
Also, the minimum and maximum concentrations of inoculum for each of the cases were tested.
EXAMPLE 3
Determination of Lactose and Ethanol Concentrations and β-Galactosidase Activity
For the determination of lactose and ethanol both commercial tests from Roche and other suppliers (always following the recommendations of the Supplier) and High Resolution Liquid Chromatography were used.
The determination of the β-galactosidase activity was performed following the method of Guarente. The Enzyme Unit (EU) was defined as the amount of enzyme that releases one nmol of o-nitrophenol per minute in test conditions. The units are given as E.U./ml of culture medium or E.U./mg protein.
EXAMPLE 4
β-Galactosidase and Ethanol Production of the Strain of the Invention in Whey in Culture in Glass Tubes
The control strain and the strain of the invention were cultured in whey in sealed glass tubes and kept at 30° C. with gentle stirring (about 50 rpm) tor the 72 hours that the experiment lasted. FIG. 1 shows the production of ethanol of the strain of the invention and of the wild strain (used as a control) over a 72 hours culture. The strain of the invention obtained 13.8 g of ethanol per gram of cells, while the control strain obtained 6.4 g of ethanol per gram of cells.
EXAMPLE 5
β-Galactosidase and Ethanol Production of the Strain of the Invention in Whey in Culture in Fermenter
The strain of the invention was cultured in whey in a fermenter at 30° C. FIG. 2 shows a culture of the strain of the invention in whey, the amount of lactose consumed, the amount of ethanol produced, as well as the growth after 68 hours of culture.
EXAMPLE 6
β-Galactosidase and Ethanol Production of the Strain of the Invention in 5% YPL Culture Medium
The strain of the invention was cultured in 5% YPL culture medium (1% yeast extract, 0.5% bactopeptone, and 5% lactose) in flasks at 30° C. and 150 rpm stirring. FIG. 3 shows an example of the ability to produce β-galactosidase of the straits of the invention.

## Claims

1. A Kluyveromyces lactis yeast strain deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Culture Collection with deposit number DSM 24900, comprising the sequence identified by SEQ ID NO: 1.

2. Yeast strain according to claim 1, wherein said yeast strain further comprises the sequences identified by SEQ ID NO: 3, 4 and 5.

3. A method for obtaining sugars comprising culturing the Kluyveromyces lactis yeast strain of claim 1, in the presence of a lactose-containing medium.

4. Method according to claim 3, wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.

5. Method according to claim 3, wherein said sugars are glucose and/or galactose.

6. A method for obtaining ethanol comprising culturing the Kluyveromyces lactis yeast strain of claim 1, in the presence of a lactose-containing medium.

7. Method according to claim 6, wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.

8. A method for obtaining biomass comprising culturing the Kluyveromyces lactis yeast strain of claim 1, in the presence of a lactose-containing medium.

9. Method according to claim 8, wherein said lactose-containing medium is selected from the group consisting of milk, whey, whey resulting from the preparation of butter, whey resulting after casein precipitation, milk permeate, whey permeate, acid whey and YPL culture medium.