Yeast strains for reducing contamination by lactic acid bacteria

Abstract

Described herein is a yeast strain wherein activity of one or more membrane transporters of the DHA1 family is reduced relative to a wild type strain or to a parental strain from which it is derived.

Claims

1. A yeast strain wherein activity of one or more membrane transporters of the DHA1 family is reduced relative to a wild type strain or to a parental strain from which it is derived.

2. The yeast strain according to claim 1, wherein one or more nucleotide sequences encoding the one or more membrane transporters of the DHA1 family are deleted or inactivated, or wherein expression of said one or more nucleotide sequences is downregulated, preferably inducibly downregulated.

3. The yeast strain according to claim 1, wherein the one or more membrane transporters of the DHA1 family are selected from the group consisting of AQR1, QDR1, QDR2, QDR3, TPO1, TPO2, TPO3, TPO4, FLR1, YHK8, DTR1, HOL1 and/or homologues thereof.

4. The yeast strain according to claim 1, wherein the one or more membrane transporters of the DHA1 family are encoded by a nucleotide sequence comprising, consisting essentially of or consisting of a sequence having at least 60% sequence identity with any of the nucleotide sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24; or comprise, consist essentially of or consist of an amino acid sequence having at least 60% identity or similarity with any of the amino acid sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23.

5. The yeast strain according to claim 1, having reduced activity of at least two, at least three, at least four, at least five, at least six, or at least seven transporters of the DHA1 family relative to a wild type strain or to a parental strain from which it is derived.

6. The yeast strain according to claim 1, wherein the one or more membrane transporters comprise: AQR1, QDR2 and QDR3 or homologues thereof; QDR1, QDR2 and QDR3 or homologues thereof; AQR1, QDR2, QDR1 and TPO4 or homologues thereof; or AQR1, QDR3, QDR2, QDR1, DTR1, HOL1 and TPO1, or homologues thereof.

7. The yeast strain according to claim 1, wherein the yeast is a Saccharomyces species, preferably Saccharomyces cerevisiae.

8. A composition comprising, consisting essentially of, or consisting of a yeast strain according to claim 1; and optionally one or more formulation excipients, preferably wherein the composition is a dry yeast composition or an instant yeast composition.

9. A method for the production of yeast cells, comprising culturing a yeast strain as defined in claim 1 in a suitable culture medium.

10. A method for producing a fermentation product, comprising: culturing a yeast strain as defined in claim 1 on a fermentable substrate; and optionally, recovering the fermentation product.

11. The method according to claim 10, wherein the fermentation substrate comprises, consists essentially of or consists of an unrefined or low-processed source of sugars.

12. The method according to claim 10, wherein the fermentation product is an alcohol, preferably a lower alkyl alcohol such as ethanol, propanol or butanol, more preferably ethanol.

13. A method for the production of a product of interest, comprising: a) optionally, transforming a yeast cell according to claim 1 with a vector encoding the product of interest or encoding one or more enzymes capable of producing the product of interest; b) culturing the yeast cell in a culture medium to produce the product of interest; and c) optionally, isolating the product of interest from the yeast cell or culture medium.

14. A method for eliminating, reducing or preventing contamination of lactic acid bacteria in yeast cultures, the method comprising culturing a yeast strain as defined in claim 1.

15. The method according to claim 14, wherein the lactic acid bacteria comprise a Lactobacillus species.

Description

DESCRIPTION OF THE FIGURES

[0150] FIG. 1. Relative growth in co-cultures of L. fermentum (LAB) and S. cerevisiae (yeast) (wild-type and mutant with aqr1, qdr2, qdr1 and tpo4 deletions). The graph shows the mean of three independent experiments. The error bars represent standard deviations. Student's t-test, * P value=0.029.

[0151] FIG. 2. Relative growth in co-cultures of L. fermentum (LAB) and S. cerevisiae (yeast) (wild-type and mutant with aqr1, qdr3, qdr2, qdr1, dtr1, hol1 and tpo1 deletions). The graph shows the mean of three independent experiments. The error bars represent standard deviations. Student's t-test, ** P value=0.0096.

[0152] FIG. 3. Relative growth in co-cultures of L. fermentum (L.f.) and S. cerevisiae (wild-type (w-t), mutant with triple aqr1 qdr3 qdr2 deletions, and mutants with single aqr1, qdr3, or qdr2 deletions) (A). Cell densities of L. fermentum (estimated by counting the number of colony forming units (CFU) per ml of culture) just after inoculation (time 0) and 48 hours after incubation at 29° C. in the absence or presence of amino acids. The graph shows the mean of three to four independent experiments. The error bars represent standard deviations. (B) Left. Initial cell densities (number of colony forming units per ml of culture) of L. fermentum (black bars) and yeast (grey bars) just after inoculation of the co-cultures. Right. Cell densities of the co-cultures after 48 hours of incubation at 29° C. The graph shows the mean of four to five independent experiments. The error bars represent standard deviations. Student's t-test, ** P value=0.0014. (C) Propagation ratios of L. fermentum vs. yeast after 48 h of co-culture (calculated from data shown in B). Error bars represent standard deviations. Student's t-test, ** P value=0.0014. (D) Propagation ratios of L. fermentum vs. yeast (wild-type vs. mutants with single aqr1, qdr3, or qdr2 deletion) after 48 h of co-culture (experimental conditions as in B and C). Error bars represent standard deviations. Student's t-test (NS: not significant, p>0.05).

[0153] FIG. 4. Propagation ratios of L. fermentum and S. cerevisiae (wild-type Ethanol Red strain (w-t) vs. derivative mutant with qdr1, qdr2, and qdr3 deletions). All conditions as in FIG. 3B. The graph shows the mean of two independent experiments. Error bars represent standard deviations. Student's t-test, ** P value=0.0181.

EXAMPLES

Example 1: Reduced Expression of Four DHA1-Family Genes in Yeast Causes Reduced Amino Acid Excretion and Reduced Propensity to Support Growth of Cocultivated Lactic Acid Bacteria (LAB)

[0154] Yeast S. cerevisiae strains 23344c (wild-type, wt), GK097 (aqr1Δ qdr2Δ qdr1Δ tpo4Δ) were cocultivated with Lactobacillus fermentum (auxotroph for several amino acids) on a MES-buffered minimal glucose medium (169) devoid of amino acids and containing NH.sub.4.sup.+ as sole nitrogen source. The number of cells per ml of culture was measured just after inoculation (time 0) and 64 h after growth in order to calculate the LAB to yeast cell expansion ratios. As can be seen in FIG. 1, growth of L. fermentum is significantly reduced when cocultivated with S. cerevisiae strain GK097 compared to cocultivation with S. cerevisiae strain 23344c, indicating reduced amino acid excretion by S. cerevisiae strain GK097.

Example 2: Reduced Expression of Seven DHA1-Family Genes in Yeast Causes Reduced Amino Acid Excretion and Reduced Propensity to Support Growth of Cocultivated Lactic Acid Bacteria (LAB)

[0155] Yeast S. cerevisiae strains 23344c (wild-type, wt) and FV1170 (aqr1Δ qdr3Δ qdr2Δ qdr1Δ dtr1Δ hol1Δ tpo1Δ) were cocultivated with Lactobacillus fermentum (auxotroph for several amino acids) on a MES-buffered minimal glucose medium (169) devoid of amino acids and containing NH.sub.4.sup.+ as sole nitrogen source. The number of cells per ml of culture was measured just after inoculation (time 0) and 64 h after growth in order to calculate the LAB to yeast cell expansion ratios. As can be seen in FIG. 2, growth of L. fermentum is significantly reduced when cocultivated with S. cerevisiae strain FV1170 compared to cocultivation with S. cerevisiae strain 23344c, indicating reduced amino acid excretion by S. cerevisiae strain FV1170.

Example 3. Reduced Expression of Three DHA1-Family Genes in Yeast Causes Reduced Amino Acid Excretion and Reduced Propensity to Support Growth of Cocultivated Lactic Acid Bacteria

[0156] (A). The MES-buffered minimal glucose medium (169) containing NH.sub.4.sup.+ as sole nitrogen source, and to which amino acids have been added or not (as amino acid dropout mixture), was inoculated with Lactobacillus fermentum (auxotroph for several amino acids). Samples of the cultures were withdrawn just after inoculation (time 0) and after 48 hours of incubation at 29° C. Cell densities were measured by counting the number of colony-forming units per ml of culture (CFU/ml). The results presented in FIG. 3A confirm that amino acids represent a limiting nutrient for propagation of the bacterium (which is auxotroph for several amino acids). (B). Yeast S. cerevisiae strains 23344c (wild-type, w-t) and GK121 (aqr1Δ qdr3Δ qdr2Δ) were co-cultivated with Lactobacillus fermentum on the MES-buffered minimal glucose medium (169) devoid of amino acids and containing NH.sub.4.sup.+ as sole nitrogen source. The number of colony-forming units per ml of culture (CFU/ml) was measured just after inoculation (time 0) and 48 h after incubation at 29° C. The results illustrated in FIG. 3B show L. fermentum is capable of propagating when co-cultivated with S. cerevisiae, and that this propagation is significantly reduced when S. cerevisiae carries the aqr1Δ, qdr3Δ, and qdr2Δ mutations. (C). Data of (B) were used to calculate the L. fermentum to yeast propagation ratios. As can be seen in FIG. 3C, this propagation ratio is reduced by more than 50% when S. cerevisiae contains the aqr1Δ, qdr3Δ, and qdr2Δ mutations, indicating reduced amino acid excretion by S. cerevisiae strain GK122. (D). Yeast S. cerevisiae strains 23344c (wild-type, w-t), FV812 (aqr1Δ), CM005 (qdr3Δ), or GK089 (qdr2Δ) were co-cultivated with Lactobacillus fermentum as detailed in B. The data were used to calculate the L. fermentum to yeast propagation ratios. As can be seen in FIG. 3D, the propagation ratios were not significantly reduced when S. cerevisiae contains the single aqr1Δ, qdr3Δ, or qdr2Δ mutations.

Example 4. Reduced Expression of Three DHA1-Family Genes in an Industrial Yeast Strain Causes Reduced Amino Acid Excretion and Reduced Propensity to Support Growth of Cocultivated Lactic Acid Bacteria

[0157] Industrial yeast S. cerevisiae strain Ethanol Red (wild-type, w-t) and the derivative strain CF171 (qdr1Δ qdr2Δ qdr3Δ) were cocultivated with Lactobacillus fermentum on a MES-buffered minimal glucose medium (169) devoid of amino acids and containing NH.sub.4.sup.+ as sole nitrogen source. The number of colony forming units per ml of culture (CFU/ml) was measured just after inoculation (time 0) and 48 h after growth in order to calculate the L. fermentum to yeast cell propagation ratios. As can be seen in FIG. 4, growth of L. fermentum is significantly reduced when cocultivated with S. cerevisiae strain CF171 compared to cocultivation with the parental S. cerevisiae Ethanol Red strain, indicating reduced amino acid excretion by S. cerevisiae strain CF171.

[0158] Composition of the 169 Medium Used for Cocultures of S. cerevisiae and L. fermentum

[0159] The medium is prepared by mixing basal 169 medium with samples of trace metals (×1000) and vitamins (×100) solutions. One liter of basal 169 medium contains: 0.7 g MgSO.sub.4.7 H.sub.2O, 1 g KH.sub.2PO.sub.4, 0.4 g CaCl.sub.2.Math.2H.sub.2O, 0.5 g NaCl, 5 g (NH.sub.4).sub.2SO.sub.4, 19.5 g 2-(N-morpholino)ethanesulfonic acid (MES). This medium was adjusted to pH 6.1 with NaOH 10M and sterilized. 100 ml of vitamin solution (×100) contains 15 μg D-biotin, 10 mg thiamine.Math.HCl, 100 mg inositol, 20 mg calcium D-panthothenate, 10 mg pyridoxin.Math.HCl, 5.6 mg folic acid, 9 mg nicotinic acid, 0.5 mg 4-aminobenzoic acid, 9 mg riboflavin, and 150 mg glutathione. 100 ml of trace metals solution (×1000) contains 10 mg H.sub.3BO.sub.4, 1 mg CuSO.sub.4.Math.5H.sub.2O, 2 mg KI, 4 mg Na.sub.2MoO.sub.4.Math.2H.sub.2O, 14 mg ZnSO.sub.4.Math.7H.sub.2O, 10 g citric acid.Math.H.sub.2O, 400 mg MnSO.sub.4.Math.H.sub.2O, 5 g FeCl.sub.3.Math.6H.sub.2O, and 190 mg CoCl.sub.2.Math.H.sub.2O.

[0160] Methods for Isolating Yeast Mutants.

[0161] The genes in the above-described yeast mutants were deleted by either the classical PCR-based replacement with antibiotic resistance genes (Wach et al., 1994) or a recently described CRISPR/Cas9-based method (Mans et al., 2018).

REFERENCES

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