Yeast strains exhibiting prolonged persistence during a plurality of fermentation cycles
The present disclosure provides yeasts, which can be recombinant yeast host cells, exhibiting prolonged persistence when submitted to a plurality of fermentation cycles. The yeasts exhibit at least one of the following phenotypic trait: a fast settling phenotype, a rugose phenotype, an improved invertase activity, triploidy, increased signaling in a RAS/cAMP/PKA pathway or combinations thereof.
1. A recombinant yeast host cell expressing a heterologous sugar transporter protein (STL1) and/or having, when compared to a parental cell, a decreased expression of a native NAD-dependent glycerol-3-phosphate dehydrogenase gene,
wherein the heterologous sugar transporter protein comprises the amino acid sequence of any one of SEQ ID NO: 8 and 31 to 69, is a variant having at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 8 and 31 to 69 and glycerol transport activity, or is a fragment having at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 8 and 31 to 69 and glycerol transport activity; and
wherein the recombinant yeast host cell has at least one of phenotypic trait providing persistence of the recombinant yeast host cell in a plurality of fermentation cycles, and wherein the at least one phenotypic trait is triploidy.
2. The recombinant yeast host cell of claim 1 , wherein the recombinant yeast host cell expresses the heterologous sugar transporter protein (STL1).
3. The recombinant yeast host cell of claim 1 , wherein the recombinant yeast host cell has decreased expression of the native NAD-dependent glycerol-3-phosphate dehydrogenase gene.
4. The recombinant yeast host cell of claim 3 , wherein the native NAD-dependent glycerol-3-phosphate dehydrogenase gene is a native glycerol-3-phosphate dehydrogenase 1 (GPD1) gene and/or a native glycerol-3-phosphate dehydrogenase 2 (GPD2) gene.
5. The recombinant yeast host cell of claim 1 further exhibiting a fast settling phenotype.
6. The recombinant yeast host cell of claim 5 , wherein:
(i) at least 5% of a population consisting essentially of the recombinant yeast host cells is able to sediment by gravity after 5 minutes and/or
(ii) the population consisting essentially of the recombinant yeast host cells is able to sediment by gravity in 5 minutes in a proportion equal to or higher than a control population consisting essentially of control yeast cells lacking the fast settling phenotype, and wherein the control yeast cells are from a Saccharomyces cerevisiae PE-2 strain.
7. The recombinant yeast host cell of claim 1 further exhibiting a rugose phenotype.
8. The recombinant yeast host cell of claim 7 , wherein at least 90% of a population consisting essentially of the recombinant yeast host cells, after exponential growth in a medium inoculated at low recombinant yeast host cell density, has at least two daughter cells attached.
9. The recombinant yeast host cell of claim 7 being capable of:
reducing the transcription factor activity of an Activator of CUP1 Expression (ACE2) polypeptide; and/or
expressing a mutated ACE2 polypeptide, wherein the mutated ACE2 polypeptide has decreased activity when compared to a wild type ACE2 polypeptide.
10. The recombinant yeast host cell of claim 1 further exhibiting improved invertase activity.
11. The recombinant yeast host cell of claim 10 , wherein a population consisting essentially of the recombinant yeast host cells is able to hydrolyze more than 0.05 gram of sucrose per gram of dry cell weight per minute and/or exhibits more than 1.0 times invertase activity than a control population consisting essentially of control yeast cells lacking the improved invertase activity phenotypic trait, wherein the control yeast cells are from a Saccharomyces cerevisiae PE-2 strain, and wherein invertase activity is measured after exponential growth of the population diluted to a concentration of 9 mg/mL on a wet cell weight in a buffer and wherein the buffer comprises 40 g/L of sucrose, is at of pH 5 and at a temperature of 35° C.
12. The recombinant yeast host cell of claim 1 being capable of increasing the enzymatic activity of at least one polypeptide having invertase activity.
13. The recombinant yeast host cell of claim 12 , wherein the at least one polypeptide having invertase activity comprises SUC1, SUC2, SUC3, SUC4, SUC5, SUC6, SUC7, SUC8 or SUC9.
14. The recombinant yeast host cell of claim 1 further exhibiting increased signaling in the RAS/cAMP/PKA pathway.
15. The recombinant yeast host cell of claim 14 , wherein a population consisting essentially of the recombinant yeast host cells is able to exhibit a fold increase in the production of cAMP of equal to or less than 1.7 and/or a fold increase in the production of cAMP production of less than 70% when compared a control population consisting essentially of control yeast cells lacking the increased signaling in the RAS/cAMP/PKA pathway phenotypic trait, wherein the control yeast cells are from a Saccharomyces cerevisiae PE-2 strain, and wherein the production of cAMP is measured in the population having been glucose depleted and at 5 minutes after a glucose spike.
16. The recombinant yeast host cell of claim 14 being capable of expressing a mutated polypeptide involved in the RAS/cAMP/PKA pathway.
17. The recombinant yeast host cell of claim 16 , wherein the mutated polypeptide involved in the RAS/cAMP/PKA pathway comprises a mutated RAS2 polypeptide having increased activity when compared to a wild-type RAS2 polypeptide, a mutated IRA2 polypeptide having a reduced inhibitory activity towards a wild-type RAS1 and/or a wild-type RAS2 polypeptide when compared to a wild-type IRA2 polypeptide.
18. The recombinant yeast host cell of claim 1 being from the genus Saccharomyces sp. or from the species Saccharomyces cerevisiae.
19. The recombinant yeast host cell of claim 2 comprising a heterologous nucleic acid encoding a sugar transporter protein (STL1).
20. The recombinant yeast host cell of claim 4 having a deletion in the native glycerol-3-phosphate dehydrogenase 1 gene (GPD1).
21. The recombinant yeast host cell of claim 4 having a deletion in the native glycerol-3-phosphate dehydrogenase 2 gene (GPD2).
22. The recombinant yeast host cell of claim 1 , wherein the heterologous sugar transporter protein (STL1) comprises the amino acid sequence of SEQ ID NO: 8, is the variant having at least 70% identity to the amino acid sequence of SEQ ID NO: 8 and glycerol transport activity, or is the fragment having at least 70% identity to the amino acid sequence of SEQ ID NO: 8 and glycerol transport activity.
23. The recombinant yeast host cell of claim 1 , wherein the heterologous sugar transporter protein (STL1) comprises the amino acid sequence of any one of SEQ ID NO: 31 to 56, 68, or 69 is the variant having at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 31 to 56, 68, and 69 and glycerol transport activity, or is the fragment having at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 31 to 56, 68, and 69 and glycerol transport activity.