Genetically modified trehalase-expressing yeasts and fermentation processes using such genetically modified yeasts

Patent metadata

Field	Value
Publication number	US-11041218-B2
Application number	US-201816609548-A
Country	US
Kind code	B2
Filing date	May 4, 2018
Priority date	May 4, 2017
Publication date	Jun 22, 2021
Grant date	Jun 22, 2021

Abstract

The present invention relates to genetically engineered yeasts having a heterologous trehalase gene and fermentation processes for using such yeasts. The yeasts can express trehalase in a quantity sufficient to convert significant amounts of trehalose to glucose, thereby improving the yield of the product in a fermentation, and/or reducing or eliminating the need to add exogenous trehalase to the fermentation. The yeasts can also include other heterologous genes for expressing enzymes useful for improving yield and/or for reducing or eliminating the need to add exogenous enzymes to the fermentation.

First claim

The invention claimed is:

1. A genetically modified yeast comprising a heterologous gene encoding a trehalase (EC 3.2.1.28) polypeptide having a sequence identity of at least 80% to at least one of the following polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 87,
wherein the yeast is capable of producing ethanol when the yeast is present in a fermentation medium comprising trehalose and the yeast secretes the trehalase polypeptide in an amount sufficient to reduce the trehalose content of a fermentation broth to less than 2 g/L when the ethanol titer is at least 110 g/L.

2. The yeast of claim 1 , wherein the trehalase polypeptide encoded by the heterologous gene comprises a sequence that has a sequence identity of at least 90%, to SEQ ID NO: 83.

3. The yeast of claim 1 , wherein the trehalase polypeptide encoded by the heterologous gene comprises a sequence that has a sequence identity of to SEQ ID NO: 84 or SEQ ID NO: 85.

4. The yeast of claim 1 , wherein the yeast is a genetically modified S. cerevisiae.

5. The yeast of claim 1 , wherein the trehalase polypeptide encoded by the heterologous gene comprises a MFα2 signal sequence.

6. The yeast of claim 5 , wherein the MFα2 signal sequence is SEQ ID NO: 4.

7. The yeast of claim 5 , wherein the MFα2 signal sequence has a sequence identity of at least 89%, to SEQ ID NO: 4.

8. The yeast of claim 1 , wherein the trehalase polypeptide encoded by the heterologous gene has a sequence identity of at least 85% to at least one of the following polypeptide sequences: SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO:
14, or SEQ ID NO: 92.

9. The yeast of claim 1 , further comprising a heterologous gene encoding a glucoamylase (EC 3.2.1.3) polypeptide.

10. The yeast of claim 1 , wherein the yeast secretes trehalase in an amount sufficient to reduce the trehalose content of a fermentation broth to less than 1 g/L when the ethanol titer is at least 110 g/L.

11. The yeast of claim 1 , wherein the trehalase polypeptide encoded by the heterologous gene comprises a sequence that has a sequence identity of at least 92% to SEQ ID NO: 86.

12. The yeast of claim 1 , wherein the yeast is capable of producing ethanol at a titer of at least 100 g/L as measured 36 h after inoculation in a fermentation medium.

13. A process for manufacturing ethanol comprising:
fermenting a medium using a genetically modified yeast, wherein the yeast comprises a heterologous trehalase gene encoding a trehalase (EC 3.2.1.28) polypeptide having a sequence identity of at least 80% to at least one of the following polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 87,
wherein the ethanol titer is at least 105 g/L as measured 36 h after inoculation and the trehalose content of the fermentation broth at the end of fermentation is less than 2 g/L.

14. The process of claim 13 , wherein the ethanol titer is at least 120 g/L as measured 48 h after inoculation and the trehalase content of the fermentation broth at the end of fermentation is less than 1 g/L.

15. The yeast of claim 1 , wherein the trehalase polypeptide comprises a sequence at least 90% identical to SEQ ID NO:1.

16. The yeast of claim 1 , wherein the trehalase polypeptide comprises a sequence at least 90% identical to SEQ ID NO:2.

17. The yeast of claim 1 , wherein the trehalase polypeptide comprises a sequence at least 90% identical to SEQ ID NO:3.

18. The yeast of claim 1 , wherein the trehalase polypeptide comprises a sequence at least 90% identical to SEQ ID NO:87.

19. The yeast of claim 1 , wherein the trehalase polypeptide comprises a sequence at least 90% identical to at least one of SEQ ID NOs: 8, 11, 14, and 92.

20. The yeast of claim 1 , wherein the yeast has reduced or eliminated expression of one or more genes encoding a glycerol-3-phosphate phosphatase.

Assignees

• Cargill Inc

Inventors

• Jauert Peter Alan
• Poynter Gregory Michael
• Rush Brian J

Classifications

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12R2001/87

  Saccharomyces lactis {; Kluyveromyces lactis} · CPC title

• C12R2001/865

  Saccharomyces cerevisiae · CPC title

• C12R2001/72

  Candida · CPC title

• C12R2001/645

  Fungi {; Processes using fungi} · CPC title