Pan-yeast autonomously replicating sequence

Patent metadata

Field	Value
Publication number	US-9670495-B2
Application number	US-201414770074-A
Country	US
Kind code	B2
Filing date	Feb 25, 2014
Priority date	Feb 25, 2013
Publication date	Jun 6, 2017
Grant date	Jun 6, 2017

Abstract

A DNA sequence that functions as an origin in many different yeast species. From 1 to 17 mutations can be introduced into this sequence to improve its function across multiple yeasts. The resulting synthetic DNA sequence confers stable plasmid replication function in all yeast species tested, including but not limited to Saccharomyces cerevisiae, Lachancea kluyveri, Kluyveromyces lactis, Kluyveromyces wickerhammii, Hansenula polymorpha , and Pichia pastoris . Also provided are sequences that function as an optimal origin in the industrially useful Pichia pastoris.

First claim

What is claimed is:

1. A recombinant or synthetic nucleic acid molecule selected from the group consisting of SEQ ID NO:1-3, 5-10 and SEQ ID NO:12-19, wherein the nucleic acid molecule includes one or more mutations relative to the corresponding wild type sequence (SEQ ID NO: 11-13, or 16), and wherein the one or more mutations are selected from the optional bases of SEQ ID NO:1-3, 5, 14-15, and 17-18.

2. The nucleic acid molecule of claim 1 , which comprises SEQ ID NO: 1 and wherein the nucleic acid sequence contains at least 2 mutations selected from the optional bases presented in SEQ ID NO: 1 relative to the corresponding wild type sequence shown in SEQ ID NO: 10.

3. The nucleic acid molecule of claim 1 , which is SEQ ID NO: 6 or 7.

4. The nucleic acid molecule of claim 1 , which is SEQ ID NO: 5 and wherein the nucleic acid sequence contains at least 2 mutations selected from the optional bases presented in SEQ ID NO: 5 relative to the corresponding wild type sequence shown in SEQ ID NO: 13.

5. The nucleic acid molecule of claim 1 , which is SEQ ID NO: 9 or 19.

6. A plasmid comprising the nucleic acid molecule of claim 1 .

7. A yeast cell comprising the nucleic acid molecule of claim 1 .

8. The yeast cell of claim 7 , which is a member of the genera consisting of Saccharomyces, Lachancea, Kluyveromyces , and Pichia ( Komagataella ).

9. The yeast cell of claim 7 , which is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces paradoxus, Lachancea kluyveri, Lachancea waltii, Kluyveromyces lactis, Kluyveromyces wickerhammii, Hansenula polymorpha, Pichia pastoris , and Naumovozyma castellii.

10. The yeast cell of claim 9 , which is Pichia pastoris , and wherein the nucleic acid molecule is SEQ ID NO: 9 or 19.

11. A method of producing stable plasmid replication in a yeast cell, the method comprising transforming the plasmid of claim 6 into the yeast cell.

12. The method of claim 11 , wherein the yeast cell is a member of the genera consisting of Saccharomyces, Lachancea, Kluyveromyces , and Pichia ( Komagataella ).

13. The method of claim 11 , wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces paradoxus, Lachancea kluyveri, Lachancea waltii, Kluyveromyces lactis, Kluyveromyces wickerhammii, Hansenula polymorpha, Pichia pastoris , and Naumovozyma castellii.

14. The method of claim 13 , wherein the yeast cell is Pichia pastoris , and wherein the nucleic acid molecule is SEQ ID NO: 9 or 19.

15. A method of enhancing plasmid replication in a yeast cell, the method comprising transforming the plasmid of claim 6 into the yeast cell.

16. The method of claim 15 , wherein the yeast cell is a member of the genera consisting of Saccharomyces, Lachancea, Kluyveromyces , and Pichia ( Komagataella ).

17. The method of claim 15 , wherein the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces paradoxus, Lachancea kluyveri, Lachancea waltii, Kluyveromyces lactis, Kluyveromyces wickerhammii, Hansenula polymorpha, Pichia pastoris , and Naumovozyma castellii.

18. The method of claim 17 , wherein the yeast cell is Pichia pastoris , and wherein the nucleic acid molecule is SEQ ID NO: 9 or 19.

19. A method of producing a shuttle vector for use in multiple species of yeast, the method comprising introducing the nucleic acid molecule of claim 1 into a plasmid.

20. The method of claim 19 , wherein the plasmid is pRS406.

Assignees

• Univ Washington Through Its Center For Commercialization

Inventors

• Dunham Maitreya
• Liachko Ivan

Classifications

• C12N15/69

  Increasing the copy number of the vector · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C12N15/815

  for yeasts other than Saccharomyces · CPC title