Isobutanol production using yeasts with modified transporter expression

Patent metadata

Field	Value
Publication number	US-9657315-B2
Application number	US-201514976563-A
Country	US
Kind code	B2
Filing date	Dec 21, 2015
Priority date	May 31, 2010
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

The present invention relates to recombinant microorganisms comprising an isobutanol producing metabolic pathway and methods of using said recombinant microorganisms to produce isobutanol. In various aspects of the invention, the recombinant microorganisms may comprise one or more modifications resulting in the reduction in the expression and/or activity of an endogenous transporter protein. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

First claim

What is claimed is:

1. A recombinant microorganism comprising enzymes of an isobutanol producing metabolic pathway, wherein said recombinant microorganism is a yeast microorganism and comprises at least one exogenous gene encoding a polypeptide that catalyzes a step in the conversion of pyruvate to isobutanol, and wherein said recombinant microorganism is engineered to disrupt, partially delete, or completely delete a gene encoding an endogenous transporter protein selected from AQR1 and PDR12, and wherein said recombinant microorganism produces more isobutanol than a corresponding yeast microorganism that has not been engineered to disrupt, partially delete, or completely delete a gene encoding an endogenous transporter protein selected from AQR1 and PDR12.

2. The recombinant microorganism of claim 1 , wherein said recombinant microorganism is engineered to disrupt, partially delete, or completely delete, or inactivate via one or more point mutations, a gene encoding pyruvate decarboxylase.

3. The recombinant microorganism of claim 1 , wherein said recombinant microorganism is engineered to disrupt, partially delete, or completely delete, or inactivate via one or more point mutations, a gene encoding glycerol-3-phosphate dehydrogenase.

4. The recombinant microorganism of claim 1 , wherein said recombinant microorganism is of the Saccharomyces clade.

5. The recombinant microorganism of claim 4 , wherein said recombinant microorganism is a Saccharomyces sensu stricto microorganism.

6. The recombinant microorganism of claim 5 , wherein said Saccharomyces sensu stricto microorganism is selected from the group consisting of S. cerevisiae, S. kudriavzevii, S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids thereof.

7. The recombinant microorganism of claim 1 , wherein said recombinant microorganism is a Crabtree-negative yeast microorganism.

8. The recombinant microorganism of claim 7 , wherein said Crabtree-negative yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Pichia, Hansenula, Issatchenkia and Candida.

9. The recombinant microorganism of claim 8 , wherein said Crabtree-negative yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala, Pichia stipitis, Pichia kudriavzevii, Issatchenkia orientalis, Hansenula anomala, Candida utilis and Kluyveromyces waltii.

10. The recombinant microorganism of claim 1 , wherein said recombinant microorganism is a Crabtree-positive yeast microorganism.

11. The recombinant microorganism of claim 10 , wherein said Crabtree-positive yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichia, Candida , and Schizosaccharomyces.

12. The recombinant microorganism of claim 11 , wherein said Crabtree-positive yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli, Kluyveromyces thermotolerans, Candida glabrata, Zygosaccharomyces bailli, Zygosaccharomyces rouxii, Debaryomyces hansenii, Pichia pastorius, Schizosaccharomyces pombe , and Saccharomyces uvarum.

13. The recombinant microorganism of claim 1 , wherein said recombinant yeast microorganism is a post-WGD (whole genome duplication) yeast microorganism.

14. The recombinant microorganism of claim 13 , wherein said post-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces or Candida.

15. The recombinant microorganism of claim 14 , wherein said post-WGD yeast microorganism is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli , and Candida glabrata.

16. The recombinant microorganism of claim 1 , wherein said recombinant yeast microorganism is a pre-WGD (whole genome duplication) yeast microorganism.

17. The recombinant microorganism of claim 16 , wherein said pre-WGD yeast microorganism is classified into a genus selected from a group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Debaryomyces, Hansenula, Issatchenkia, Pachysolen, Yarrowia and Schizosaccharomyces.

18. The recombinant microorganism of claim 17 , wherein said pre-WGD yeast microorganism is selected from the group consisting of Saccharomyces kluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus, Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichia pastoris, Pichia anomala, Pichia Pichia kudriavzevii, Issatchenkia orientalis, Debaryomyces hansenii, Hansenula anomala, Pachysolen tannophilis, Yarrowia hpolytica , and Schizosaccharomyces pombe.

19. A method of producing isobutanol, comprising:
(a) providing a recombinant microorganism of claim 1 ;
(b) cultivating the recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of isobutanol is produced.

Assignees

• Vib Vzw
• Univ Leuven Kath

Inventors

• Dundon Catherine Asleson
• Smith Christopher
• Nahreini Piruz
• Thevelein Johan
• Saerens Sofie

Classifications

• C12N15/81

  for yeasts · CPC title

• C12N1/16

  Yeasts; Culture media therefor · CPC title

• C12N1/00

  Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor · CPC title

• C07K14/395

  from Saccharomyces · CPC title

• C12P7/16 Primary

  Butanols · CPC title