Acid-resistance in Kluyveromyces marxianus by engineering transcriptional factor

Patent metadata

Field	Value
Publication number	US-9605284-B2
Application number	US-201414531508-A
Country	US
Kind code	B2
Filing date	Nov 3, 2014
Priority date	Nov 1, 2013
Publication date	Mar 28, 2017
Grant date	Mar 28, 2017

Abstract

A microorganism with improved acid-resistance. A microorganism capable of efficiently producing 3-HP, and methods for producing an organic acid.

First claim

What is claimed is:

1. A recombinant Kluyveromyces marxianus yeast cell comprising a gene encoding a mutated subunit of the RNA polymerase II (RNAP II) complex, wherein the mutated subunit increases the acid resistance of the yeast cell, and the mutated subunit is a mutated TATA-box binding protein (TBP) comprising a sequence selected from the group consisting of SEQ ID NO: 2, 3, and 4.

2. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus yeast cell is capable of surviving or growing at a pH of 2.5 or lower.

3. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus yeast cell further comprises a heterologous polynucleotide encoding a protein needed to synthesize an organic acid or 1,4-butandiol, and the organic acid is lactic acid, succinic acid, malic acid, 3-hydroxypropionic acid or 4-hydroxybutyric acid.

4. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus yeast cell comprises at least one pathway selected from the group consisting of malonyl CoA pathway, β-alanine pathway, and glycerol pathway.

5. The recombinant Kluyveromyces marxianus yeast cell of claim 3 , wherein the heterologous polynucleotide encodes a protein selected from the group consisting of 3-hydroxyisobutyl-CoA hydrolase, 3-hydroxyisobutyrate dehydrogenase, 3-hydroxypropionyl-CoA hydrolase, 3-hydroxypropionyl-CoA dehydratase, acetyl-CoA carboxylase, aspartate decarboxylase, CoA transferase, malonyl-CoA reductase, phosphoenolpyruvate (PEP) carboxylase, 3-oxopropanoate:NADP+ oxidoreductase, malonate semialdehyde reductase, 3-hydroxypropionate dehydrogenase, 4-aminobutyrate aminotransferase, acetyl-CoA carboxylase, aspartate aminotransferase, aspartate decarboxylase, glutamate dehydrogenase, OS17 enzyme, pyruvate carboxylase, β-alanyl-CoA ammonia lyase, glycerol dehydratase, and aldehyde dehydrogenase.

6. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus further comprises a heterologous polynucleotide encoding a protein selected from the group consisting of 3-hydroxyisobutryl-CoA hydrolase, 3-hydroxyisobutyrate dehydrogenase, 3-hydroxypropionyl-CoA hydrolase, 3-hydroxypropionyl-CoA dehydratase, acetyl-CoA carboxylase, aspartate decarboxylase, CoA transferase, malonyl-CoA reductase, PEP carboxylase, 3-oxopropanoate:NADP+ oxidoreductase, malonate semialdehyde reductase, and 3-hydroxypropionate dehydrogenase.

7. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus further comprises one or more heterologous polynucleotides encoding 3-oxopropanoate:NADP+ oxidoreductase and 3-hydroxypropionate dehydrogenase.

8. The recombinant Kluyveromyces marxianus yeast cell of claim 6 , wherein the Kluyveromyces marxianus further comprises one or more heterologous polynucleotides encoding malonyl-CoA reductase and malonate semialdehyde reductase.

9. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus yeast cell further comprises a heterologous polynucleotide encoding a protein selected from the group consisting of 3-hydroxyisobutyrate dehydrogenase, 4-aminobutyrate aminotransferase, acetyl-CoA carboxylase, aspartate aminotransferase, aspartate decarboxylase, glutamate dehydrogenase, OS17 enzyme, pyruvate carboxylase, β-alanyl-CoA ammonia lyase, and 3-hydroxypropionate dehydrogenase.

10. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus yeast cell further comprises a heterologous polynucleotide encoding 3-hydroxypropionate dehydrogenase.

11. The recombinant Kluyveromyces marxianus yeast cell of claim 1 , wherein the Kluyveromyces marxianus yeast cell further comprises one or more heterologous polynucleotides encoding glycerol dehydratase and aldehyde dehydrogenase.

12. A method of producing an organic acid comprising culturing a recombinant Kluyveromyces marxianus yeast cell of claim 1 , whereby the yeast cell produces an organic acid; and
recovering the organic acid from the culture solution.

13. The method of claim 12 , wherein the organic acid is selected from the group consisting of 3-hydroxypropionate (3-HP), 1,4-butandiol, lactic acid, and succinic acid.

14. The method of claim 12 , wherein the organic acid is a 3-hydroxypropionate (3-HP) and the heterologous polynucleotide encodes a protein selected from the group consisting of 3-hydroxyisobutryl-CoA hydrolase, 3-hydroxyisobutyrate dehydrogenase, 3-hydroxypropionyl-CoA hydrolase, 3-hydroxypropionyl-CoA dehydratase, acetyl-CoA carboxylase, aspartate decarboxylase, CoA transferase, malonyl-CoA reductase, PEP carboxylase, 3-oxopropanoate:NADP+ oxidoreductase, malonate semialdehyde reductase, and 3-hydroxypropionate dehydrogenase.

15. The method of claim 12 , wherein the organic acid is a 3-hydroxypropionate (3-HP) and the heterologous polynucleotide encodes a protein selected from the group consisting of 3-hydroxyisobutyrate dehydrogenase, 4-aminobutyrate aminotransferase, acetyl-CoA carboxylase, aspartate aminotransferase, aspartate decarboxylase, glutamate dehydrogenase, OS17 enzyme, pyruvate carboxylase, β-alanyl-CoA ammonia lyase, and 3-hydroxypropionate dehydrogenase.

16. The method of claim 12 , wherein the organic acid is a 3-hydroxypropionate (3-HP) and the Kluyveromyces marxianus yeast cell further comprises one or more heterologous polynucleotides encoding glycerol dehydratase and aldehyde dehydrogenase.

17. The method of claim 12 , wherein the recombinant Kluyveromyces marxianus yeast cell is cultured under aerobic conditions.

Assignees

• Samsung Electronics Co Ltd

Inventors

• Lee Juyoung
• Koo Hyunmin
• Park Jaechan
• Kim Jieun
• Kim Jinha
• Park Joonsong
• Chung Soonchun
• Cho Byungkwan

Classifications

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title

• C12P7/56

  Lactic acid · CPC title

• C12P7/46

  Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid · CPC title

• C07K14/4705

  stimulating, promoting or activating activity · CPC title

• C12P7/42 Primary

  Hydroxy-carboxylic acids · CPC title