Genetically modified microorganisms capable of producing β-glucans and methods for producing β-glucans

Patent metadata

Field	Value
Publication number	US-9388441-B1
Application number	US-201615074330-A
Country	US
Kind code	B1
Filing date	Mar 18, 2016
Priority date	Jul 4, 2012
Publication date	Jul 12, 2016
Grant date	Jul 12, 2016

Abstract

The present invention relates to genetically modified microorganisms capable of producing beta-glucans, characterized in that the genetically modified microorganism overexpresses (i) a polynucleotide encoding a polypeptide having 1,3-β-D-glucan synthase-activity, and/or (ii) a polypeptide having 1,3-β-D-glucan synthase-activity, compared to a corresponding non-modified control microorganism of the same strain. The present invention also relates to the use of a polynucleotide encoding a polypeptide having 1,3-β-D-glucan synthase-activity or the use of such a polypeptide for producing β-glucans. Furthermore, the present invention relates to methods for producing β-glucans comprising the introduction of a promoter upstream of a polynucleotide encoding a polypeptide having 1,3-β-D-glucan synthase-activity thereby increasing the expression of the polynucleotide, or a polynucleotide encoding a polypeptide having 1,3-β-D-glucan synthase-activity into a microorganism being able to synthesize β-glucans.

First claim

The invention claimed is:

1. A genetically modified microorganism capable of producing a polymer consisting of a linear main chain of β-D-(1-3)-glucopyranosyl units having a single β-D-glucopyranosyl unit (1-6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, characterized in that said genetically modified microorganism overexpresses (i) a polynucleotide encoding a polypeptide having 1,3-β-D-glucan synthase activity, wherein said polynucleotide comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15, and/or (ii) a polypeptide having 1,3-β-D-glucan synthase activity, wherein said polypeptide comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16, compared to a corresponding non-modified control microorganism of the same strain.

2. The genetically modified microorganism of claim 1 , wherein said polymer is selected from the group consisting of schizophyllan, scleroglucan, pendulan, cinerian, laminarin, lentinan and pleuran.

3. The genetically modified microorganism of claim 1 , wherein said polynucleotide is a 1,3-β-D-glucan synthase gene.

4. The genetically modified microorganism of claim 1 , wherein said polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15.

5. The genetically modified microorganism of claim 1 , wherein said polynucleotide comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15.

6. The genetically modified microorganism of claim 1 , wherein said polypeptide is a 1,3-β-D-glucan synthase.

7. The genetically modified microorganism of claim 1 , wherein said polypeptide comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

8. The genetically modified microorganism of claim 1 , wherein said polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

9. The genetically modified microorganism of claim 1 , wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15, or wherein said polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16.

10. The genetically modified microorganism of claim 1 , wherein said microorganism is selected from the group consisting of Schizophyllum commune, Sclerotium rolfsii, Sclerotium glucanicum, Sclerotium delphinii, Porodisculus pendulus, Botrytis cinerea, Laminaria sp., Lentinula edoles , and Monilinia fructigena.

11. The genetically modified microorganism of claim 1 , wherein said modified microorganism is able to produce at least 1.5 times more of said polymer compared to said non-modified control microorganism.

12. A method of producing a polymer consisting of a linear main chain of β-D-(1-3)-glucopyranosyl units having a single β-D-glucopyranosyl unit (1-6) linked to a β-D-glucopyranosyl unit of the linear main chain with an average branching degree of about 0.3, said method comprising the steps of:
(a) introducing a polynucleotide encoding a polypeptide having 1,3-β-D-glucan synthase-activity into a microorganism being able to synthesize said polymer, wherein said polynucleotide comprises:
(i) a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15; or
(ii) a nucleotide sequence encoding a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16,
and wherein said polynucleotide is optionally downstream of a strong promoter thereby increasing the expression of said polynucleotide;

(b) culturing said microorganism of (a) in a medium, thereby allowing said microorganism to produce said polymer; and
(c) optionally recovering said polymer from the medium.

13. The method of claim 12 , wherein said polymer is selected from the group consisting of schizophyllan, scleroglucan, pendulan, cinerian, laminarin, lentinan and pleuran.

14. The method of claim 12 , wherein said polynucleotide is a 1,3-β-D-glucan synthase gene.

15. The method of claim 12 , wherein said polynucleotide comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15.

16. The method of claim 12 , wherein said polynucleotide encodes a 1,3-β-D-glucan synthase.

17. The method of claim 12 , wherein said polynucleotide encodes a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

18. The method of claim 12 , wherein said polynucleotide comprises:
(a) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15; or
(b) a nucleotide sequence encoding a polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 16.

19. The method of claim 12 , wherein said polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15, or wherein said polynucleotide encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, or SEQ ID NO: 16.

20. The method of claim 12 , wherein said microorganism is selected from the group consisting of Schizophyllum commune, Sclerotium rolfsii, Sclerotium glucanicum, Sclerotium delphinii, Porodisculus pendulus, Botrytis cinerea, Laminaria sp., Lentinula edoles , and Monilinia fructigena.

Assignees

• Basf Se
• Wintershall Holding GmbH

Inventors

• Brockmann Beata
• Herold Andrea
• Zelder Oskar
• Haefner Stefan
• Fleck Christian
• Schröder Hartwig
• Granström Mari
• Schmidt Julia Kristiane

Classifications

• C08B37/0024

  beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof · CPC title

• C12N15/80

  for fungi · CPC title

• C12Y204/01034

  1,3-Beta-glucan synthase (2.4.1.34) · CPC title

• C12N9/1051

  Hexosyltransferases (2.4.1) · CPC title

• Y02P20/52

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