Modified microorganisms comprising an optimized system for oligosaccharide utilization and methods of using same

Patent metadata

Field	Value
Publication number	US-11345908-B2
Application number	US-201515327616-A
Country	US
Kind code	B2
Filing date	May 29, 2015
Priority date	May 30, 2014
Publication date	May 31, 2022
Grant date	May 31, 2022

Abstract

The present disclosure generally relates to modified microorganisms comprising an optimized system for oligosaccharide utilization comprising one or more polynucleotides coding for one or more energy independent oligosaccharide transporters for transporting an oligosaccharide into the microorganism, one or more polynucleotides coding for enzymes that catalyze the conversion of the oligosaccharide into at least one phosphorylated saccharide, and one or more polynucleotides coding for enzymes that catalyze the conversion of the phosphorylated saccharide into an isomer of the phosphorylated saccharide that is utilized in one or more enzymatic pathways in the microorganism for the production of an organic molecule such as acetic acid, acrylic acid, 3-hydroxypropionic acid, lactic acid, etc. The present disclosure also generally relates to methods of using the optimized system for oligosaccharide utilization.

First claim

What is claimed is:

1. A method of optimizing utilization of an oligosaccharide in a Saccharomyces microorganism comprising:
a) providing an oligosaccharide source, wherein the oligosaccharide is sucrose;
b) expressing one or more polynucleotides in the Saccharomyces microorganism that encode an energy-independent oligosaccharide transporter for transporting the oligosaccharide into the Saccharomyces microorganism, wherein the oligosaccharide transporter is selected from ‘sugars will eventually be exported transporter’ (SWEET) proteins, wherein the SWEET protein is SWEET14 from Arabidopsis thaliana;
c) disrupting or deleting all energy-dependent transporters which transport the oligosaccharide into the Saccharomyces microorganism;
d) expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphorylase for catalyzing a conversion of the oligosaccharide into at least one phosphorylated saccharide;
e) expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphoglucomutase for catalyzing a conversion of the phosphorylated saccharide into an isomer of the phosphorylated saccharide, wherein the isomer of the phosphorylated saccharide is utilized in one or more enzymatic pathways in the Saccharomyces microorganism; and
f) contacting the oligosaccharide source with the Saccharomyces microorganism.

2. The method of claim 1 , wherein the polynucleotides encoding the phosphorylase are genes selected from the group consisting of: spl of Bifidobacterium adolescentis , sucP of Leuconostoc mesenteroides , LVIS_0358 of Lactobacillus brevis , mapA of Lactobacillus sanfranciscensis , and cbp from Clostridium thermocellum.

3. The method of claim 1 , wherein the phosphoglucomutase is selected from the group consisting of: α-phosphoglucomutase and β-phosphoglucomutase.

4. The method of claim 1 , wherein the phosphoglucomutase is selected from the group consisting of: pgm1 and pgm2 from Saccharomyces cerevisiae , pgmA from Lactobacillus sanfranciscensis , and pgmB from Lactococcus lactis.

5. The method of claim 1 , wherein the oligosaccharide source is contacted with the Saccharomyces microorganism prior to expressing one or more polynucleotides in the Saccharomyces microorganism that encode an energy-independent oligosaccharide transporter for transporting the oligosaccharide into the Saccharomyces microorganism; expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphorylase for catalyzing a conversion of the oligosaccharide into at least one phosphorylated saccharide; and expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphoglucomutase for catalyzing a conversion of the phosphorylated saccharide into an isomer of the phosphorylated saccharide, wherein the isomer of the phosphorylated saccharide is utilized in one or more enzymatic pathways in the Saccharomyces microorganism.

6. The method of claim 1 , wherein the isomer of the phosphorylated saccharide is utilized in a pathway for the production of an organic molecule.

7. The method of claim 1 , wherein the microorganism is S. cerevisiae and one or more of suc2, mal 11, mal12, mal31, or mal32 may be knocked out, or one or more of suc2, mal11, mal12, mal31, mal32, mph2, or mph3 may be knocked out.

8. A method of optimizing utilization of an oligosaccharide in a Saccharomyces microorganism to produce an organic molecule comprising:
a) providing an oligosaccharide source, wherein the oligosaccharide is sucrose;
b) expressing one or more polynucleotides in the Saccharomyces microorganism that encode an energy-independent oligosaccharide transporter for transporting the oligosaccharide into the Saccharomyces microorganism, wherein the oligosaccharide transporter is selected from ‘sugars will eventually be exported transporter’ (SWEET) proteins, wherein the SWEET protein is SWEET14 from Arabidopsis thaliana;
c) disrupting or deleting all energy-dependent transporters which transport the oligosaccharide into the Saccharomyces microorganism;
d) expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphorylase for catalyzing a conversion of the oligosaccharide into at least one phosphorylated saccharide;
e) expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphoglucomutase for catalyzing a conversion of the phosphorylated saccharide into an isomer of the phosphorylated saccharide;
f) expressing one or more polynucleotides in the Saccharomyces microorganism for catalyzing a conversion of the isomer into an organic molecule, wherein the organic molecule is isopropanol; and
g) contacting the oligosaccharide source with the Saccharomyces microorganism.

9. A method of increasing the yield of an organic molecule produced by a Saccharomyces microorganism under anaerobic conditions, the method comprising:
a) providing an oligosaccharide source for use by the Saccharomyces microorganism, wherein the oligosaccharide is sucrose;
b) expressing one or more polynucleotides in the Saccharomyces microorganism that encode an energy-independent oligosaccharide transporter for transporting the oligosaccharide into the Saccharomyces microorganism, wherein the oligosaccharide transporter is selected from ‘sugars will eventually be exported transporter’ (SWEET) proteins, wherein the SWEET protein is SWEET14 from Arabidopsis thaliana;
c) disrupting or deleting all energy-dependent transporters which transport the oligosaccharide into the Saccharomyces microorganism;
d) expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphorylase for catalyzing a conversion of the oligosaccharide into at least one phosphorylated saccharide;
e) expressing one or more polynucleotides in the Saccharomyces microorganism that encode a phosphoglucomutase for catalyzing a conversion of the phosphorylated saccharide into an isomer of the phosphorylated saccharide;
f) expressing one or more polynucleotides in the Saccharomyces microorganism for catalyzing a conversion of the isomer into an organic molecule, wherein the organic molecule is isopropanol; and
g) contacting the oligosaccharide source with the Saccharomyces microorganism.

10. The method of claim 1 , wherein the phosphorylase is sucP of Leuconostoc mesenteroides.

11. The method of claim 8 , wherein the phosphorylase is sucP of Leuconostoc mesenteroides.

12. The method of claim 9 , wherein the phosphorylase is sucP of Leuconostoc mesenteroides.

13. The method of claim 1 , wherein the oligosaccharide transporter, phosphorylase, and phosphoglucomutase are expressed using a constitutive strong promoter.

Assignees

• Braskem Sa

Inventors

• Slovic Avram Michael
• Koch Daniel Johannes

Classifications

• C12P5/007

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title

• C12Y504/02002

  Phosphoglucomutase (5.4.2.2) · CPC title

• C12P19/32

  having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide · CPC title

• C12N9/1048

  Glycosyltransferases (2.4) · CPC title

• C12N9/90 Primary

  Isomerases (5.) · CPC title