Enhanced protein expression

Patent metadata

Field	Value
Publication number	US-10683528-B2
Application number	US-201515529948-A
Country	US
Kind code	B2
Filing date	Dec 3, 2015
Priority date	Dec 16, 2014
Publication date	Jun 16, 2020
Grant date	Jun 16, 2020

Abstract

The present invention relates in general to bacterial cells having genetic alterations that result in increased expression of a protein of interest and methods of making and using such cells. Aspects of the present invention include altered Gram positive microorganisms having one or more a genetic alterations that reduce the expression of a gene in the sin operon, thereby resulting in the enhanced expression of one or more proteins of interest.

First claim

The invention claimed is:

1. A method for increasing expression of a protein of interest (POI) in a Bacillus spp. bacterial cell comprising:
(a) obtaining an altered Bacillus cell producing a protein of interest, wherein the altered Bacillus cell comprises a genetic alteration of the sinR gene comprising a G to A silent mutation at a nucleotide position corresponding to nucleotide position 330 of SEQ ID NO: 1; and
(b) culturing the altered Bacillus cell under conditions such that the POI is expressed, wherein the increased amount of the POI is relative to the expression of the same POI in an unaltered Bacillus spp. cell.

2. The method of claim 1 , wherein the Bacillus cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. sonorensis, B. halodurans, B. pumilus, B. lautus, B. pabuli, B. cereus, B. agaradhaerens, B akibai, B. clarkii, B. pseudofirmus, B. lehensis, B. megaterium, B. coagulans, B. circulans, B. gibsonii , and B. thuringiensis.

3. The method of claim 1 , wherein the POI is encoded by a gene heterologous to the altered bacterial cell or a gene endogenous to the altered bacterial cell.

4. The method of claim 1 , wherein the POI is an enzyme is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, α-galactosidases, β-galactosidases, α-glucanases, glucan lysases, endo-β-glucanases, glucoamylases, glucose oxidases, α-glucosidases, β-glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

5. The method of claim 1 , further comprising recovering the POI.

6. The method of claim 1 , wherein the increased amount of an expressed POI relative to unaltered Bacillus spp. cell is at least 10%.

7. An altered Bacillus spp. bacterial cell expressing an increased amount of a protein of interest (POI) relative to the expression of the same POI in an unaltered Bacillus spp. cell, wherein the altered cell comprises a genetic alteration of the sinR gene comprising a G to A silent mutation at a nucleotide position corresponding to nucleotide position 330 of SEQ ID NO: 1.

8. The altered cell of claim 7 , wherein the Bacillus cell is selected from the group consisting of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. sonorensis, B. halodurans, B. pumilus, B. lautus, B. pabuli, B. cereus, B. agaradhaerens, B akibai, B. clarkii, B. pseudofirmus, B. lehensis, B. megaterium, B. coagulans, B. circulans, B. gibsonii , and B. thuringiensis.

9. The altered cell of claim 7 , wherein the POI is encoded by a gene heterologous to the altered bacterial cell or a gene endogenous to the altered bacterial cell.

10. The altered cell of claim 7 , wherein the POI is an enzyme is selected from the group consisting of acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, carboxypeptidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, α-galactosidases, β-galactosidases, α-glucanases, glucan lysases, endo-β-glucanases, glucoamylases, glucose oxidases, α-glucosidases, β-glucosidases, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, laccases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, perhydrolases, polyol oxidases, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidases, and combinations thereof.

11. The altered cell of claim 7 , wherein the increased amount of an expressed POI relative to the unaltered Bacillus spp. cell is at least 10%.

12. A polynucleotide comprising a G to A silent mutation at a nucleotide position corresponding to nucleotide position 330 of SEQ ID NO: 1.

13. A vector comprising the polynucleotide sequence of claim 12 .

Assignees

• Danisco Us Inc

Inventors

• Bongiorni Cristina
• Gonzales Caguiat Rodante
• Fioresi Carol Marie
• Schmidt Brian F
• Van Kimmenade Anita
• Zhu Chao

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/75

  for Bacillus · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C07K14/32 Primary

  from Bacillus (G) · CPC title