Recombinant host cells having an increase in buoyant density

Patent metadata

Field	Value
Publication number	US-9273336-B2
Application number	US-201113164990-A
Country	US
Kind code	B2
Filing date	Jun 21, 2011
Priority date	Feb 21, 2011
Publication date	Mar 1, 2016
Grant date	Mar 1, 2016

Abstract

Methods are provided to obtain recombinant microbial cells having at least one genetic modification that increase the buoyant density of a recombinant microbial cell or the buoyant density of inclusion bodies produced within a recombinant microbial cell. Exemplified are genetic modifications that increase the buoyant density of a recombinant microbial cell expressing heterologous peptides and polypeptides. Increasing expression of the genes ysaB, glyQ, glyS or a combination thereof within the recombinant microbial cell produces cells or inclusion bodies having higher buoyant density. A similar effect was achieved by decreasing or disrupting expression of the endogenous gltA gene. Increases in buoyant density render peptide production more efficient with respect to time and costs.

First claim

What is claimed is:

1. A method comprising:
a) providing a population of recombinant microbial cells, the recombinant microbial cells in the population comprising;
i) at least one introduced genetic modification that increases expression of GlyS, GlyQ, YsaB, or a combination thereof; and
ii) a chimeric genetic construct encoding a polypeptide of interest, which polypeptide is not GlyS, GlyQ, or YsaB;

b) growing the recombinant microbial cells under suitable conditions whereby the polypeptide of interest is produced and accumulates within the recombinant microbial cells ;
c) fractionating the population of recombinant microbial cells grown in (b) by a density gradient centrifugation;
d) isolating a subpopulation of the recombinant microbial cells from a fraction having a higher buoyant density; and
e) optionally repeating steps (a) through (d).

2. The method of claim 1 wherein the polypeptide of interest accumulates within the recombinant microbial cells in the form of at least one inclusion body.

3. The method of claim 1 wherein polypeptide of interest is 14 to 600 amino acids in length.

4. The method of claim 3 wherein the polypeptide of interest is expressed as a fusion protein.

5. The method of claim 4 wherein the fusion protein comprises the general structure:
IBT-CL-POI
or
POI-CL-IBT
wherein;
IBT =at least one inclusion body tag;
CL =at least one cleavable peptide linker; and POI=the polypeptide of interest.

6. The method of claim 1 wherein the recombinant microbial cells are bacterial cells, yeast cells or fungal cells.

7. The method of claim 6 wherein the recombinant microbial cells are selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida, Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella , and Myxococcus.

8. The method of claim 7 wherein the recombinant microbial cells are Escherichia coli .

9. The method of claim l further comprising introducing at least one genetic modification that decreases or disrupts expression of GltA.

10. The method of claim 1 wherein the isolated subpopulation of cells of step (d) has a buoyant density of at least 1.1 g/mL.

11. A method comprising:
a) providing a population of recombinant microbial cells, the recombinant microbial cells in the population comprising;
i) at least one introduced genetic modification that decreases or disrupts expression of GltA; and
ii) a chimeric genetic construct encoding a polypeptide of interest;

b) growing the recombinant microbial cells under suitable conditions whereby the polypeptide of interest is produced and accumulates within the recombinant microbial cells ;
c) fractionating the population of recombinant microbial cells grown in (b) by a density gradient centrifugation;
d) isolating a subpopulation of the recombinant microbial cells from a fraction having a higher buoyant density; and
e) optionally repeating steps (a) through (d).

12. The method of claim 11 wherein the polypeptide of interest accumulates within the recombinant microbial cells in the form of at least one inclusion body.

13. The method of claim 11 wherein the at least one introduced genetic modification is a knockout mutation.

14. The method of claim 11 wherein polypeptide of interest is 14 to 600 amino acids in length.

15. The method of claim 11 wherein the polypeptide of interest is expressed as a fusion protein.

16. The method of claim 15 wherein the fusion protein comprises the general structure:
IBT-CL-POI
or
POI-CL-IBT
wherein;
IBT=at least one inclusion body tag;
CL=at least one cleavable peptide linker; and POI=the polypeptide of interest.

17. The method of claim 11 wherein the recombinant microbial cells are bacterial cells, yeast cells or fungal cells.

18. The method of claim 17 wherein the recombinant microbial cells are selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida, Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella , and Myxococcus.

19. The method of claim 18 wherein the recombinant microbial cells are Escherichia coli.

20. The method of claim 11 further comprising introducing at least one genetic modification that increases expression of GlyS, GlyQ, YsaB, or a combination thereof.

21. The method of claim 11 wherein the isolated subpopulation of cells of step (d) has a buoyant density of at least 1.1 g/mL.

Assignees

• Bhalla Ritu
• Chen Qi
• Cheng Qiong
• Pandey Neeraj
• Rouviere Pierre E
• Ruebling-Jass Kristin
• Sachan Annapurna
• Du Pont

Inventors

• Bhalla Ritu
• Chen Qi
• Cheng Qiong
• Pandey Neeraj
• Rouviere Pierre E
• Ruebling-Jass Kristin
• Sachan Annapurna

Classifications

• C12N1/02

  Separating microorganisms from their culture media · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12P21/02 Primary

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

• C12N9/1025

  Acyltransferases (2.3) · CPC title