Directed evolution of microorganisms

• US 7,125,695 B2
• Filed: 11/20/2003
• Issued: 10/24/2006
• Est. Priority Date: 05/19/1999
• Status: Expired due to Fees

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Schellenberger, Volker, Liu, Amy D., Selifonova, Olga V.
• Primary Examiner(s): Nguyen, Dave Trong
• Assistant Examiner(s): Marvich, Maria
• Application Number:
  US10/719,571
• Publication Number:
  US 20040086972A1
• Time in Patent Office: 1,069 Days
• Field of Search: 435/170, 435/252.3, 435/440, 435/441, 435/233, 435/234, 435/195, 435/183, 435/173.8, 435/471, 435/479, 435/481, 435/482, 435/69.1
• US Class Current: 435/170
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12R 2001/01 Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• CPC Class Codes: C12R 2001/19 Escherichia coli

Abstract

The present invention provides methods for directing the evolution of microorganisms comprising the use of mutator genes and growth under conditions of selective pressure. The method discloses mutator genes which can be used in the methods of the present invention and provides ATCC deposits which exemplify the evolved microorganisms produced by the methods.

Claims

1. A method for producing a protein from an evolved microorganism comprising the steps of:

a) obtaining a microorganism comprising at least one heterologous mutator gene and at least one introduced nucleic acid encoding at least one heterologous protein;

b) culturing said microorganism for at least 20 doublings under conditions suitable for selection of an evolved microorganism, wherein said heterologous mutator gene generates a mutation rate of at least 5–

100,000 fold relative to wild type, and wherein said heterologous protein is expressed by said microorganism; and

c) restoring said evolved microorganism to a wild type mutation rate.

2. The method of claim 1, further comprising the step of isolating said at least one heterologous protein from said evolved microorganism.

3. The method of claim 1, wherein said at least one heterologous protein is a hydrolase.

4. The method of claim 3, wherein said hydrolase is selected from the group consisting of proteases, esterases, lipases, phenol oxidase, permeases, amylases, pullulananses, cellulases, glucose isomerase, laccases, and protein disulfide isomerases.

5. The method of claim 1, wherein said microorganism comprises at least one copy of said mutator gene in its chromosome and said step of restoring said evolved microorganism to wild-type mutation rate comprises excision of said mutator gene.

6. The method of claim 1, wherein said mutator gene is a mutD gene comprising at least one mutation.

7. The method of claim 6, wherein said at least one mutation is selected from the group of mutD mutations set forth in Table 1.

8. The method of claim 1, wherein said microorganism to be evolved is selected from the group consisting of E. coli and E. blattae.

9. The method of claim 1, wherein said microorganism comprises a plasmid comprising the heterologous mutator gene and said step of restoring said evolved microorganism to a wild type mutation rate comprises curing the evolved microorganism of said plasmid.

10. The method of claim 9, wherein said plasmid comprises a temperature sensitive origin of replication.

11. A method for producing a heterologous protein in an evolved microorganism comprising the steps of:

a) obtaining a microorganism comprising at least one heterologous mutator gene and at least one introduced nucleic acid encoding at least one heterologous protein, wherein said at least one heterologous protein is an enzyme necessary for an enzymatic pathway in a microorganism;

b) culturing said microorganism for at least 20 doublings under conditions suitable for selection of an evolved microorganism, wherein said heterologous mutator gene generates a mutation rate of at least 5 to 100,000-fold relative to wild type, and wherein said heterologous protein is expressed by said microorganism; and

c) restoring said evolved microorganism to a wild type mutation rate.

12. The method of claim 11, wherein said enzyme is selected from the group consisting of reductases and dehydrogenases, and further wherein said enzymatic pathway results in the production of at least one compound selected from the group consisting of ascorbic acid or ascorbic acid intermediates.

13. The method of claim 12, wherein said enzyme is selected from the group consisting of glycerol dehydratase and 1,3-propanediol dehydrogenase, and further wherein said enzymatic pathway results in the production of at least one compound selected from the group consisting of 1,3-propanediol, 1,3-propanediol precursors, and 1,3-propanediol derivatives.

14. The method of claim 12, wherein said enzyme is selected from the group consisting of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatases, and further wherein said enzymatic pathway results in the production of at least one compound selected from the group consisting of glycerol and glycerol derivatives.

15. The method of claim 11, further comprising the step of isolating said at least one heterologous protein from said evolved microorganism.

16. The method of claim 11, wherein said microorganism is selected from the group consisting of E. coli and E. blattae.

17. The method of claim 11, wherein said microorganism comprises a plasmid comprising the heterologous mutator gene and said step of restoring said evolved microorganism to a wild type mutation rate comprises curing the evolved microorganism of said plasmid.

18. The method of claim 11, wherein said plasmid comprises a temperature sensitive origin of replication.

19. The method of claim 18, wherein said mutator gene is a mutD gene comprising at least one mutation.

20. The method of claim 11, wherein said mutator gene comprises mutations or.