Directed evolution of microorganisms

• US 6,706,503 B2
• Filed: 10/23/2001
• Issued: 03/16/2004
• 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: Liu, Amy D., Selifonova, Olga V., Schellenberger, Volker
• Primary Examiner(s): McKelvey, Terry
• Assistant Examiner(s): Marvich, Maria
• Application Number:
  US10/037,677
• Publication Number:
  US 20020173003A1
• Time in Patent Office: 875 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 preparing 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;

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 at least 5-100,000 fold relative to wild type; and

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

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

3. The method of claim 2, wherein said hydrolase is selected from the group consisting of proteases, esterases, lipases, phenol, oxidase, permeases, amylases, pullufanaces, cellulases, glucose isomerase, laccases, and protein disulfide isomerases.

4. The method of claim 1, wherein said mocroorganism 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 siad mutator gene.

5. The method of claim 4, wherein said mutator gene comprises mutD mutations.

6. The method of claim 4, wherein said mutator gene comprises mutD mtutations selected form the group of mutD mutations set forth in table 1.

7. A method for preparing an evolved microorganism comprising the step 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;

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

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

8. The method of claim 7, 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.

9. The method of claim 7, 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 precursors, and 1,3-propanadiol derivatives.

10. The method of claim 7, 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.