Mutated enzyme of flavin containing monooxygenase with increased indigo production and recombinant microorganism producing the same

• US 10,626,427 B2
• Filed: 04/22/2019
• Issued: 04/21/2020
• Est. Priority Date: 04/24/2018
• Status: Active Grant

• Current Assignee: Gyeongbuk Institute For Marine Bioindustry
• Original Assignee: Gyeongbuk Institute For Marine Bioindustry
• Inventors: Woo, Jung Hee, Kim, Hae Seon, Park, Nyun-Ho
• Primary Examiner(s): Monshipouri, Maryam
• Application Number:
  US16/390,083
• Publication Number:
  US 20190323047A1
• Time in Patent Office: 365 Days
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0073 with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
• CPC Class Codes: C12P 17/165 Heterorings having nitrogen atoms as the only ring heteroatoms
• CPC Class Codes: C12Y 114/13008 Flavin-containing monooxygenase (1.14.13.8), i.e. dimethylaniline-monooxygenase

Abstract

The present invention relates to a mutated protein of FMO derived from Celeribacter sp. and a gene encoding the same, a vector comprising the gene, a recombinant cell transformed by the vector, a composition for producing indigo comprising them, and a method for increasing indigo production in the recombinant cell using the transformed recombinant cell.

Claims

1. A mutated enzyme having flavin containing monooxygenase activity (FMO), comprising the amino acid sequence of SEQ ID NO:

1.

2. A nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

2.

4. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

3.

5. A recombinant vector comprising a nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1.

6. A recombinant microorganism which comprises a nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1.

7. The recombinant microorganism of claim 6, wherein the recombinant microorganism is E. coli or yeast.

8. The recombinant microorganism of claim 6, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

2 or SEQ ID NO;

3.

9. A method for production of indigo, comprising a step of culturing a recombinant microorganism which comprises a nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1 and produces indigo in the presence of indole or tryptophan.

10. The method for production of indigo of claim 9, wherein the concentration of tryptophan is 0.1 to 0.4% (w/v).

11. The method for production of indigo of claim 9, wherein the recombinant microorganism is cultured at the temperature range of 20 to 40°

C.

12. The method for production of indigo of claim 9, wherein the recombinant microorganism is cultured for 20 to 60 hours.

13. The method for production of indigo of claim 9, wherein the culturing is carried out in a medium containing 0.5 to 1.5% (w/v) of NaCl and 0.2 to 0.8% (w/v) of yeast extract.

14. The method for production of indigo of claim 9, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:

2 or SEQ ID NO;

3.

15. The method for production of indigo of claim 9, wherein the step of culturing a recombinant microorganism is performed under the condition in which the air supply is 0.3 to 1.5 vvm and the stirring speed is 300 to 600 rpm.

16. The method for production of indigo of claim 15, wherein the air supply and the stirring speed are increased with the culture time.