Genome-wide rationally-designed mutations leading to enhanced lysine production in E. coli

Patent metadata

Field	Value
Publication number	US-11078458-B2
Application number	US-202117159137-A
Country	US
Kind code	B2
Filing date	Jan 26, 2021
Priority date	Jun 21, 2019
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

The present disclosure relates to various different types of variants in E. coli coding and noncoding regions leading to enhanced lysine production for, e.g., supplements and nutraceuticals.

First claim

We claim:

1. An engineered E. coli cell comprising the following variant sequences: a promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of a dapA gene, and further comprising one of the following proteins expression of which is not driven by a promoter sequence having a nucleic acid of SEQ ID NO: 2: a mfdY5 protein having the amino acid sequence of SEQ ID NO: 21, a nupXR5 protein having the amino acid sequence of SEQ ID NO: 22, a pck protein having the amino acid sequence of SEQ ID NO: 23, a rlmL protein having the amino acid sequence of SEQ ID NO: 26, a wzxB protein having the amino acid sequence of SEQ ID NO: 27, a ytfP protein having the amino acid sequence of SEQ ID NO: 33, a marA protein having the amino acid sequence of SEQ ID NO: 34, a marA protein having the amino acid sequence of SEQ ID NO: 35, a metL protein having the amino acid sequence of SEQ ID NO: 36, a metL protein having the amino acid sequence of SEQ ID NO: 37, a pck protein having the amino acid sequence of SEQ ID NO: 38.

2. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the mfdY5 protein having the amino acid sequence of SEQ ID NO: 21.

3. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the nupXR5 protein having the amino acid sequence of SEQ ID NO: 22.

4. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the pck protein having the amino acid sequence of SEQ ID NO: 23.

5. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the rlmL protein having the amino acid sequence of SEQ ID NO: 26.

6. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the wzxB protein having the amino acid sequence of SEQ ID NO: 27.

7. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the ytfP protein having the amino acid sequence of SEQ ID NO: 33.

8. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the marA protein having the amino acid sequence of SEQ ID NO: 34.

9. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the marA protein having the amino acid sequence of SEQ ID NO: 35.

10. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the metL protein having the amino acid sequence of SEQ ID NO: 36.

11. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the metL protein having the amino acid sequence of SEQ ID NO: 37.

12. The engineered E. coli cell of claim 1 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the pck protein having the amino acid sequence of SEQ ID NO: 38.

13. An engineered E. coli cell comprising the following variant sequences: a promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of a dapA gene, and further comprising one of the following: a promoter sequence having the nucleic acid sequence of SEQ ID NO: 24 driving expression of a phoB protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 25 driving expression of a purM protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 28 driving expression of a ydgl protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 29 driving expression of a ydgE protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 30 driving expression of a yicL protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 31 driving of a yliE protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 32 driving expression of a yohF protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 39 driving expression of a setB protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 40 driving expression of a ydfO protein; a promoter sequence having the nucleic acid sequence of SEQ ID NO: 41 driving expression of a ydgD protein; or a promoter sequence having the nucleic acid sequence of SEQ ID NO: 42 driving expression of a yejD protein.

14. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 24 driving expression of the phoB protein.

15. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 25 driving expression of the purM protein.

16. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 28 driving expression of the ydg1 protein.

17. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 29 driving expression of the ydgE protein.

18. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 30 driving expression of the yicL protein.

19. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 31 driving expression of the yliE protein.

20. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 32 driving expression of the ydg1 protein.

21. The engineered E. coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID NO: 2 driving transcription of the dapA gene and further comprising the promoter sequence having the nucleic acid sequence of SEQ ID NO: 39 driving expression of the setB protein.

22. The engineered E coli cell of claim 13 comprising the promoter sequence having the nucleic acid SEQ ID

Assignees

• Inscripta Inc

Inventors

• Fox Richard
• Held Daniel
• Abbate Eric
• Clay Michael
• Krouse Katherine
• Krishnamurthy Nandini
• Yerramsetty Krishna

Classifications

• C12N9/88

  Lyases (4.) · CPC title

• C12N2510/02

  Cells for production · CPC title

• C12P13/08 Primary

  Lysine; Diaminopimelic acid; Threonine; Valine · CPC title

• C12N2511/00

  Cells for large scale production · CPC title

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title