Genetic perturbation of the RNA degradosome protein complex

Patent metadata

Field	Value
Publication number	US-11549096-B2
Application number	US-201716314953-A
Country	US
Kind code	B2
Filing date	Jun 27, 2017
Priority date	Jul 5, 2016
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

The present disclosure provides novel bacterial strains with altered expression or start codon modification of one or more RNA degradation/processing genes. The RNA degradation genes of the present disclosure are controlled by heterologous promoters. The present disclosure further describes methods for generating microbial strains comprising heterologous promoter sequences operably linked to RNA degradation/processing genes.

First claim

The invention claimed is:

1. A genetically engineered prokaryotic host cell with enhanced industrial performance, said host cell comprising:
a. a heterologous promoter polynucleotide, and
b. a polynucleotide encoding an RNA degradation gene;

wherein the heterologous promoter polynucleotide is operably linked to the polynucleotide encoding the RNA degradation gene, wherein the heterologous promoter comprises a promoter from a promoter ladder; wherein the polynucleotide encoding the RNA degradation gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 9, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 22.

2. The genetically engineered host cell of claim 1 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1-8.

3. The genetically engineered host cell of claim 1 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 5, and wherein the polynucleotide encoding the RNA degradation gene encodes for SEQ ID NO: 45.

4. The genetically engineered host cell of claim 3 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 1.

5. The genetically engineered host cell of claim 3 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

6. The genetically engineered host cell of claim 1 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 5, and wherein the polynucleotide encoding the RNA degradation gene comprises SEQ ID NO: 20.

7. The genetically engineered host cell of claim 6 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 1.

8. The genetically engineered host cell of claim 6 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

9. The genetically engineered host cell of claim 1 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5, and SEQ ID NO: 2, and wherein the polynucleotide encoding the RNA degradation gene encodes for SEQ ID NO: 36.

10. The genetically engineered host cell of claim 9 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

11. The genetically engineered host cell of claim 1 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5, and SEQ ID NO: 2, and wherein the polynucleotide encoding the RNA degradation gene comprises SEQ ID NO: 11.

12. The genetically engineered host cell of claim 11 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

13. The genetically engineered host cell of claim 1 , wherein the genetically engineered host cell belongs to the genus Corynebacterium.

14. The genetically engineered host cell of claim 1 , wherein the heterologous promoter is derived from Corynebacterium glutamicum , is less than 100 base pairs in length, is able to constitutively express genes across different growth conditions, and is able to co-operate to form a ladder of promoters comprising a plurality of promoters with incrementally increasing levels of promoter activity.

15. A genetically engineered prokaryotic host cell with enhanced industrial performance, said host cell comprising:
a. a heterologous promoter polynucleotide, and
b. a polynucleotide encoding an RNA degradation gene;

wherein the heterologous promoter polynucleotide is operably linked to the polynucleotide encoding the RNA degradation gene, wherein the heterologous promoter comprises a promoter from a promoter ladder; wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1-8.

16. The genetically engineered host cell of claim 15 , wherein the polynucleotide encoding the RNA degradation gene encodes for an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 34, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, and SEQ ID NO: 47.

17. The genetically engineered host cell of claim 15 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 5, and wherein the polynucleotide encoding the RNA degradation gene encodes for SEQ ID NO: 45.

18. The genetically engineered host cell of claim 17 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 1.

19. The genetically engineered host cell of claim 17 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

20. The genetically engineered host cell of claim 15 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 5, and wherein the polynucleotide encoding the RNA degradation gene comprises SEQ ID NO: 20.

21. The genetically engineered host cell of claim 20 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 1.

22. The genetically engineered host cell of claim 20 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

23. The genetically engineered host cell of claim 15 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5, and SEQ ID NO: 2, and wherein the polynucleotide encoding the RNA degradation gene encodes for SEQ ID NO: 36.

24. The genetically engineered host cell of claim 23 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

25. The genetically engineered host cell of claim 15 , wherein the heterologous promoter is a promoter comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 5, and SEQ ID NO: 2, and wherein the polynucleotide encoding the RNA degradation gene comprises SEQ ID NO: 11.

26. The genetically engineered host cell of claim 25 , wherein the heterologous promoter is a promoter comprising the nucleotide sequence of SEQ ID NO: 3.

27. The genetically engineered host cell of claim 15 , wherein the genetically engineered host cell belongs to the genus Corynebacterium.

28. A genetically engineered prokaryotic host cell with enhanced industrial performance, said host cell comprising:
a. a heterologous promoter polynucleotide, and
b. a polynucleotide encoding an RNA degradation gene;

wherein the heterologous promoter is derive

Assignees

• Zymergen Inc

Inventors

• Manchester Shawn

Classifications

• C12P7/56

  Lactic acid · CPC title

• C12P7/46

  Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid · CPC title

• C12P13/04

  Alpha- or beta- amino acids {(other amino acids C12P13/005)} · CPC title

• C12P7/06

  Ethanol, i.e. non-beverage · CPC title

• C12P7/04

  acyclic · CPC title