Nucleic acid molecules comprising a variant inc coding strand

Patent metadata

Field	Value
Publication number	US-11549116-B2
Application number	US-201916534132-A
Country	US
Kind code	B2
Filing date	Aug 7, 2019
Priority date	Aug 7, 2018
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

A nucleic acid molecule comprising a variant inc coding strand is disclosed as a regulator of plasmid copy number. Also disclosed is a replicon comprising the nucleic acid molecule, a promoter, and an origin of replication. Also disclosed is a vector comprising the replicon. Also disclosed is a recombinant microorganism comprising the vector.

First claim

What is claimed is:

1. A nucleic acid molecule comprising a variant inc coding strand that comprises SEQ ID NO: 15, wherein:
the variant inc coding strand encodes a variant inc RNA; and
the variant inc RNA is a regulator of plasmid copy number.

2. The nucleic acid molecule according to claim 1 , wherein expression of the variant inc RNA increases copy number of a vector relative to expression of wild-type inc RNA encoded by an inc sequence comprising SEQ ID NO: 19.

3. The nucleic acid molecule according to claim 1 , wherein the variant inc coding strand comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 17.

4. A replicon comprising the nucleic acid molecule of claim 1 , a promoter, and an origin of replication, wherein:
the promoter is operably linked to the variant inc coding strand; and
the variant inc RNA regulates copy number of the replicon.

5. The replicon according to claim 4 , wherein the replicon comprises a variant repFIC replicon, and the nucleic acid molecule is part of the variant repFIC replicon.

6. A vector comprising the replicon of claim 4 .

7. The vector according to claim 6 , wherein expression of the variant inc RNA increases copy number of the vector relative to expression of wild-type inc RNA encoded by an inc sequence comprising SEQ ID NO: 19.

8. The vector according to claim 6 , wherein the vector comprises a plasmid.

9. The vector according to claim 6 , further comprising a target gene for making a target product.

10. The vector according to claim 9 , wherein the target product comprises one or more of (i) a target RNA, (ii) a target protein, (iii) a target biomaterial, (iv) a target polymer, precursor thereof, and/or enzyme for production thereof, (v) a target sweetener, precursor thereof, and/or enzyme for production thereof, (vi) a target oil, precursor thereof, and/or enzyme for production thereof, (vii) a target fat, precursor thereof, and/or enzyme for production thereof, (viii) a target polysaccharide, precursor thereof, and/or enzyme for production thereof, (ix) a target amino acid, precursor thereof, and/or enzyme for production thereof, (x) a target nucleotide, precursor thereof, and/or enzyme for production thereof, (xi) a target vaccine, precursor thereof, and/or enzyme for production thereof, or (xii) a target pharmaceutical product, precursor thereof, and/or enzyme for production thereof.

11. A recombinant microorganism comprising the replicon of claim 4 .

12. A recombinant microorganism comprising the vector of claim 6 .

13. The recombinant microorganism according to claim 12 , wherein expression of the variant inc RNA in the recombinant microorganism increases copy number of the vector relative to expression of wild-type inc RNA encoded by an inc sequence comprising SEQ ID NO: 19 in a control microorganism.

14. The recombinant microorganism according to claim 12 , wherein the variant inc coding strand comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 17.

15. The recombinant microorganism according to claim 12 , wherein the recombinant microorganism was prepared by introducing the vector into a precursor microorganism by one or more of transformation, conjugation, or transduction.

16. The recombinant microorganism according to claim 12 , wherein the recombinant microorganism was prepared from one or more of a bacterium of the genus Escherichia or a bacterium of the species Escherichia coli.

17. The recombinant microorganism according to claim 12 , wherein the recombinant microorganism further comprises another nucleic acid molecule comprising a variant rpoC RNA-polymerase β′ subunit protein coding sequence (variant rpoC coding sequence), wherein:
the variant rpoC coding sequence encodes a variant RpoC RNA-polymerase β′ subunit protein (variant RpoC); and
the variant RpoC comprises an R47C substitution, with numbering of the R47C substitution defined based on wild-type RpoC RNA-polymerase β′ subunit protein (wild-type RpoC) of Escherichia coli of SEQ ID NO: 44.

18. A method for regulating copy number of the vector of claim 6 in a recombinant microorganism, the method comprising the steps of:
(1) introducing the vector into a precursor microorganism, thereby obtaining the recombinant microorganism; and
(2) cultivating the recombinant microorganism in a culture medium under conditions sufficient for replication of the vector, thereby regulating copy number of the vector.

19. The method according to claim 18 , wherein expression of the variant inc RNA in the recombinant microorganism increases copy number of the vector relative to expression of wild-type inc RNA encoded by an inc sequence comprising SEQ ID NO: 19 in a control microorganism.

20. A method for making a target product by use of the vector of claim 7 in a recombinant microorganism, wherein the vector further comprises a target gene for making the target product, the method comprising the steps of:
(1) introducing the vector into a precursor microorganism, thereby obtaining the recombinant microorganism;
(2) cultivating the recombinant microorganism in a culture medium under conditions under which the recombinant microorganism expresses the target gene, thereby making the target product; and
(3) recovering the target product from the recombinant microorganism and/or the culture medium.

21. The method according to claim 20 , wherein expression of the variant inc RNA in the recombinant microorganism increases copy number of the vector relative to expression of wild-type inc RNA encoded by an inc sequence comprising SEQ ID NO: 19 in a control microorganism.

22. The method according to claim 20 , wherein the variant inc coding strand comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 17.

23. The method according to claim 20 , wherein the target product comprises one or more of (i) a target RNA, (ii) a target protein, (iii) a target biomaterial, (iv) a target polymer, precursor thereof, and/or enzyme for production thereof, (v) a target sweetener, precursor thereof, and/or enzyme for production thereof, (vi) a target oil, precursor thereof, and/or enzyme for production thereof, (vii) a target fat, precursor thereof, and/or enzyme for production thereof, (viii) a target polysaccharide, precursor thereof, and/or enzyme for production thereof, (ix) a target amino acid, precursor thereof, and/or enzyme for production thereof, (x) a target nucleotide, precursor thereof, and/or enzyme for production thereof, (xi) a target vaccine, precursor thereof, and/or enzyme for production thereof, or (xii) a target pharmaceutical product, precursor thereof, and/or enzyme for production thereof.

24. The method according to claim 18 , wherein: the recombinant microorganism further comprises another nucleic acid molecule comprising a variant rpoC coding sequence; the variant rpoC coding sequence encodes a variant RpoC; and the variant RpoC comprises an R47C substitution, with numbering of the R47C substitution defined based on wild-type RpoC of Escherichia coli of SEQ ID NO: 44.

25. The method according to claim 20 , wherein: the recombinant microorganism further comprises another nucleic acid molecule comprising a variant rpoC coding sequence; the variant rpoC coding sequence encodes a variant RpoC; and the variant RpoC comprises an R47C substitution, with numbering of the R47C substitution defined based on wild-type RpoC of Escherichia coli of SEQ ID

Assignees

• Cj Cheiljedang Corp

Inventors

• Lee Ji Sun
• Chang Dong Eun
• Kim Sang Jun
• Kim So Young

Classifications

• C12N2800/101

  for bacteria · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N2830/42

  being an intron or intervening sequence for splicing and/or stability of RNA · CPC title

• C12N9/1247

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12N15/69 Primary

  Increasing the copy number of the vector · CPC title