Expression vector for cyanobacteria

Patent metadata

Field	Value
Publication number	US-9719097-B2
Application number	US-201514860373-A
Country	US
Kind code	B2
Filing date	Sep 21, 2015
Priority date	Dec 10, 2014
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

The present disclosure discloses a vector that can be used for both cyanobacteria and E. coli , which contains, sequentially, a pUC replication origin as a replication origin; a spectinomycin-resistant gene as a selection marker; and a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter. An industrially useful substance may be produced effectively using a host cell transformed with the vector. Also, the vector may be used to insert a variety of target genes through simple combination and, as a result, various vectors can be prepared effectively.

First claim

The invention claimed is:

1. An expression vector which is operable in cyanobacteria comprising, sequentially:
a pUC replication origin as a replication origin;
a spectinomycin-resistant gene as a selection marker; and
a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter,
wherein the modified tetA promoter comprises a sequence of SEQ ID NO 6.

2. The vector according to claim 1 , wherein the vector further comprises a repressor selected from a group consisting of a lad repressor, a tetR repressor and an AraC repressor upstream of the promoter.

3. The vector according to claim 1 , wherein the vector further comprises a green fluorescent protein (GFP) gene downstream of the promoter.

4. The vector according to claim 1 , wherein the vector further comprises a neutral site (NSI) comprising the neutral site comprising:
NSIa comprising a sequence of SEQ ID NO 7; and
NSIb comprising a sequence of SEQ ID NO 8.

5. The vector according to claim 1 , wherein the vector further comprises a BglII site and a BamHI site as restriction enzyme sites.

6. The vector according to claim 5 , wherein the vector further comprises a target DNA sequence which encodes a target protein desired to be overexpressed.

7. The vector according to claim 6 , wherein the BglII site and the BamHI site are located on both sides of the target DNA sequence.

8. The vector according to claim 7 , wherein the target DNA sequence encodes two or more proteins.

9. The vector according to claim 8 , wherein the target DNA sequence is included in the vector by complementary binding between the BglII site of the upstream side of a first portion of the target DNA sequence and the BamHI site of the downstream side of a second portion of the target DNA sequence.

10. The vector according to claim 1 , wherein the vector is
a pSe1Bb1s-GFP vector comprising a lad repressor and a trc promoter;
a pSe1Bb2s-GFP vector comprising a tetR repressor and a tetA promoter;
a pSe1Bb8s-GFP vector comprising an AraC repressor and a BAD promoter;
a pSe1Bb cbbL s-GFP vector comprising a cbbL promoter; or
a pSe1Bb2 O s-GFP vector comprising a tetR repressor and a modified tetA promoter comprising a sequence of SEQ ID NO 6.

11. The vector according to claim 10 , wherein the vector comprises a sequence of SEQ ID NO 1; SEQ ID NO 2; SEQ ID NO 3; SEQ ID NO 4; or SEQ ID NO 5.

12. A host cell transformed with the vector according to claim 1 .

13. The host cell according to claim 12 , wherein the host cell is cyanobacterium.

14. The host cell according to claim 13 , wherein the cyanobacterium is Synechococcus elongatus.

15. A host cell transformed with the vector according to claim 10 .

16. A host cell transformed with the vector according to claim 11 .

17. Transformed Synechococcus elongates PCC 7942 in which the vector according to claim 4 is inserted onto the genome of Synechococcus elongates PCC 7942 via the neutral site between NSIa and NSIb.

Assignees

• Korea Inst Sci & Tech

Inventors

• Woo Han Min
• Chwa Jun Won
• Um Youngsoon
• Gong Gyeong Taek
• Kim Yunje

Classifications

• C12N15/74 Primary

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title

• C12N2510/02

  Cells for production · CPC title

• C12N15/635

  Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title