Expression vector for cyanobacteria

Patent metadata

Field	Value
Publication number	US-2016168580-A1
Application number	US-201514860373-A
Country	US
Kind code	A1
Filing date	Sep 21, 2015
Priority date	Dec 10, 2014
Publication date	Jun 16, 2016
Grant date	—

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

What is claimed is:

1 . An expression vector for cyanobacteria which comprises, 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.

2 . The vector according to claim 1 , wherein the vector further comprises a repressor selected from a group consisting of a lacI 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 cyanobacterium is Synechococcus elongatus.

5 . The vector according to claim 4 , wherein the vector further comprises a neutral site (NSI) derived from Synechococcus elongatus PCC 7942 upstream or downstream of the replication origin.

6 . The vector according to claim 5 , wherein the neutral site comprises:
NSIa comprising a sequence of SEQ ID NO 7; and NSIb comprising a sequence of SEQ ID NO 8.

7 . The vector according to claim 5 , wherein the vector is inserted onto the genome of Synechococcus elongatus PCC 7942 via the neutral site.

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

9 . The vector according to claim 8 , wherein the vector further comprises a target gene which encodes a target protein desired to be overexpressed.

10 . The vector according to claim 9 , wherein the BglII site and the BamHI site are located on both sides of the target gene.

11 . The vector according to claim 10 , wherein the vector comprises two or more target genes.

12 . The vector according to claim 11 , wherein the two or more target genes are included in one vector by complementary binding between the BglII site of the upstream side of one target gene and the BamHI site of the downstream side of the other target gene.

13 . The vector according to claim 1 , wherein the modified tetA promoter comprises a sequence of SEQ ID NO 6.

14 . The vector according to claim 1 , wherein the vector is
a pSe1Bb1s-GFP vector comprising a lacI 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.

15 . The vector according to claim 14 , 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.

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

17 . The host cell according to claim 16 , wherein the host cell is cyanobacterium.

18 . The host cell according to claim 17 , wherein the cyanobacterium is Synechococcus elongatus.

19 . A host cell transformed with the vector according to claim 14 .

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

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

• 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

• 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