Orotate transporter encoding maker genes

a technology of orotate transporter and maker genes, which is applied in the field of orotate transporter polypeptide encoding marker genes to achieve the effect of increasing the production of gene products and increasing the copy number of nucleotide sequences

Inactive Publication Date: 2007-04-19
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0080] More than one copy of a nucleotide sequence of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the host cell geno

Problems solved by technology

In particular, there is concern about the cultivation of recombinant cells carrying antibiotic genetic

Method used

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  • Orotate transporter encoding maker genes

Examples

Experimental program
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Effect test

example 1

Construction Outline for L. lactis ssp. Cremoris Strain ED79.1175

[0174] The L. lactis ssp. cremoris strain ED79.1175 is a MG1363 ΔpyrDa ΔpyrDb mutant, harboring deletions in the pyrDa and pyrDb genes. These two deletions result in a pyrimidine-requirement of the strain, which can be bypassed by addition of uracil to the growth medium of the strain. The parent strain MG1363 (Gasson, 1983) is prototrophic for pyrimidines. Both pyrDa and pyrDb encode a functional dihydroorotate dehydrogenase that catalyzes the conversion of dihydroorotate into orotate, the fourth step in the de novo pyrimidine biosynthesis shown in FIG. 1 (Andersen et al., 1994, 1996). Both pyrDa and pyrDb open reading frames are 933 basepairs (bp) long, and their respective gene products comprise 311 amino acid (aa) residues each.

[0175] A DNA fragment of 261 bp was deleted in the C-terminal part of the pyrDa gene in MG1363 resulting in a truncated and incomplete ΔpyrDa gene product of only 224 aa residues. An inter...

example 2

pED301—Cloning of the ysbC Gene in the Vector pDG268

[0205] The vector pDG268 (C. W. Price) is an integrative vector for Bacillus subtilis that specifically integrates into the chromosomal amyE gene by a double cross-over recombination through the amyE N-terminal, and amyE C-terminal parts, located on pDG268. The plasmid also contains an E. coli origin, neomycin and ampicillin resistance markers, and the LacZ operon for blue / white distinction of recombinant clones in Escherichia coli. The cloning sites used for the molecular cloning of the ysbC gene are BamHI and EcoRI located in the N-terminal end of LacZ. The KpnI site is used for linearization in order to augment chromosomal integration.

[0206] A DNA fragment containing the ysbC gene (2048 bp) between primer DBORO22BamHI (SEQ ID NO:17) and primer DBORO23EcoRI (SEQ ID NO:18) was PCR amplified using ELONGASE™ polymerase, and QIAGEN™-purified plasmid DNA of plasmid pDBORO. The PCR fragment was cleaved by BamHI and EcoRI and ligated...

example 3

Construction of Plasmid pED307 (ysbC)

[0214] The recombinant plasmid pED307 is constructed like pED301 (see example 2), except that primer DBORO24EcoRI (SEQ ID NO:19) was used as downstream primer instead of primer DBORO23EcoRI (SEQ ID NO:18), resulting in the amplication of a longer ysbC PCR fragment of 2693 bp.

ysbC as a Selection Marker in Bacillus subtilis

[0215]Bacillus subtilis HH180 is a 168 pyrB strain (Potvin et al., 1975) with a pyrimidine-requirement.

[0216] QIAGEN™-purified plasmid DNA of pED307 (ysbC) was linearized by cleavage with KpnI. Transformation of HH180 cells with lineair pED307 / KpnI was carried out exactly as described in materials and methods. To satisfy the pyrimidine requirement of the HH180 cells, 20 micro-g / ml uracil was added to the GM1 and GM2 medium.

[0217] Homologous recombination of pED307 by a double cross-over event into the amyE gene of HH180 was verified by streaking the transformants on 1% starch plates. The plates were incubated at 37° C. and...

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Abstract

A recombinant marker gene encoding an orotate transporter polypeptide comprising an amino acid sequence at least 60% identical to SEQ ID NO: 2, a polynucleotide construct comprising at least one copy of the recombinant marker gene, a cell comprising at least one exogenous copy of the marker gene, and a method of selecting or identifying a cell comprising at least one copy of the recombinant marker gene, and/or selecting or identifying a cell which has been cured of the recombinant marker gene.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a pioneering new class of orotate transporter polypeptide encoding marker genes. One orotate transporter of the invention is derived from a Lactococcus lactis strain, and is encoded by the ysbC gene. It is demonstrated herein that the orotate transporter gene is suitable to be used as a versatile non-antibiotic marker gene, both in selection, counter-selection, screening, and bi-directional selection protocols. BACKGROUND [0002] A putative gene, denoted ysbC, was previously identified by genome sequencing of a Lactococcus lactis strain, but the gene was not annotated, and no function of the predicted encoded polypeptide was identified (WO 200177334, Bolotin et al., 2001, The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. Lactis IL1403, Genome Res. 11:731). The amino acid sequence of the predicted YsbC protein is available from the database GeneSeqP, accession number: ABB55104. [0003] There ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/04C12P21/06C07K14/705C07K14/195C12N15/65C12Q1/02G01N33/50
CPCC07K14/195G01N33/5005G01N2800/52C12Q1/04
Inventor DEFOOR, ELSE MARIE CELINEMARTINUSSEN, JAN
Owner NOVOZYMES AS
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