Orotate Transporter Encoding Marker Genes

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.

1 : A cell comprising at least one exogenous marker gene encoding an orotate transporter polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 2, wherein the cell is pyrimidine auxotrophic. 
     
     
         2 : The cell of  claim 1 , wherein the at least one marker gene is a selection marker, a screening marker, a counter-selection marker, or a bi-directional selection marker. 
     
     
         3 : The cell of  claim 1 , wherein the marker is a bi-directional marker. 
     
     
         4 : The cell of  claim 1 , wherein the at least one marker gene encoded orotate transporter polypeptide also transports one or more orotate analogues. 
     
     
         5 : The cell of  claim 1 , wherein the at least one marker gene encoded orotate transporter polypeptide also transports the orotate analogue 5-fluoroorotate (FOA). 
     
     
         6 : The cell of  claim 1 , wherein the at least one marker gene is transcribed from at least one heterologous and/or artificial promoter. 
     
     
         7 : The cell of  claim 1 , which is a microbial cell. 
     
     
         8 : The cell of  claim 1 , which is a bacterial cell. 
     
     
         9 : The cell of  claim 1 , which is a Gram-negative or Gram-positive bacterial cell. 
     
     
         10 : The cell of  claim 1 , which is of the genus  Lactobacillus, Bacillus , or  Escherichia.    
     
     
         11 : The cell of  claim 1 , wherein said marker gene encodes an orotate transporter polypeptide comprising an amino acid sequence at least 96% identical to SEQ ID NO: 2. 
     
     
         12 : The cell of  claim 1 , wherein said marker gene encodes an orotate transporter polypeptide comprising an amino acid sequence at least 97% identical to SEQ ID NO: 2. 
     
     
         13 : The cell of  claim 1 , wherein said marker gene encodes an orotate transporter polypeptide comprising an amino acid sequence at least 99% identical to SEQ ID NO: 2. 
     
     
         14 : The cell of  claim 1 , wherein said marker gene encodes an orotate transporter polypeptide comprising an amino acid sequence that has one substitution, deletion or insertion as compared to SEQ ID NO:2. 
     
     
         15 : The cell of  claim 1 , wherein said marker gene encodes an orotate transporter polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. 
     
     
         16 : The cell of  claim 1 , wherein the cell lacks a functional orotate transporter protein in the absence of the marker gene. 
     
     
         17 : The cell of  claim 1 , wherein the cell is pyrimidine auxotrophic due to a mutation in at least one gene encoding an enzyme which converts dihydro-orotate to orotate. 
     
     
         18 : The cell of  claim 1 , wherein the cell is pyrimidine auxotrophic due to a mutation in one or more genes encoding dihydroorotate dehydrogenase, dihydroorotate dehydrogenase A, dihydroorotate dehydrogenase B or dihydroorotate dehydrogenase B electron transfer subunit. 
     
     
         19 : The cell of  claim 1 , wherein the cell is resistant to 5-fluoroorotate (FOA) and lacks a functional orotate transporter protein in the absence of the recombinant marker. 
     
     
         20 : A method of identifying the cell of  claim 1 , comprising the steps of culturing the cell under suitable conditions; and identifying the cell with the marker gene.