Non-revertible beta-oxidation blocked candida tropicalis

Abstract

Genetically modified strains of C. tropicalis, which will not revert to wild-type activity at the POX 4 and/or POX 5 locus, are disclosed. The strains are β-oxidation blocked and have been transformed through homologous recombination with a construct which deletes a portion of the POX 4 and/or POX 5 gene. The modified strains may be used to increase yields of dicarboxylic acids produced in host cells of the strains. Methods for blocking the β-oxidation pathway in a C. tropicalis host cell are also provided.

What is claimed is:  
     
         1 . A  Candida tropicalis  cell having a disrupted chromosomal POX 4 gene wherein a portion of said POX 4 gene has been deleted through homologous recombination with a DNA fragment comprising a selectable marker gene, wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to non-contiguous portions of said POX 4 gene and wherein the presence of the deleted portion of the POX 4 gene prevents recombination to a functional POX 4 gene.  
     
     
         2 . The  Candida tropicalis  cell of  claim 1  wherein the POX 4 gene is POX 4A.  
     
     
         3 . The  Candida tropicalis  cell of  claim 1  wherein the POX 4 gene is POX 4B.  
     
     
         4 . The  Candida tropicalis  cell of  claim 1  wherein the POX 4 gene comprises both POX 4A and POX 4B.  
     
     
         5 . The  Candida tropicalis  cell of  claim 1  wherein the DNA fragment comprises a URA 3 selectable marker gene.  
     
     
         6 . The  Candida tropicalis  cell of  claim 1  comprising the strain H53.  
     
     
         7 . A process for producing a substantially pure α, Ω-dicarboxylic acid which comprises culturing the Candida tropicalis cell of  claim 1  in a culture medium containing a nitrogen source, an organic substrate and a cosubstrate.  
     
     
         8 . A  Candida tropicalis  cell having a disrupted chromosomal POX 5 gene wherein a portion of said POX 5 gene has been deleted through homologous recombination with a DNA fragment comprising a selectable marker gene, wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to non-contiguous portions of said POX 5 gene and wherein the presence of the deleted portion of the POX 5 gene prevents recombination to a functional POX 4 gene.  
     
     
         9 . The  Candida tropicalis  cell of  claim 8  comprising both copies of a chromosomal POX 5 gene.  
     
     
         10 . The Candida tropicalis cell of  claim 8  wherein the DNA fragment comprises a URA 3 selectable marker gene.  
     
     
         11 . The  Candida tropicalis  cell of  claim 8  comprising the strain H53.  
     
     
         12 . A process for producing a substantially pure α, Ω-dicarboxylic acid comprising culturing the Candida tropicalis cell of  claim 8  in a culture medium containing a nitrogen source, an organic substrate and a cosubstrate.  
     
     
         13 . A  Candida tropicalis  cell having disrupted chromosomal POX 4 and POX 5 genes wherein a portion of both of said POX 4 and POX 5 genes has been deleted through homologous recombination with a DNA fragment comprising a selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to non-contiguous portions of both of said POX 4 and POX 5 genes and wherein the presence of the deleted portions of both of the POX 4 and POX 5 genes prevent recombination to functional POX 4 and POX 5 genes.  
     
     
         14 . The  Candida tropicalis  cell of  claim 13  wherein the POX 4 gene is POX 4A.  
     
     
         15 . The  Candida tropicalis  cell of  claim 13  wherein the POX 4 gene is POX 4B.  
     
     
         16 . The  Candida tropicalis  cell of  claim 13  wherein the POX 4 gene comprises both POX 4A and POX 4B.  
     
     
         17 . The  Candida tropicalis  cell of  claim 13  comprising both copies of a chromosomal POX 5 gene.  
     
     
         18 . The  Candida tropicalis  cell of  claim 13  wherein the DNA fragment comprises a URA 3 selectable marker gene.  
     
     
         19 . The  Candida tropicalis  cell of  claim 13  comprising the strain H53.  
     
     
         20 . The  Candida tropicalis  cell of  claim 17  wherein the POX 4 gene is POX 4A.  
     
     
         21 . The  Candida tropicalis  cell of  claim 17  wherein the POX 4 gene is POX 4B.  
     
     
         22 . The  Candida tropicalis  cell of  claim 17  wherein the POX 4 gene is both POX 4A and POX 4B.  
     
     
         23 . A process for producing a substantially pure α, Ω-dicarboxylic acid which comprises culturing the Candida tropicalis cell of  claim 13  in a culture medium containing a nitrogen source, an organic substrate and a cosubstrate.  
     
     
         24 . A process for completely blocking the β-oxidation pathway in a  Candida tropicalis  host cell which comprises disrupting through homologous recombination a portion of a POX 4 gene with a DNA fragment comprising a selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to non-contiguous portions of said POX 4 gene and wherein the presence of the deleted portion of the POX 4 gene prevents recombination to a functional POX 4 gene.  
     
     
         25 . The process of  claim 24  wherein the POX 4 gene is POX 4A.  
     
     
         26 . The process of  claim 24  wherein the POX 4 gene is POX 4B.  
     
     
         27 . The process of  claim 24  wherein the POX 4 gene comprises both POX 4A and POX 4B.  
     
     
         28 . The process of  claim 24  wherein the DNA fragment comprises a URA 3 selectable marker gene.  
     
     
         29 . The process of  claim 24  wherein the  Candida tropicalis  host cell comprises strain H53.  
     
     
         30 . A process for completely blocking the β-oxidation pathway in a Candida tropicalis host cell which comprises disrupting through homologous recombination a portion of a POX 5 gene with a DNA fragment comprising a selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to non-contiguous parts of said POX 5 gene and wherein the presence of the deleted portion of the POX 5 gene prevents recombination to a functional POX 5 gene.  
     
     
         31 . The process of  claim 30  wherein the host cell comprises both copies of a chromosomal POX 5 gene.  
     
     
         32 . The process of  claim 30  wherein the DNA fragment comprises a URA 3 selectable marker gene.  
     
     
         33 . The process of  claim 30  wherein the  Candida tropicalis  host cell comprises strain H53.  
     
     
         34 . A process for completely blocking the β-oxidation pathway in a  Candida tropicalis  host cell which comprises disrupting through homologous recombination a portion of both of POX 4 and POX 5 genes with a DNA fragment comprised of a selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to non-contiguous portions of both of said POX 4 and POX 5 genes and wherein the deleted portions of both of the POX 4 and POX 5 genes prevent recombination to functional POX 4 and POX 5 genes.  
     
     
         35 . The process of  claim 34  wherein the POX 4 gene is POX 4A.  
     
     
         36 . The process of  claim 34  wherein the POX 4 gene is POX 4B.  
     
     
         37 . The process of  claim 34  wherein the POX 4 gene comprises both POX 4A and POX 4B.  
     
     
         38 . The process of  claim 34  wherein the host cell comprises both copies of the chromosomal POX 5 gene.  
     
     
         39 . The process of  claim 34  wherein the DNA fragment comprises a URA 3 selectable marker gene.  
     
     
         40 . The process of  claim 34  wherein the  Candida tropicalis  host cell comprises strain H53.  
     
     
         41 . The process of  claim 38  wherein the POX 4 gene is POX 4A.  
     
     
         42 . The process of  claim 38  wherein the POX 4 gene is POX 4B.  
     
     
         43 . The process of  claim 38  wherein the POX 4 gene comprises both POX 4A and POX 4B.