Site-specific modification of the candida tropicals genome

A process for restoring uracil auxotrophy to C. tropicalis cells previously transformed to prototrophy with a URA 3 selectable marker comprising the steps of:

• (a) selecting or screening for spontaneous mutations which inactivate said selectable marker, (b) confirming the uracil auxotrophy of said mutants, (c) confirming the parental genotype of the uracil auxotrophs by Southern hybridization to appropriate gene probes.

A process for restoring uracil auxotrophy to C. tropicalis cells previously transformed to prototrophy with a URA 3 selectable marker comprising the steps of:

• (a) transforming prototrophic host cells with a non-functional URA3 selectable marker gene which has been made non-functional by an in-vitro deletion of a portion of the central coding sequence of said gene to produce auxotrophic mutants, (b) confirming the uracil auxotrophy of said mutants, (c) confirming the uracil auxotrophy of said mutants, (c) confirming the parental geneotype of the uracil auxotrophs by Southern hybridization to appropriate gene probes.

A process for completely blocking the .beta.-oxidation pathway in C. tropicalis at its first committed reaction comprising disrupting through homologous recombination of the POX4A, POX4B, and both POX5 target genes with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to said target genes of a C. tropicalis host strain.

A Candida tropicalis cell having a disrupted chromosomal POX4A gene wherein said POX4A gene is disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to said POX4A gene of a C. tropicalis host strain.

A Candida tropicalis cell having a disrupted chromosomal POX4B gene wherein said POX4B gene is disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to said POX4B gene of a C. tropicalis host strain.

A Candida tropicalis cell having a disrupted chromosomal POX5 gene wherein said POX5 gene is disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to said POX5A gene of a C. tropicalis host strain.

A Candida tropicalis cell wherein the chromosomal POX4A and one of the chromosomal POX5 genes are disrupted wherein said POX4A and one of said chromosomal POX5 genes are disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to said POX4A and one of said chromosomal POX5 genes of a C. tropicalis host strain.

A Candida tropicalis cell having a disrupted chromosomal POX4A and POX4B genes wherein said POX4A and POX4B genes are disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to said POX4A and POX4B genes of a C. tropicalis host strain.

A Candida tropicalis cell wherein both copies of the chromosomal POX5 gene are disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to both of said POX5 genes of a C. tropicalis host strain.

A Candida tropicalis cell wherein both copies of the chromosomal POX5 gene and the chromosomal POX4A genes are disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to both of said POX5 genes and said POX4A gene of a C. tropicalis host strain.

A Candida tropicalis cell wherein both copies of the chromosomal POX5 gene and the chromosomal POX4B genes are disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to both of said POX5 genes and said POX4B gene of a C. tropicalis host strain.

A Candida tropicalis cell wherein both copies of the chromosomal POX5 gene and the chromosomal POX4A and POX4B genes are disrupted through homologous recombination with a linear DNA fragment comprised of a URA3A selectable marker gene wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to both of said POX5 genes and both of said POX4A and POX4B genes of a C. tropicalis host strain.

A process for producing a substantially pure alpha, omega-dicarboxylic acid in substantially quantitative yield comprising culturing C. tropicalis strain H5343 in a culture medium containing a nitrogen source, an organic substrate and a cosubstrate.