Methods For Increasing Homologous Recombination Of A Nucleic Acid Sequence
The present invention relates to methods for increasing homologous recombination of a nucleic acid sequence introduced into a host cell, comprising: (a) introducing into a population of filamentous fungal host cells a first nucleic acid sequence encoding a recombination protein and a second nucleic acid sequence comprising one or more regions which are homologous with the genome of the filamentous fungal host cell, wherein (i) the recombination protein promotes the recombination of the one or more regions with the corresponding homologous region in the host's genome to incorporate the second nucleic acid sequence by homologous recombination, and (ii) the number of host cells comprising the incorporated second nucleic acid sequence in the population is increased at least 20% compared to the same population without the first nucleic acid sequence; (b) and isolating from the population a filamentous fungal cell comprising the incorporated second nucleic acid sequence.
1 . A nucleic acid construct, comprising a nucleic acid encoding a recombination protein selected from the group consisting of: (a) a nucleic acid encoding a recombination protein comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; (b) a nucleic acid comprising a nucleotide sequence having at least 90% homology to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5; and (c) a nucleic acid comprising a nucleotide sequence which hybridizes under high stringency conditions with (i) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (ii) the full-length complement thereof; wherein the nucleic acid encoding the recombination protein is operably linked to one or more heterologous control sequences that direct the production of the recombination protein in an expression host and wherein high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.
2 . The nucleic acid construct of claim 1 , wherein the recombination protein encoded by the nucleic acid has at least 95% identity with SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
3 . The nucleic acid construct of claim 2 , wherein the recombination protein encoded by the nucleic acid has at least 97% identity with SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
4 . The nucleic acid construct of claim 1 , wherein the recombination protein encoded by the nucleic acid comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
5 . The nucleic acid construct of claim 4 , wherein the recombination protein encoded by the nucleic acid consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6; or a fragment thereof which has recombination activity.
6 . The nucleic acid construct of claim 1 , wherein the nucleic acid comprising a nucleotide sequence has at least 95% homology with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
7 . The nucleic acid construct of claim 6 , wherein the nucleic acid comprising a nucleotide sequence has at least 97% homology with SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.
8 . The nucleic acid construct of claim 1 , wherein the nucleic acid encoding a recombination protein hybridizes under very high stringency conditions with (i) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (ii) the full-length complement thereof, wherein very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50% formamide and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.
9 . The nucleic acid construct of claim 4 , wherein the recombination protein is encoded by the nucleic acid sequence contained in plasmid pZL1rdhA13 which is contained in Escherichia coli NRRL B-30503; plasmid pZL1rdhB6 which is contained in Escherichia coli NRRL B-30504; or plasmid pZL1rdhD17 which is contained in Escherichia coli NRRL B-30505 and plasmid pZL1rdhD10 which is contained in Escherichia coli NRRL B-30506.
10 . An expression vector comprising the nucleic acid construct of claim 1 .
11 . An isolated recombinant host cell comprising the nucleic acid construct of claim 1 .
12 . An isolated recombinant host cell comprising the expression vector of claim 10 .
13 . A method of producing a recombination protein, comprising cultivating the recombinant host cell of claim 11 under conditions conducive for production of the recombination protein.
14 . A method of producing a recombination protein, comprising cultivating the recombinant host cell of claim 12 under conditions conducive for production of the recombination protein.