Formaldehyde dehydrogenase genes from methylotrophic yeasts

An isolated nucleic acid comprising a formaldehyde dehydrogenase gene (FLD) from a methylotrophic yeast wherein said isolated nucleic acid hybridizes under low stringency conditions to at least one of the nucleotide sequence set forth in SEQ ID NO:1, the nucleotide sequence set forth in SEQ ID NO:5, or a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NOs:

• 1 or 5.

An isolated nucleic acid comprising an FLD gene having the restriction map as set forth in FIG. 7 or having the restriction map shown in the cross hatched area of FIG. 10.

An isolated nucleic acid comprising an FLD gene from a methylotrophic yeast, said isolated nucleic acid having a coding sequence with a sequence homology of about 70% to about 85% when compared to the nucleotide sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising an FLD gene from a methylotrophic yeast, said isolated nucleic acid having a coding sequence with a sequence homology of about 85% to about 95% when compared to the nucleotide sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising an FLD gene from a methylotrophic yeast, said isolated nucleic acid having a coding sequence with a sequence homology of greater than about 95% when compared to the nucleotide sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising the sequence set forth in SEQ ID NO:1 or SEQ ID NO:5.

An isolated nucleic acid from a methylotrophic yeast comprising an FLD promoter, said promoter upstream from the translational start codon of an FLD gene, wherein the coding sequence of said FLD gene has a sequence homology of about 70% to about 85% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5.

An isolated nucleic acid from a methylotrophic yeast comprising an FLD promoter, said promoter located upstream from the translational start codon of an FLD gene, wherein the coding sequence of said FLD gene has a sequence homology of about 85% to about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5.

An isolated nucleic acid from a methylotrophic yeast comprising an FLD promoter, said promoter located upstream from the translational start codon of an FLD gene, wherein the coding sequence of said FLD gene has a sequence homology of greater than about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising the sequence set forth in SEQ ID NO:3.

An isolated nucleic acid comprising an FLD promoter having the restriction map set forth in FIG. 8.

An isolated nucleic acid comprising an FLD 3′ termination sequence from a methylotrophic yeast, said 3′ termination sequence located downstream from the translational stop codon of an FLD gene, wherein the coding sequence of said FLD gene has a sequence homology of at about 70% to about 85% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising an FLD 3′ termination sequence from a methylotrophic yeast, said 3′ termination sequence located downstream from the translational stop codon of an FLD gene, wherein the coding sequence of said FLD gene has a sequence homology of at about 85% to about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising an FLD 3′ termination sequence from a methylotrophic yeast, said 3′ termination sequence located downstream from the translational stop codon of an FLD gene, wherein the coding sequence of said FLD gene has a sequence homology of greater than about 95% when compared to the nucleotide sequence of the FLD coding sequence set forth in SEQ ID NO:5.

An isolated nucleic acid comprising the sequence set forth in SEQ ID NO:4.

An isolated nucleic acid comprising an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of about 30% to about 49% when compared to the amino acid sequence as set forth in SEQ ID NO:2.

An isolated nucleic acid comprising an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of about 50% to about 90% when compared to the amino acid sequence as set forth in SEQ ID NO:2.

An isolated nucleic acid comprising an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of greater than about 90% when compared to the amino acid sequence as set forth in SEQ ID NO:2.

An isolated nucleic acid comprising at least one of a promoter, coding sequence or 3′ termination sequence from an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of about 30% to about 49% when compared to the amino acid sequence as set forth in SEQ ID NO:2.

An isolated nucleic acid comprising at least one of a promoter, coding sequence or 3′ termination sequence from an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of about 50% to about 90% when compared to the amino acid sequence as set forth in SEQ ID NO:2.

An isolated nucleic acid comprising at least one of a promoter, coding sequence or 3′ termination sequence from an FLD gene wherein said FLD gene encodes a product having an amino acid sequence identity of greater than about 90% when compared to the amino acid sequence as set forth in SEQ ID NO:2.

An isolated nucleic acid comprising at least one of a promoter, coding sequence or 3′ termination sequence of an FLD gene, wherein said FLD gene encodes a product having the amino acid sequence set forth in SEQ ID NO:2.

A method for directing expression of a heterologous gene in a methylotrophic yeast which comprises:

• a) introducing into a methylotrophic yeast cell an isolated nucleic acid comprising an FLD promoter isolated from a methylotrophic yeast, said promoter operably linked at its.3′ end to the 5′ end of a heterologous gene, said heterologous gene operably linked at its 3′ end to the 5′ end of a termination sequence which functions in a methylotrophic yeast;
• b) growing said methylotrophic yeast cell in a medium having a suitable carbon source such as glycerol, or glucose, and having a suitable nitrogen source such as an ammonium salt or ammonium hydroxide, and after the carbon or nitrogen source is depleted;
• c) inducing expression of said heterologous gene by addition of methanol or methylamine or both methanol and methylamine.

A method for directing expression of a heterologous gene in a methylotrophic yeast which comprises:

• a) introducing into a methylotrophic yeast cell an isolated nucleic acid comprising an FLD promoter isolated from a methylotrophic yeast, said promoter operably linked at its 3′ end to the 5′ end of a heterologous gene, said heterologous gene operably linked at its 3′ end to the 5′ end of a termination sequence which functions in a methylotrophic yeast;
• b) growing said methylotrophic yeast cell in a medium having a suitable carbon source such as glycerol or glucose, having a suitable nitrogen source such as an ammonium salt or ammonium hydroxide, and after the carbon or nitrogen source is depleted;
• c) inducing expression of said heterologous gene by addition of formaldehyde, formate, or a methylated amine.

A strain of methylotrophic yeast which is defective in an FLD gene (fld).

A strain of methylotrophic yeast which is defective in an FLD gene and auxotrophic for another biosynthetic gene.

A kit which comprises:

• a) an expression cassette comprising an FLD promoter and a 3′ termination sequence which functions in a methylotrophic yeast, wherein at least one restriction site is located between the FLD promoter and 3′ termination sequence so that a heterologous gene may be inserted and operably linked to said promoter and said 3′ termination sequence;
• and
• b) a vector which replicates in a methylotrophic yeast or which integrates into the genome of a methylotrophic yeast, said vector comprising a marker gene and one or more restriction sites for insertion of said cassette.

A kit which comprises:

• an expression vector comprising an FLD gene from a methylotrophic yeast as a marker gene and an expression cassette wherein said expression cassette comprises a promoter and a 3′ termination sequence which functions in a methylotrophic yeast, and wherein at least one restriction site is located between the promoter and 3′ termination sequence so that a heterologous gene may be inserted and operably linked to said promoter and said 3′ termination sequence.