Biological method for the production of tuliposide A and its intermediates
OwnerHoward Glenn Damude, Vikram Prabhu +2inventors
InventorsHoward Glenn Damude, Vikram Prabhu, Hong Wang, Dennis H. Flint
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Abstract
This invention relates to genes encoding key enzymes in the biosynthesis of α-methylene-γ-butyrolactone (tulipalin A). The key enzymes are glutamate decarboxylase, γ-aminobutyrate aminotransferase, γ-hydroxybutyrate dehydrogenase and UDP-glucosyltransferase. The genes and their expression products are useful for the creation of recombinant organisms that have the ability to synthesize tulipalin A, tuliposide A or any tuliposide A pathway intermediates.
Claims50
An isolated nucleic acid fragment encoding a tuliposide A synthesizing protein selected from the group consisting of:
- (a) an isolated nucleic acid fragment encoding the amino acid sequence set forth in SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22 and SEQ ID NO:
- 24;
- (b) an isolated nucleic acid fragment that hybridizes with SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 14, SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22 and SEQ ID NO:
- 24 Under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS at 65° C., and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid fragment that is completely complementary to (a) or (b).
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 498 amino acids that has at least 78% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 2 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has glutamate decarboxylase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 509 amino acids that has at least 74% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 4 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has glutamate decarboxylase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 529 amino acids that has at least 74% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 6 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has glutamate decarboxylase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 471 amino acids that has at least 77% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 8 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has □-aminobutyrate aminotransferase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 507 amino acids that has at least 80% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 10 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has □-aminobutyrate aminotransferase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 290 amino acids that has at least 81% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 14 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has γ-hydroxybutyrate dehydrognease activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 454 amino acids that has at least 51% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 18 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has UDP-glucosyltransferase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 454 amino acids that has at least 51% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 20 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has UDP-glucosyltransferase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 459 amino acids that has at least 49% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 22 or a second nucleotide sequence comprising the complement of the first nucleotide sequence;
- wherein said enzyme has UDP-glucosyltransferase activity.
An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a polypeptide of at least 459 amino acids that has at least 50% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:
- 24 or a second nucleotide sequence comprising the complement of the first nucleotide sequence, wherein said enzyme has UDP-glucosyltransferase activity.
A host cell comprising a partial or complete knockout of at least one tuliposide A synthesizing protein, the protein having an amino acid sequence selected from the group consisting of:
- (a) SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22, and SEQ ID NO:
- 24;
- (b) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22, and SEQ ID NO:
- 24 under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- or
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b).
A compound α-methylenesuccinate seminaldehyde, represented by Formula I.
- 1
A method of producing a nucleic acid fragment encoding a tuliposide A synthesizing protein, the method comprising:
- (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 19, SEQ ID NO:
- 21 and SEQ ID NO:
- 23, and
- (b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a), wherein the amplified insert encodes a portion of an amino acid sequence encoding a tuliposide A synthesizing protein.
A method of producing α-methylene-γ-aminobutyrate comprising contacting a transformed host cell with an effective amount of γ-methyleneglutamate, said transformed host cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of:
- (a) SEQ ID NO:
- 2, SEQ ID NO:
- 4, and SEQ ID NO:
- 6;
- (b) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:
- 2, SEQ ID NO:
- 4, and SEQ ID NO:
- 6 under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b);
- operably linked to suitable regulatory sequences.
A method of producing α-methylenesuccinate seminaldehyde comprising contacting a transformed host cell with an effective amount of α-methylene-γ-aminobutyrate, said transformed host cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of:
- (a) SEQ ID NO:
- 8, SEQ ID NO:
- 10, and SEQ ID NO:
- 12, operably linked to suitable regulatory sequences;
- (b) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:
- 8, SEQ ID NO:
- 10, and SEQ ID NO:
- 12 under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b);
- operably linked to suitable regulatory sequences.
A method of producing α-methylene-γ-hydroxybutyrate comprising contacting a transformed host cell with an effective amount α-methylenesuccinate seminaldehyde, said transformed host cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of:
- (a) SEQ ID NO:
- 14 and SEQ ID NO:
- 16;
- (b) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:
- 14 and SEQ ID NO:
- 16 under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b);
- operably linked to suitable regulatory sequences.
A method of producing tuliposide A comprising contacting a transformed host cell with an effective amount of α-methylene-γ-hydroxybutyrate, said transformed host cell comprising a nucleic acid fragment encoding the polypeptide selected from the group consisting of:
- (a) SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22 and SEQ ID NO:
- 24;
- (b) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22, and SEQ ID NO:
- 24 under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b);
- operably linked to suitable regulatory sequences.
A method for producing tuliposide A and tuliposide A pathway intermediates, the method comprising contacting a transformed host cell under suitable growth conditions with an effective amount of γ-methyleneglutamate, said transformed host cell comprising a tuliposide A synthesizing protein selected from the group consisting of:
- (a) SEQ ID NO:
- 2, SEQ ID NO:
- 4, SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22,
- SEQ ID NO:
- 24 and mixtures thereof;
- (b) an isolated nucleic acid molecule that hybridizes with SEQ ID NO:
- 2, SEQ ID NO:
- 4, and SEQ ID NO:
- 6, SEQ ID NO:
- 8, SEQ ID NO:
- 10, SEQ ID NO:
- 12, SEQ ID NO:
- 14, SEQ ID NO:
- 16, SEQ ID NO:
- 18, SEQ ID NO:
- 20, SEQ ID NO:
- 22, SEQ ID NO:
- 24 under the following hybridization conditions:
- 0.1× SSC, 0.1% SDS, 65° C. and washed with 2× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b);
- operably linked to suitable regulatory sequences.
A method of producing a mutated microbial gene sequence encoding a protein having an altered biological activity, the method comprising the steps of:
- (a) digesting a mixture of nucleotide sequences with restriction endonucleases wherein said mixture comprises:
- (i) a native microbial gene selected from the group consisting of SEQ ID NO:
- 1, SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 19, SEQ ID NO:
- 21, and SEQ ID NO:
- 23;
- (ii) a first population of nucleotide fragments which hybridizes to the native microbial gene sequence;
- (iii) a second population of nucleotide fragments which does not hybridize to said native microbial sequence;
- wherein a mixture of restriction fragments are produced;
- (b) denaturing the mixture of restriction fragments obtained in step (a);
- (c) incubating the denatured mixture of restriction fragments of step (b) with a polymerase;
- and (d) repeating steps (b) and (c) wherein a mutated microbial gene sequence is produced encoding a protein having an altered biological activity.
A mutated microbial gene sequence encoding a protein having an altered biological activity produced by the method of claim 44.
A method for producing tuliposide A, in an aqueous reaction mixture, comprising the steps of:
- (a) contacting γ-methyleneglutamate with γ-methyleneglutamate decarboxylase;
- (b) contacting the product of step (a) with {tilde over (α)}-methylene-γ-aminobutyrate aminotransferase;
- (c) contacting the product of step (b) with α-methylene-γ-hydroxybutyrate dehydrogenase;
- (d) contacting the product of step (c) with α-methylene-γ-hydroxybutyrate/UDP-glucose glucosyltransferase to produce tuliposide A;
- and
- (e) isolating the tuliposide A produced in step (d).
A method of producing α-methylene-γ-aminobutyrate comprising contacting γ-methyleneglutamate with γ-methyleneglutamate decarboxylase under suitable enzymatic conditions in an aqueous reaction mixture and isolating the α-methylene-γ-aminobutyrate produced.
A method of producing α-methylenesuccinate semialdehyde comprising contacting α-methylene-γ-aminobutyrate with {tilde over (α)}methylene-γ-aminobutyrate aminotransferase under suitable enzymatic conditions in an aqueous reaction mixture and isolating the α-methylenesuccinate semialdehyde produced.
A method of producing {tilde over (α)}methylene-γ-hydroxybutyrate comprising contacting {tilde over (α)}methylenesuccinate semialdehyde with α-methylene-γ-hydroxybutyrate dehydrogenase under suitable enzymatic conditions in a suitable aqueous reaction mixture and isolating the α-methylene-{tilde over (γ)}hydroxybutyrate produced.
A method of producing tuliposide A comprising contacting α-methylene-γ-hydroxybutyrate with α-methylene-γ-hydroxybutyrate/UDP-glucose glucosyltransferase under suitable enzymatic conditions in an aqueous reaction mixture and isolating the tuliposide A produced.
A method of producing tulipalin A comprising contacting α-methylene-γ-hydroxybutyrate with a catalytic amount of a strong acid catalyst and isolating the tulipalin A produced.
Dependencies are inferred from the claim text.