POLYUNSATURATED FATTY ACID SYNTHASE NUCLEIC ACID MOLECULES AND POLYPEPTIDES, COMPOSITIONS, AND METHODS OF MAKING AND USES THEREOF

• US 20150152450A1
• Filed: 12/10/2014
• Published: 06/04/2015
• Est. Priority Date: 03/19/2009
• Status: Active Grant

• Current Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Original Assignee: DSM IP Assets BV (DSM-Firmenich AG)
• Inventors: APT, Kirk E., SIMPSON, David, ZIRKLE, Ross, RICHTER, Leslie
• Granted Patent: US 9,540,666 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/001 acting on the CH-CH group of donors (1.3)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/6427 Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12P 7/6432 Eicosapentaenoic acids [EPA]
• CPC Class Codes: C12P 7/6434 Docosahexenoic acids [DHA]
• CPC Class Codes: C12P 7/6472 containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
• CPC Class Codes: C12Y 101/01 with NAD+ or NADP+ as acceptor (1.1.1)
• CPC Class Codes: C12Y 103/01 with NAD+ or NADP+ as acceptor (1.3.1)
• CPC Class Codes: C12Y 203/01039 [Acyl-carrier-protein] S-malonyltransferase (2.3.1.39)
• CPC Class Codes: C12Y 203/01041 Beta-ketoacyl-acyl-carrier-protein synthase I (2.3.1.41)
• CPC Class Codes: C12Y 402/01 Hydro-lyases (4.2.1)
• CPC Class Codes: C12Y 402/01059 3-Hydroxyacyl-[acyl-carrier-protein] dehydratase (4.2.1.59)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The present invention is directed to isolated nucleic acid molecules and polypeptides of thraustochytrid polyunsaturated fatty acid (PUFA) synthases involved in the production of PUFAs, including PUFAs enriched in docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), or a combination thereof. The present invention is directed to vectors and host cells comprising the nucleic acid molecules, polypeptides encoded by the nucleic acid molecules, compositions comprising the nucleic acid molecules or polypeptides, and methods of making and uses thereof.

Claims

1-120. -120. (canceled)

121. A recombinant nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:

72 and a transcription control sequence, wherein the polynucleotide sequence encodes a polypeptide comprising a polyunsaturated fatty acid (PUFA) synthase activity selected from the group consisting of beta-hydroxyacyl-ACP dehydrase (DH) activity, and enoyl-ACP reductase (ER) activity, and combinations thereof, wherein said polynucleotide sequence is heterologous to said transcription control sequence.

122. The recombinant nucleic acid molecule of claim 121, wherein the polynucleotide sequence is at least 95% identical to SEQ ID NO:

72.

123. The recombinant nucleic acid molecule of claim 121, wherein the nucleic acid molecule comprises the polynucleotide sequence sequences of SEQ ID NO:

72.

127. A host cell that expresses the nucleic acid molecule of claim 121 or claim 124, wherein said nucleic acid molecule is heterologous to the host cell, and wherein said host cell is not a human cell.

128. The host cell of claim 127, wherein the host cell is selected from the group consisting of a plant cell, a microbial cell, and an isolated animal cell.

131. A method of increasing production of DHA, EPA, or a combination thereof in an organism having PUFA synthase activity, comprising:

expressing the recombinant nucleic acid molecule of claim 121 or claim 124 in the organism under conditions effective to produce DHA, EPA, or a combination thereof, wherein the PUFA synthase activity replaces an inactive or deleted activity, introduces a new activity, or enhances an existing activity in the organism, and wherein production of DHA, EPA, or a combination thereof in the organism is increased.

124. An recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide and a transcription control sequence, wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:

73, wherein the polypeptide comprises a PUFA synthase activity selected from the group consisting of beta-hydroxyacyl-ACP dehydrase (DH) activity, and enoyl-ACP reductase (ER) activity, and combinations thereof, wherein said polynucleotide sequence is heterologous to said transcription control sequence.

125. The recombinant nucleic acid molecule of claim 124, wherein the amino acid sequence is at least 95% identical to SEQ ID NO:

73.

126. The recombinant nucleic acid molecule of claim 125, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

73.

129. A method to produce at least one PUFA, comprising:

expressing a PUFA synthase gene in a host cell under conditions effective to produce PUFA, wherein the PUFA synthase gene comprises a nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to SEQ ID NO;

72 or a polynucleotide sequence encoding a polypeptide which comprises an amino acid sequence at least 90% identical to SEQ ID NO;

73, wherein said nucleic acid molecule is heterologous to the host cell, wherein said polynucleotide sequence encodes a PUFA synthase polypeptide comprising beta-hydroxyacyl-ACP dehydrase (DH) activity and enoyl-ACP reductase (ER) activity, and wherein at least one PUFA is produced.

130. A method to produce lipids enriched for DHA, EPA, or a combination thereof, comprising:

expressing a PUFA synthase gene in a host cell under conditions effective to produce lipids, wherein the PUFA synthase gene comprises a nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to SEQ ID NO;

72 or a polynucleotide sequence encoding a polypeptide which comprises an amino acid sequence at least 90% identical to SEQ ID NO;

73, wherein said nucleic acid molecule is heterologous to the host cell, wherein said polynucleotide sequence encodes a PUFA synthase polypeptide comprising beta-hydroxyacyl-ACP dehydrase (DH) activity and enoyl-ACP reductase (ER) activity, and wherein lipids enriched with DHA, EPA, or a combination thereof are produced.

134. An recombinant nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:

110 and a transcription control sequence, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity, said polynucleotide sequence is heterologous to said transcription control sequence.

135. The recombinant nucleic acid molecule of claim 134, wherein the polynucleotide sequence is at least 95% identical to SEQ ID NO:

110.

136. The recombinant nucleic acid molecule of claim 134, wherein the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO:

110.

148. A host cell that expresses any one of the nucleic acid molecule of claim 134, 137, 140, 143, or 146, wherein said nucleic acid molecule is heterologous to the host cell, and wherein said host cell is not a human cell.

149. The host cell of claim 148, wherein the host cell is selected from the group consisting of a plant cell, a microbial cell, and an isolated animal cell.

150. A method to produce at least one PUFA, comprising:

expressing a PUFA synthase gene in a host cell under conditions effective to produce PUFA, wherein the PUFA synthase gene comprises any one of the nucleic acid molecule of claim 134, 137, 140, 143, or 146, and wherein at least one PUFA is produced, and wherein the nucleic acid molecule is heterologous to the host cell.

137. An recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide and a transcription control sequence, wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:

111, wherein the polypeptide comprises DH activity, and wherein said polynucleotide sequence is heterologous to said transcription control sequence.

138. The recombinant nucleic acid molecule of claim 137, wherein the amino acid sequence is at least 95% identical to SEQ ID NO:

111.

139. The recombinant nucleic acid molecule of claim 137, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

111.

140. An recombinant nucleic acid molecule comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:

112 and a transcription control sequence, wherein the polynucleotide sequence encodes a polypeptide comprising DH activity, said polynucleotide sequence is heterologous to said transcription control sequence.

141. The recombinant nucleic acid molecule of claim 140, wherein the polynucleotide sequence is at least 95% identical to SEQ ID NO:

112.

142. The recombinant nucleic acid molecule of claim 140, wherein the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO:

112.

143. An recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide and a transcription control sequence, wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:

113, wherein the polypeptide comprises DH activity, and wherein said polynucleotide sequence is heterologous to said transcription control sequence.

144. The recombinant nucleic acid molecule of claim 143, wherein the amino acid sequence is at least 95% identical to SEQ ID NO:

113.

145. The recombinant nucleic acid molecule of claim 143, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

115.

146. An recombinant nucleic acid molecule comprising a polynucleotide sequence encoding a polypeptide and a transcription control sequence, wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:

115, wherein the polypeptide comprises ER activity, and wherein said polynucleotide sequence is heterologous to said transcription control sequence.

147. The recombinant nucleic acid molecule of claim 146, wherein the amino acid sequence is at least 95% identical to SEQ ID NO:

115.