Product and process for transformation of Thraustochytriales microorganisms
First Claim
1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
- a. a nucleic acid sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO;
15, SEQ ID NO;
19, SEQ ID NO;
22 and SEQ ID NO;
24 wherein said protein is an acetolactate synthase;
b. a nucleic acid sequence encoding a protein having an amino acid sequence that is at least 95% identical to an amino acid sequence of (a), wherein said protein is an acetolactate synthase; and
, c. a nucleic acid sequence that is fully complementary to said nucleic acid sequence of (a) or (b).
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Abstract
Disclosed are nucleic acid and amino acid sequences for acetolactate synthase, acetolactate synthase regulatory regions, α-tubulin promoter, a promoter from a Thraustochytriales polyketide synthase (PKS) system, and fatty acid desaturase promoter, each from a Thraustochytriales microorganism. Also disclosed are recombinant vectors useful for transformation of Thraustochytriales microorganisms, as well as a method of transformation of Thraustochytriales microorganisms. The recombinant nucleic acid molecules of the present invention can be used for the expression of foreign nucleic acids in a Thraustochytriales microorganism as well as for the deletion, mutation, or inactivation of genes in Thraustochytriales microorganisms.
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Citations
37 Claims
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1. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
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a. a nucleic acid sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO;
15, SEQ ID NO;
19, SEQ ID NO;
22 and SEQ ID NO;
24 wherein said protein is an acetolactate synthase;
b. a nucleic acid sequence encoding a protein having an amino acid sequence that is at least 95% identical to an amino acid sequence of (a), wherein said protein is an acetolactate synthase; and
,c. a nucleic acid sequence that is fully complementary to said nucleic acid sequence of (a) or (b). - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. A recombinant vector for transformation of microorganisms of the Order Thraustochytriales, comprising a nucleic acid sequence encoding an acetolactate synthase that confers reduced sensitivity to compounds selected from the group consisting of:
- sulfonylurea compounds, imidazolinone-class inhibitors, and pyimidinyl oxybenzoates, onto a microorganism of the order Thraustochytriales, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;
a. an amino acid sequence selected from the group consisting of SEQ ID NO;
19, SEQ ID NO;
22 and SEQ ID NO;
24; and
,b. an amino acid sequence that is at least 95% identical to an amino acid sequence of (a);
wherein said nucleic acid sequence encoding an acetolactate synthase is operatively linked to a transcription control sequence. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19)
- sulfonylurea compounds, imidazolinone-class inhibitors, and pyimidinyl oxybenzoates, onto a microorganism of the order Thraustochytriales, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;
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20. A method for transformation of cells of a microorganism of the Order Thraustochytriales, said method comprising:
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a. introducing into cells of a microorganism of the Order Thraustochytriales a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an acetolactate synthase that confers onto said cells reduced sensitivity to compounds selected from the group consisting of;
sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;
i. an amino acid sequence selected from the group consisting of SEQ ID NO;
19, SEQ ID NO;
22 and SEQ ID NO;
24; and
,ii. an amino acid sequence that is at least 95% identical to an amino acid sequence of (i); and
,b. selecting cells that have been successfully transformed with said recombinant nucleic acid molecule by culturing said cells of (a) in a medium containing at least one compound that is inhibitory to untransformed cells, said compound being selected from the group consisting of;
a sulfonylurea compound, an imidazolinone-class inhibitor, and pyrimidinyl oxybenzoates. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A recombinant microorganism of the order Thraustochytriales, transformed with a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding an acetolactate synthase that confers onto said microorganism reduced sensitivity to compounds selected from the group consisting of:
- sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;
a. an amino acid sequence selected from the group consisting of SEQ ID NO;
19, SEQ ID NO;
22 and SEQ ID NO;
24; and
,b. an amino acid sequence that is at least 95% identical to an amino acid sequence of (a). - View Dependent Claims (31, 32, 33, 34, 35, 36, 37)
- sulfonylurea compounds, imidazolinone-class inhibitors, and pyrimidinyl oxybenzoates, wherein said acetolactate synthase has an amino acid sequence selected from the group consisting of;
Specification