Product and Process for Transformation of Thraustochytriales Microorganisms

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.

1 - 35 . (canceled) 
     
     
         36 . A method for production of a protein, comprising:
 culturing a recombinant microorganism of the Order Thraustochytriales in a medium, wherein the recombinant microorganism comprises an isolated nucleic acid molecule comprising a foreign gene that encodes the protein, to produce the protein; and   recovering the protein.   
     
     
         37 . The method of  claim 36 , wherein the isolated nucleic acid molecule further comprises one or more transcription control sequences and the foreign gene is operatively linked to the one or more transcription control sequences. 
     
     
         38 . The method of  claim 37 , wherein the one or more transcription control sequences comprise a Thraustochytriales transcription control sequence. 
     
     
         39 . The method of  claim 38 , wherein the Thraustochytriales transcription control sequence is selected from the group consisting of Thraustochytriales promoter sequences, Thraustochytriales enhancer sequences, Thraustochytriales operator sequences, Thraustochytriales repressor sequences, and combinations thereof. 
     
     
         40 . The method of  claim 39 , wherein at least one Thraustochytriales transcription control sequence is a Thraustochytriales promoter sequence. 
     
     
         41 . The method of  claim 40 , wherein the Thraustochytriales promoter sequence is selected from the group consisting of a Thraustochytriales acetolactate synthase promoter sequence, Thraustochytriales α-tubulin promoter sequence, Thraustochytriales polyketide synthase system promoter sequence, and Thraustochytriales fatty acid desaturase promoter sequence. 
     
     
         42 . The method of  claim 36 , wherein the isolated nucleic acid molecule further comprises a signal segment nucleic acid sequence. 
     
     
         43 . The method of  claim 36 , wherein the protein is secreted into the medium, and wherein the protein is recovered from the medium. 
     
     
         44 . The method of  claim 36 , wherein the microorganism is from a genus selected from the group consisting of  Thraustochytrium, Labyrinthuloides, Japonochytrium,  and  Schizochytrium.    
     
     
         45 . The method of  claim 44 , wherein the microorganism is from a species selected from the group consisting of  Schizochytrium  sp.,  Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium minutum, Thraustochytrium  sp.,  Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum,  and  Japonochytrium  sp. 
     
     
         46 . A method for production of a protein, comprising:
 culturing a recombinant microorganism of the Order Thraustochytriales in a medium, wherein the recombinant microorganism comprises an isolated nucleic acid molecule comprising a foreign gene that encodes the protein, to produce the protein; and   harvesting the microorganism comprising the protein.   
     
     
         47 . The method of  claim 46 , wherein the microorganism is harvested as a biomass. 
     
     
         48 . A method for transforming a microorganism cell, comprising:
 introducing into the microorganism cell of the Order Thraustochytriales a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a protein to be expressed and a selection marker; and   selecting a microorganism cell that has been successfully transformed with the recombinant nucleic acid molecule.   
     
     
         49 . The method of  claim 48 , wherein the nucleic acid sequence encoding a protein to be expressed is integrated in the genome of the microorganism by homologous recombination. 
     
     
         50 . The method of  claim 48 , wherein the recombinant nucleic acid molecule further comprises one or more transcription control sequences and the nucleic acid sequence encoding the protein to be expressed is operatively linked to the one or more transcription control sequences. 
     
     
         51 . The method of  claim 50 , wherein the one or more transcription control sequences comprises one or more Thraustochytriales transcription control sequences. 
     
     
         52 . The method of  claim 51 , wherein the one or more Thraustochytriales transcription control sequences are selected from the group consisting of Thraustochytriales promoter sequences, Thraustochytriales enhancer sequences, Thraustochytriales operator sequences, Thraustochytriales repressor sequences, and combinations thereof. 
     
     
         53 . The method of  claim 52 , wherein at least one Thraustochytriales transcription control sequence is a Thraustochytriales promoter sequence. 
     
     
         54 . The method of  claim 53 , wherein the Thraustochytriales promoter sequence is selected from the group consisting of a Thraustochytriales acetolactate synthase promoter sequence, Thraustochytriales α-tubulin promoter sequence, Thraustochytriales polyketide synthase system promoter sequence, and Thraustochytriales fatty acid desaturase promoter sequence. 
     
     
         55 . The method of  claim 48 , wherein the recombinant nucleic acid molecule further comprises a signal segment nucleic acid sequence. 
     
     
         56 . A method for inactivating, deleting, or mutating a native gene of a microorganism, comprising:
 introducing into a microorganism cell of the Order Thraustochytriales a recombinant nucleic acid molecule comprising a nucleic acid sequence that hybridizes with a target nucleic acid sequence in the microorganism, wherein a gene in the microorganism is inactivated, deleted, or mutated by homologous recombination between the nucleic acid sequence that hybridizes and the target nucleic acid sequence; and   selecting a microorganism cell comprising the inactivated, deleted, or mutated gene.   
     
     
         57 . The method of  claim 56 , wherein the gene encodes or regulates expression of a protein involved in fatty acid biosynthesis, a protein involved in carbohydrate biosynthesis, a protein involved in synthesis of products of an isoprenoid pathway, or a protein involved in synthesis of a cell wall component. 
     
     
         58 . The method of  claim 57 , wherein the gene encodes or regulates expression of a protein involved in the synthesis of a lipase, a fatty acid oxidation enzyme, a protein having an objectionable flavor or odor, a fatty acid, a carotenoid, a sterol, or a vitamin.