Method to transform algae, materials therefor, and products produced thereby

• US 5,661,017 A
• Filed: 03/15/1995
• Issued: 08/26/1997
• Est. Priority Date: 09/14/1993
• Status: Expired due to Fees

• Current Assignee: Midwest Research Institute
• Original Assignee: Midwest Research Institute
• Inventors: Roessler, Paul G., Jarvis, Eric E., Dunahay, Terri Goodman
• Primary Examiner(s): Fox, David T.
• Application Number:
  US08/404,732
• Time in Patent Office: 895 Days
• Field of Search: 435/69.1, 435/70.1, 435/71.1, 435/172.3, 435/240.4, 435/320.1, 435/257.2, 536/24.1, 536/23.6, 536/23.7, 800/205
• US Class Current: 435/470
• CPC Class Codes: C12N 15/8241 Phenotypically and genetically modified plants via recombinant DNA technology
• CPC Class Codes: C12N 9/93 Ligases (6)

Abstract

Disclosed is a method to transform chlorophyll C-containing algae which includes introducing a recombinant molecule comprising a nucleic acid molecule encoding a dominant selectable marker operatively linked to an algal regulatory control sequence into a chlorophyll C-containing alga in such a manner that the marker is produced by the alga. In a preferred embodiment the algal regulatory control sequence is derived from a diatom and preferably Cyclotella cryptica. Also disclosed is a chimeric molecule having one or more regulatory control sequences derived from one or more chlorophyll C-containing algae operatively linked to a nucleic acid molecule encoding a selectable marker, an RNA molecule and/or a protein, wherein the nucleic acid molecule does not normally occur with one or more of the regulatory control sequences. Further specifically disclosed are molecules pACCNPT10, pACCNPT4.8 and pACCNPT5.1. The methods and materials of the present invention provide the ability to accomplish stable genetic transformation of chlorophyll C-containing algae.

Claims

1. A method to transform a chlorophyll C-containing alga, comprising introducing a recombinant molecule comprising a nucleic acid molecule encoding a dominant selectable marker operatively linked to an algal regulatory control sequence into a chlorophyll C-containing alga such that said marker is produced by the transformed alga, wherein the regulatory control sequence is selected from the group consisting of a Cyclotella cryptica acetyl-CoA carboxylase 5'"'"' untranslated regulatory control sequence, a Cyclotella cryptica acetyl-CoA carboxylase 3'"'"'-untranslated regulatory control sequence, and combinations thereof.

2. A method as claimed in claim 1, wherein said chlorophyll C-containing alga is of a class selected from the group consisting of Bacillariophyceae, Chrysophyceae, Phaeophyceae, Xanthophyceae, Raphidophyceae, Prymnesiophyceae and Cryptophyceae.

3. A method as claimed in claim 1, wherein said chlorophyll C-containing alga is a diatom.

4. A method as claimed in claim 1, wherein said chlorophyll C-containing alga is of a genus selected from the group consisting of Cyclotella, Navicula, Cylindrotheca, Phaeodactylum, Amphora, Chaetoceros, Nitzschia and Thalassiosira.

5. A method as claimed in claim 1, wherein said chlorophyll C-containing alga is of a genus selected from the group consisting of Cyclotella and Navicula.

6. A method as claimed in claim 1, wherein said chlorophyll C-containing algae is of a species selected from the group consisting of Cyclotella cryptica and Navicula saprophila.

7. A method as claimed in claim 1, wherein said dominant selectable marker is selected from the group consisting of a heterologous marker capable of conferring resistance to a compound to which said alga otherwise exhibits sensitivity and a homologous marker, wherein said homologous marker is a homologous modified protein or nucleic acid, said homologous marker being capable of conferring resistance to a compound to which said alga otherwise exhibits sensitivity.

8. A method as claimed in claim 1, further comprising:

a) culturing said transformed alga in the presence of a compound to which algal cells exhibit sensitivity if not transformed by said recombinant molecule and to which said dominant selectable marker provides resistance; and

b) isolating from said culture a transformed alga that is capable of growing in the presence of said compound.

9. The method of claim 7, wherein said compound is selected from the group consisting of a compound that inhibits translation, a compound that inhibits transcription, a compound that inhibits enzyme function, a compound that inhibits cell growth, a compound that inhibits cell division, and a compound that inhibits microtubule formation.

10. A method as claimed in claim 7, wherein said dominant selectable marker is selected from the group consisting of neomycin phosphotransferase, aminoglycoside phosphotransferase, aminoglycoside acetyltransferase, chloramphenicol acetyl transferase, hygromycin B phosphotransferase, bleomycin binding protein, phosphinothricin acetyltransferase, bromoxynil nitrilase, glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase, cryptopleurine-resistant ribosomal protein S14, emetine-resistant ribosomal protein S14, sulfonylurea-resistant acetolactate synthase, imidazolinone-resistant acetolactate synthase, streptomycin-resistant 16S ribosomal RNA, spectinomycin-resistant 16S ribosomal RNA, erythromycin-resistant 23S ribosomal RNA, and methyl benzimidazole-resistant tubulin.

11. A method as claimed in claim 1, further comprising introducing a recombinant molecule comprising a nucleic acid molecule encoding a product operatively linked to an algal regulatory control sequence into said alga such that said product is produced by said alga.

12. A method as claimed in claim 11, wherein said recombinant molecule comprising a nucleic acid molecule encoding a product is different from said recombinant molecule encoding a dominant selectable marker.

13. A method of claim 11, wherein said alga is selected from the group consisting of Cyclotella cryptica and Navicula sapropila.

14. A method as claimed in claim 11, wherein one or both of said regulatory control sequences is selected from the group consisting of a C. cryptica acetyl-CoA carboxylase 5'"'"'-untranslated regulatory control sequence, a C. cryptica acetyl-CoA carboxylase 3'"'"'-untranslated regulatory control sequence, and combinations thereof.

15. A method as claimed in claim 11, wherein one or both of said regulatory control sequences is selected from the group consisting of a nucleic acid molecule comprising about 816 nucleotides immediately upstream from the translation initiation site of a C. cryptica acetyl-CoA carboxylase gene, a nucleic acid molecule comprising about 445 nucleotides immediately upstream from the translation initiation site of a C. cryptica acetyl-CoA carboxylase gene, a nucleic acid molecule comprising about 594 nucleotides immediately downstream from the translation termination site of a C. cryptica acetyl-CoA carboxylase gene, and combinations thereof.

16. A method as claimed in claim 1, wherein said regulatory control sequence is selected from the group consisting of a nucleic acid molecule comprising about 816 nucleotides immediately upstream from the translation initiation site of a C. cryptica acetyl-CoA carboxylase gene, a nucleic acid molecule comprising about 445 nucleotides immediately upstream from the translation initiation site of a C. cryptica acetyl-CoA carboxylase gene, a nucleic acid molecule comprising about 594 nucleotides immediately downstream from the translation termination site of a C. cryptica acetyl-CoA carboxylase gene, and combinations thereof.

17. The method of claim 11, wherein said product is selected from the group consisting of an RNA molecule and a protein.

18. A method as claimed in claim 11, wherein said product is acetyl-CoA carboxylase.

19. A method as claimed in claim 1, wherein said transformed alga is capable of maintaining said recombinant molecule for at least about eight months when cultured on a non-selective medium.

20. A method as claimed in claim 1, wherein said recombinant molecule comprises a molecule selected from the group consisting of pACCNPT10, pACCNPT5.1, and pACCNPT4.

21. A method as claimed in claim 1, wherein said recombinant molecule is integrated at a location selected from the group consisting of the nuclear genome of said alga, a chloroplast genome of said alga and a mitochondrial genome of said alga.

22. A chimetic molecule comprising one or more C. cryptica acetyl-CoA carboxylase regulatory control sequences operatively linked to a nucleic acid molecule encoding a compound selected from the group consisting of a selectable marker, an RNA molecule and a protein, wherein said nucleic acid molecule is not naturally associated with one or more of said regulatory control sequences, and wherein said regulatory control sequences are selected from the group consisting of 5'"'"'-untranslated regulatory control sequences, 3'"'"'-untranslated regulatory control sequences, and combinations thereof.

23. The chimeric molecule of claim 22, wherein said regulatory control sequences are selected from the group consisting of transcription control sequences, translation control sequences and combinations thereof.

24. The chimeric molecule of claim 22, wherein said molecule comprises a nucleic acid molecule operatively linked to a 5'"'"'-untranslated regulatory control sequence derived from a C. cryptica acetyl-CoA carboxylase gene and to a 3'"'"'-untranslated regulatory control sequence derived from a C. cryptica acetyl-CoA carboxylase gene.

25. The chimeric molecule of claim 22, wherein said selectable marker is neomycin phosphotransferase.

26. The chimeric molecule as claimed in claim 22, wherein said selectable marker is a dominant selectable marker operatively linked to said one or more regulatory control sequences and wherein said dominant selectable marker is selected from the group consisting of neomycin phosphotransferase, aminoglycoside phosphotransferase, aminoglycoside acetyltransferase, hygrormycin B phosphotransferase, bleomycin binding protein, phosphinothricin acetyltransferase, bromoxynil nitrilase, glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase, emetine-resistant ribosomal protein S14, cryptopleurine-resistant ribosomal protein S14, sulfonylurea-resistant acetolactate synthase, imidazolinone-resistant acetolactate synthase, streptomycin-resistant 16S ribosomal RNA, spectinomycin-resistant 16S ribosomal RNA, erythromycin-resistant 23S ribosomal RNA, and methyl benzimidazole-resistant tubulin.

27. The chimeric molecule as claimed in claim 22, wherein said protein is acetyl-CoA carboxylase.

28. The chimeric molecule as claimed in claim 22, wherein said regulatory control sequence is selected from the group consisting of a nucleic acid molecule comprising about 816 nucleotides immediately upstream from the translation initiation site of the C. cryptica acetyl-CoA carboxylase gene, a nucleic acid molecule comprising about 445 nucleotides immediately upstream from the translation initiation site of the C. cryptica acetyl-CoA carboxylase gene, a nucleic acid molecule comprising about 594 nucleotides immediately downstream from the translation termination site of the C. cryptica acetyl-CoA carboxylase gene, and combinations thereof.

29. A recombinant chlorophyll C-containing algal strain, wherein said strain is selected from the group consisting of:

(a) a chlorophyll C-containing algal strain transformed with a chimeric molecule comprising one or more C. cryptica acetyl-CoA carboxylase regulatory control sequences operatively linked to a nucleic acid molecule encoding a compound selected from the group consisting of a selectable marker, an RNA molecule and a protein, wherein said nucleic acid molecule is not naturally associated with one or more of said regulatory control sequences; and

(b) chlorophyll C-containing algal strain transformed with at least one additional copy of a homologous nucleic acid molecule;

wherein the regulatory control sequences are selected from the group consisting of a Cyclotella cryptica acetyl-CoA carboxylase 5'"'"'-untranslated regulatory control sequence, a Cyclotella cryptica acetyl-CoA carboxylase 3'"'"'-untranslated regulatory control sequence, and combinations thereof.

30. A strain as claimed in claim 29, wherein said strain is transformed with a DNA sequence encoding acetyl-CoC carboxylase.

31. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of sequences identified as SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3 and homologues thereof wherein the regulatory control sequences of said homologues are selected from the group consisting of a Cyclotella cryptica acetyl-CoA carboxylase 5'"'"'-untranslated regulatory control sequence, a Cyclotella cryptica acetyl-CoA carboxylase 3'"'"'-untranslated regulatory control sequence, and combinations thereof;

said homologues having a regulatory function of the nucleic acid sequence SEQ ID NO;

1, SEQ ID NO;

2 or SEQ ID NO;

3.