received by the International Bureau on 01 July 2019 (01 .07.2019)

What is claimed is:

1. A mutant microorganism having attenuated expression of a gene encoding a polypeptide that comprises a GAF domain, wherein the mutant microorganism:

a) produces at least about 25% more lipid than a control microorganism;

and/or

b). exhibits increased partitioning of carbon to lipid with respect to the

control microorganism;

when the mutant microorganism and control microorganism are cultured under identical conditions.

2. The mutant microorganism of claim 1, wherein the GAF domain is a GAF2 domain.

3. The mutant microorganism of claim 2, wherein the GAF2 domain has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence set forth in SEQ ID NO: 1.

4. The mutant microorganism of claim 2, wherein the GAF2 domain comprises the amino acid sequence set forth in SEQ ID NO:l, or a conservative variant thereof.

5. The mutant microorganism of claim 1, wherein the polypeptide comprises an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence set forth in SEQ ID NO:2.

6. The mutant microorganism of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2, or a conservative variant thereof.

7. The mutant microorganism of claim 1, wherein the mutant microorganism comprises one or more mutations to or affecting the expression of a gene in the Naga_l00020g79 locus or a syntenic locus in a heterokont or algal species.

8. The mutant microorganism of claim 7, wherein the mutant microorganism comprises a mutation to, or a mutation affecting the expression of, a gene comprising an open reading frame having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 3.

9. A mutant microorganism of claim 7, wherein said one or more mutations are present in or affect expression of: a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:l or SEQ ID NO:2; and/or comprising an open reading frame that comprises a nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 3 or SEQ ID NO:4.

10. The mutant microorganism of claim 7, wherein the one or more mutations are in the GAF domain of the gene in the Naga_l00020g79 locus.

11. A mutant microorganism of any one of claims 1-10, wherein the control microorganism is a wild type microorganism.

12. A mutant microorganism of any one of claims 1-11, wherein the mutant microorganism produces at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, or at least about 250% more fatty acid methyl ester-derivatizable lipids (FAME lipids) than a control microorganism.

13. The mutant microorganism of claim 12, wherein the mutant microorganism produces at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, or at least about 250% more fatty acid methyl ester-derivatizable lipids (FAME lipids) than a control microorganism when cultured in a culture medium comprising nitrate as the sole nitrogen source.

14. The mutant microorganism of claim 12 or 13, wherein the mutant microorganism is an alga and produces at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, or at least about 250% more FAME lipids than a control alga when cultured under photoautotrophic conditions.

15. The mutant microorganism of any one of claims 12-14, wherein the microorganism produces at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, or at least about 120% more storage lipids than the control microorganism.

16. The mutant microorganism of claim 15, wherein the storage lipids are triacylglycerols (TAGs).

17. The mutant microorganism according to any one of claims 1-16, wherein the mutant microorganism exhibits a FAME/TOC ratio at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 140%, at least about 160%, at least about 180%, at least about 200%, at least about 220%, at least about 240%, at least about 260%, at least about 280%, at least about 300%, at least about 320%, at least about 340%, at least about 360%, at least about 380%, or at least about 400% higher than the FAME/TOC ratio of the control microorganism.

18. The mutant microorganism according to claim 17, wherein the mutant microorganism exhibits a FAME/TOC ratio of at least about 0.35.

19. The mutant microorganism according to any one of claims 1-18, wherein lipid productivity is determined in a batch, semicontinuous, or continuous productivity assay.

20. The mutant microorganism according to any of claims 1-18, wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium comprising less than 2 mM ammonium.

21. The mutant microorganism according to claim 20, wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium comprising nitrate as substantially the sole nitrogen source.

22. The mutant microorganism of any one of claims 1-21, wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium that is nutrient replete with respect to the control microorganism.

23. A mutant microorganism of any one of claims 1-22, wherein the mutant microorganism is a classically-derived mutant or a genetically engineered mutant.

24. The mutant microorganism of claim 23, wherein the mutant microorganism has a mutation in the gene encoding a polypeptide that comprises a GAF domain, or a gene affecting the expression thereof, that results in a decrease of expression of said gene encoding a polypeptide that includes a GAF domain compared to expression of the gene in a control microorganism.

25. The mutant microorganism of claim 23, wherein the mutation is a knockdown mutation.

26. The mutant microorganism according to claim 25, wherein the knockdown mutation is generated using a Cas/CRISPR system.

27. The mutant microorganism of claim 23, wherein the mutant microorganism comprises an RNAi construct, a ribozyme construct, or an antisense construct that targets the gene encoding the polypeptide having a GAF domain, or a gene affecting the expression thereof.

28. The mutant microorganism of claim 24, wherein the mutant microorganism has a knockout mutation in the gene encoding a polypeptide that comprises a GAF domain, or a gene affecting the expression thereof.

29. The mutant microorganism according to claim 28, wherein the knockout mutation is produced by site directed homologous recombination.

30. The mutant microorganism according to claim 28 or 29, wherein the knockout mutation disrupts the gene by partial or total deletion, truncation, frameshifting, or insertional mutation.

31. The mutant microorganism according to any one of claims 28-30, wherein the knockout mutation is generated using meganuclease, zinc finger nuclease, a Transcription Activator-Like Effector Nuclease (TALEN) system, and/or a Cas/CRISPR system.

32. The mutant microorganism of any one of claims 26, 28, 30, or 31, wherein the mutant microorganism comprises a Cas/CRISPR-mediated insertion into the gene.

33. The mutant microorganism according to any one of claims 1-32, wherein the mutant microorganism comprises at least one additional genetic modification that confers herbicide resistance, toxin resistance, enhanced growth properties, enhanced photosynthetic efficiency, enhanced lipid production or accumulation, and/or production of particular lipids.

34. The mutant microorganism according to any one of claims 1-33, wherein the mutant microorganism is an algal or heterokont species.

35. The mutant microorganism according to claim 34, wherein the mutant microorganism is an algal species selected from the group consisting of Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Desmodesmus, Dunaliella, Elipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Hantzschia, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova, Pelagomonas, Phceodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria, and Volvox.

36. The mutant microorganism according to claim 34, wherein the mutant microorganism is a heterokont selected from the group consisting of bacillariophytes, eustigmatophytes, xanthophytes, phaeophytes, chrysophytes, or raphidophytes.

37. The mutant microorganism according to claim 34, wherein the mutant microorganism is a heterokont selected from the group consisting of a Labyrinthulomycite species of Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Oblongichytrium, Japonochytrium, Diplophrys , or Ulkenia.

38. The mutant microorganism according to claim 34, wherein the mutant microrganism is a heterokont alga.

39. The mutant microorganism according to claim 38, wherein the mutant microrganism is a Eustigmatophyte.

40. The mutant microorganism according to claim 39, wherein the mutant microrganism is a species of a genus selected from the group consisting of Chloridella, Chlorobptrys, Ellipsoidion, Eustigmatos, Goniochloris, Monodopsis, Monodus, Nannochloropsis, Pseudocharaciopsis, Pseudostaruastrum, Pseudotetraedriella, and Vischeria.

41. The mutant microorganism according to claim 38, wherein the mutant microrganism is a Bacilliarophyte.

42. The mutant microorganism according to claim 41, wherein the mutant microrganism is a species of of a genus selected from the group consisting of Amphora, Chaetoceros, Cyclotella, Fragilaria, Fragilaropsis, Hantzschia, Navicula, Nitzschia, Phceodactylum, and Thalassiosira.

43. A method of producing lipid, comprising culturing a mutant microorganism according to any one of claims 1-42 in a culture medium to produce lipid.

44. The method of claim 43, further comprising isolating lipid from the microorganism, the culture medium, or both.

45. The method of claim 43 or 44, wherein the microorganism is cultured using batch, continuous, or semi-continuous culture conditions.

46. The method of any one of claims 43-45, wherein the microorganism is an alga and the culturing is under photoautotrophic conditions.

47. The method of any one of claims 43-45, wherein the microorganism is Labyrinthulomycite and the culturing is under heterotrophic conditions.

48. The method of any one of claims 43-47, wherein the microorganism is cultured in a photobioreactor or fermenter.

49. The method of any one of claims 43-47, wherein the microorganism is grown in a pond, canal, sea-based growth container, trench, raceway, or channel.

50. A guide RNA of a CRISPR system, wherein the guide RNA comprises a sequence corresponding to SEQ ID NO:4.

51. The guide RNA according to claim 50, wherein the guide RNA is a chimeric guide.

52. The guide RNA according to claim 50, wherein the guide RNA does not include a tracr sequence.

53. A nucleic acid construct for homologous recombination comprising a nucleotide sequence from or adjacent to a naturally-occurring algal gene encoding a polypeptide having an amino acid sequence with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2; a gene localized to the Naga_l00020g79 locus; and/or a gene that comprises an ORF comprising a sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:3 and/or SEQ ID NO:4.

54. A nucleic acid construct for expression of an antisense RNA, shRNA, microRNA, or ribozyme comprising a nucleotide sequence complementary to at least a portion of a naturally-occurring gene encoding a polypeptide having an amino acid sequence with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: l or SEQ ID NO:2; a gene localized to the Naga_l00020g79 locus; and/or a gene that comprises an ORF comprising a sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:3 and/or SEQ ID NO:4.

55. A nucleic acid molecule encoding a guide RNA of a CRISPR system, wherein the guide RNA comprises at least a portion of a naturally-occurring algal gene encoding a polypeptide having an amino acid sequence with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO: l or SEQ ID NO:2; a gene localized to the Naga_l00020g79 locus; and/or a gene that comprises an ORF comprising a sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:3.

56. A method for producing a mutant microorganism of any one of claims 1-42 comprising introducing into said microorganism one or more mutations and/or one or more agents that attenuates the expression of a polypeptide comprising a GAF domain.

57. The method of claim 56 wherein the one or more mutations affects the expression of a polypeptide comprising the amino acid sequence of SEQ ID NO: l and/or SEQ ID NO:2; a gene localized to the Naga_l00020g79 locus; or a gene comprising an open reading frame that comprises the nucleotide sequence of SEQ ID NO:3.

58. The method of claim 57 wherein the one or more agents is selected from the group consisting of antisense RNA, RNAi, shRNA, microRNA, ribozyme, a meganuclease, a zinc finger nuclease, a component of a Cas/CRISPR system, and/ or a component of a Transcription Activator-Like Effector Nuclease (TALEN) system.

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