Algal lipid productivity via genetic modification of a signaling protein
The present invention provides mutant microorganisms having attenuated expression of a gene encoding a polypeptide that includes a GAF domain wherein the mutant microorganisms have higher lipid productivity and/or exhibit increased partitioning of carbon to lipid as compared to wild-type microorganisms from which they are derived. Also provided are methods of producing lipids using the mutant microorganisms, guide RNAs, and nucleic acid constructs used for producing mutant microorganisms.
1. A mutant Nannochloropsis microorganism comprising a complete knockout of a gene encoding a polypeptide comprising a GAF2 domain having at least 90% sequence identity to SEQ ID NO: 1; and 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; and
when the mutant microorganism and control microorganism are cultured under identical conditions.
2. The mutant Nannochloropsis microorganism of claim 1 , wherein the gene encodes a polypeptide comprising a GAF2 domain having at least 90% sequence identity to SEQ ID NO: 2.
3. The mutant Nannochloropsis microorganism of claim 1 , wherein the mutant microorganism comprises a complete knockout of a gene comprising an open reading frame having at least 90% sequence identity to SEQ ID NO: 3.
4. The mutant Nannochloropsis microorganism of claim 1 , wherein the control microorganism is a wild type microorganism.
5. The mutant Nannochloropsis microorganism of claim 1 , wherein the mutant microorganism produces at least 100% more fatty acid methyl ester-derivatizable lipids (FAME lipids) than a control microorganism over a 5 day period.
6. The mutant Nannochloropsis microorganism of claim 5 , wherein the culture medium comprises nitrate as the sole nitrogen source.
7. The mutant Nannochloropsis microorganism of claim 5 , wherein the 7 day period occurs under photoautotrophic conditions.
8. The mutant Nannochloropsis microorganism of claim 1 , wherein the mutant microorganism exhibits a FAME/TOC ratio at least 90% higher than the FAME/TOC ratio of the control microorganism.
9. The mutant Nannochloropsis microorganism according to claim 8 , wherein the mutant microorganism exhibits a FAME/TOC ratio of at least about 0.35.
10. The mutant Nannochloropsis microorganism of claim 1 , wherein lipid productivity is determined in a batch productivity assay.
11. The mutant Nannochloropsis microorganism of claim 1 , wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium comprising less than 2 mM ammonium.
12. The mutant Nannochloropsis microorganism according to claim 11 , wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium comprising nitrate as substantially the sole nitrogen source.
13. The mutant Nannochloropsis microorganism of claim 1 , wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium that is nutrient replete with respect to the control microorganism.
14. The mutant Nannochloropsis microorganism of claim 1 , wherein the mutant microorganism is a genetically engineered mutant.
15. The mutant Nannochloropsis microorganism according to claim 14 , wherein the deletion is generated using a Cas/CRISPR system.
16. The mutant Nannochloropsis microorganism of claim 14 , 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.
17. The mutant Nannochloropsis microorganism according to claim 14 , wherein the knockout is produced by site directed homologous recombination.
18. The mutant Nannochloropsis microorganism of claim 14 , wherein the knockout is a truncation, frameshifting, or insertional mutation.
19. The mutant Nannochloropsis microorganism of claim 14 , wherein the knockout is generated using meganuclease, zinc finger nuclease, a Transcription Activator-Like Effector Nuclease (TALEN) system, and/or a Cas/CRISPR system.
20. The mutant Nannochloropsis microorganism of claim 15 , wherein the mutant microorganism comprises a Cas/CRISPR-mediated insertion into the gene.
21. The mutant Nannochloropsis microorganism of claim 1 , 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.
22. A method of producing lipid, comprising culturing the mutant Nannochloropsis microorganism of claim 1 in a culture medium to produce lipid.
23. The method of claim 22 , further comprising isolating lipid from the microorganism, the culture medium, or both.
24. The method of claim 22 , wherein the microorganism is cultured using batch, continuous, or semi-continuous culture conditions.
25. The method of claim 22 , wherein the microorganism is an alga and the culturing is under photoautotrophic conditions.
26. The method of claim 22 , wherein the microorganism is cultured in a photobioreactor or fermenter.
27. The method of claim 22 , wherein the microorganism is grown in a pond, canal, sea-based growth container, trench, raceway, or channel.