Algal lipid productivity via genetic modification of a TPR domain containing protein
The present invention provides mutant microorganisms having attenuated expression of a gene encoding a polypeptide that includes a TPR 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 Eustigmatophyte microorganism having a disrupted gene encoding a polypeptide comprising a tetratricopeptide repeat (TPR) domain having at least 80% amino acid sequence identity to SEQ ID NO: 3, wherein the mutant microorganism:
a) produces at least 25% more lipid than a control microorganism not having attenuated expression of the gene encoding a polypeptide comprising a TPR domain; 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 TPR domain has at least 90%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:1.
3. The mutant microorganism of claim 1 , wherein the tetratricopeptide repeat (TPR) domain has at least 90% amino acid sequence identity to SEQ ID NO:3.
4. The mutant microorganism of claim 3 , wherein the polypeptide comprises a domain of unknown function (DUF4470) that has at least 80%, identity to the amino acid sequence set forth in SEQ ID NO: 2.
5. The mutant microorganism of claim 1 , wherein the mutant microorganism comprises one or more mutations to or affecting expression of a gene in the Naga_100148g8 locus in the mutant Eustigmatophyte microorganism.
6. The mutant microorganism of claim 5 , wherein the mutant microorganism comprises a mutation to, or a mutation affecting expression of, a gene comprising an open reading frame having at least 90% sequence identity to SEQ ID NO:4.
7. The mutant microorganism of claim 1 , wherein the control microorganism is a wild type microorganism.
8. The mutant microorganism of claim 1 , wherein the mutant microorganism produces at least 80% more fatty acid methyl ester-derivatizable lipids (FAME lipids) than a control microorganism when cultured in a medium comprising nitrate as the sole nitrogen source.
9. The mutant microorganism of claim 8 , wherein the mutant microorganism is an alga and produces at least 80% more FAME lipids than a control alga when cultured under photoautotrophic conditions.
10. The mutant microorganism according to claim 1 , wherein the mutant microorganism exhibits a FAME/TOC ratio at least 80% higher than the FAME/TOC ratio of the control microorganism.
11. The mutant microorganism according to claim 10 , wherein the mutant microorganism exhibits a FAME/TOC ratio of between about 0.25 and about 0.75 under conditions that are nitrogen replete with respect to the control microorganism.
12. The mutant microorganism according to claim 1 , wherein lipid production or productivity is determined using semi-continuous, culture conditions.
13. The mutant microorganism of claim 1 , wherein said identical conditions comprise culturing said mutant and control microorganisms in a medium comprising less than 2 mM ammonium.
14. The mutant microorganism of claim 1 , wherein the mutant microorganism is a genetically engineered mutant.
15. The mutant microorganism of claim 1 , wherein the disruption comprises a partial or total deletion, a truncation, a frameshift mutation, or an insertional mutation.
16. The mutant microorganism of claim 1 , wherein the disruption comprises a knockout mutation in the gene encoding a polypeptide that includes a TPR domain, or a gene affecting expression thereof.
17. The mutant microorganism according to 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, or enhanced lipid production or accumulation.
18. A method of producing lipid, comprising culturing a mutant microorganism according to claim 1 in a culture medium to produce lipid.
19. The method of claim 18 , wherein the microorganism is cultured using batch, continuous, or semi-continuous culture conditions.
20. The method of claim 18 or 19 , wherein the culturing is under photoautotrophic conditions.
21. The mutant Eustigmatophyte microorganism of claim 1 , wherein the microorganism is of the genus Nannochloropsis.
22. The mutant Eustigmatophyte microorganism of claim 3 , wherein the microorganism is of the genus Nannochloropsis.