Modified photosynthetic microorganisms for producing triglycerides
This disclosure describes genetically modified photosynthetic microorganisms, including Cyanobacteria, that contain one or more exogenous genes encoding a diacylglycerol acyltransferase, a phosphatidate phosphatase, and/or an acetyl-CoA carboxylase, and which are capable of producing increased amounts of fatty acids and/or synthesizing triglycerides.
1. A method of producing a triglyceride, a wax ester or both in a photosynthetic microorganism, comprising culturing a modified Cyanobacterium comprising an exogenous polynucleotide encoding a prokaryotic diacylglycerol acyltransferase (DGAT), wherein said modified Cyanobacterium produces a triglyceride, a wax ester or both.
2. The method of claim 1 , wherein said DGAT is an Acinetobacter DGAT, a Streptomyces DGAT, or an Alcanivorax DGAT.
3. The method of claim 2 , wherein said DGAT is a Streptomyces DGAT.
4. The method of claim 3 , wherein said Streptomyces DGAT is a Streptomyces coelicolor DGAT, or a biologically active fragment or variant thereof.
5. The method of claim 4 , wherein said Streptomyces coelicolor DGAT comprises a polypeptide sequence set forth in SEQ ID NO: 14.
6. The method of claim 2 , wherein said DGAT is an Alcanivorax DGAT.
7. The method of claim 6 , wherein said Alcanivorax DGAT is an Alcanivorax borkumensis DGAT, or a biologically active fragment or variant thereof.
8. The method of claim 7 , wherein said Alcanivorax borkumensis DGAT comprises a polypeptide sequence set forth in SEQ ID NO:15.
9. The method of claim 2 , wherein said DGAT is an Acinetobacter DGAT.
10. The method of claim 9 , wherein said Acinetobacter DGAT is a Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA), or a biologically active fragment or variant thereof.
11. The method of claim 10 , wherein said AtfA comprises a polypeptide sequence set forth in SEQ ID NO: 1 or SEQ ID NO:18.
12. The method of claim 1 , wherein said modified Cyanobacterium further comprises an exogenous polynucleotide encoding a phosphatidate phosphatase and/or an acetyl-CoA carboxylase (ACCase).
13. The method of claim 12 , wherein said phosphatidate phosphatase is a yeast phosphatidate phosphatase.
14. The method of claim 12 , wherein said ACCase is a Saccharomyces cerevisiae ACCase, a Synechococcus sp. 7002 ACCase, or a Triticum aestivum ACCase.
15. The method of claim 1 , wherein said exogenous polynucleotide is present in an expression construct.
16. The method of claim 15 , wherein said expression construct is stably integrated into the modified photosynthetic microorganism's genome.
17. The method of claim 15 , wherein said expression construct comprises an inducible promoter.
18. The method of claim 1 , wherein said exogenous polynucleotide is codon-optimized for expression in a Cyanobacterium.
19. The method of claim 1 , wherein said Cyanobacterium is S. elongatus PCC 7942, a salt tolerant variant of S. elongatus PCC 7942 , Synechococcus PCC 7002 or Synechocystis PCC 6803.
20. The method of claim 1 , wherein said modified Cyanobacterium comprises an exogenous polynucleotide encoding Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA), or a biologically active fragment or variant thereof, wherein said exogenous polynucleotide is codon-optimized for expression in Cyanobacterium, wherein expression of said Acinetobacter baylii ADP1 diacylglycerol acyltransferase (AtfA), or biologically active fragment or variant thereof, is regulated by an inducible promoter, and wherein said modified Cyanobacterium is S. elongatus PCC7942 or a salt-tolerant version thereof.
21. A method of producing a triglyceride, a wax ester or both in a photosynthetic microorganism, comprising culturing a modified Cyanobacterium comprising an exogenous polynucleotide encoding a prokaryotic diacylglycerol acyltransferase (DGAT) that uses acyl-ACP as a substrate, wherein said modified Cyanobacterium produces a triglyceride, a wax ester or both.