Modified photosynthetic microorganisms for producing lipids
Inventors
Roberts, James • Cross, Fred • McCormick, Margaret Mary • Munoz, Ernesto Javier • Kaiser, Brett K. • Carleton, Michael
Assignees
Publication Number
US-9523096-B2
Publication Date
2016-12-20
Expiration Date
2031-12-19
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Abstract
This disclosure describes genetically modified photosynthetic microorganisms, e.g., Cyanobacteria, that overexpress an acyl carrier protein (ACP), an acyl-ACP synthase (Aas), or both, optionally in combination with one or more overexpressed or exogenous lipid biosynthesis proteins, and/or one or more overexpressed or exogenous glycogen breakdown proteins. Exemplary biosynthesis proteins include diacyglycerol acyltransferases, thioesterases, phosphatidate phosphatases, phospholipases, triacylglycerol (TAG) hydrolases, fatty acyl-CoA synthetases, and/or acetyl-CoA carboxylases, including combinations thereof. Also included are photosynthetic microorganisms comprising mutations or deletions in a glycogen biosynthesis or storage pathway, which accumulate a reduced amount of glycogen under reduced nitrogen conditions as compared to a wild type photosynthetic microorganism. The modified photosynthetic microorganisms provided herein are capable of producing increased amounts of lipids such as fatty acids and/or synthesizing triglycerides.
Core Innovation
The invention provides genetically modified photosynthetic microorganisms, such as Cyanobacteria, that are engineered to overexpress an acyl carrier protein (ACP), an acyl-ACP synthetase (Aas), or both. These modifications may be combined with overexpression or introduction of one or more lipid biosynthesis proteins (including, for example, acyl-ACP thioesterases, diacylglycerol acyltransferases, phosphatidate phosphatases, phospholipases, triacylglycerol hydrolases, fatty acyl-CoA synthetases, or acetyl-CoA carboxylases) and/or reduced expression of genes involved in glycogen biosynthesis or storage. This design results in photosynthetic microorganisms capable of producing increased amounts of lipids such as fatty acids, free fatty acids, and triglycerides, compared to their unmodified counterparts.
The background describes that while eukaryotic microorganisms like algae produce triglycerides as energy storage molecules and can be valuable for biofuel feedstocks, they are challenging to genetically manipulate and often produce less oil under culture conditions than in the wild. Cyanobacteria, on the other hand, are genetically tractable, but naturally lack key enzymes for triglyceride synthesis and instead store carbon primarily as glycogen rather than as triglycerides or fatty acids. Thus, there is an unmet need for genetically engineered photosynthetic microorganisms that can be redirected to efficiently produce lipids—especially triglycerides—for use in producing biofuels and specialty chemicals.
Through molecular engineering, the invention addresses this need by equipping Cyanobacteria and other photosynthetic microorganisms with enhanced acyl-ACP synthesis and lipid biosynthesis pathways, and, optionally, by reducing carbon flux to glycogen. This is achieved by introducing or overexpressing ACP and/or Aas genes, together with genes for one or more lipid biosynthesis proteins, and/or manipulating expression or deletion of glycogen biosynthesis or storage pathway genes. These modifications have been shown to significantly increase lipid output—such as fatty acid and triglyceride accumulation—relative to wild-type or singly-modified organisms.
Claims Coverage
The patent includes several independent claims that define inventive features centered around genetically modified Cyanobacteria with enhanced acyl-ACP synthesis, increased lipid biosynthesis activity, and optionally altered glycogen metabolism.
Genetically modified Cyanobacterium with enhanced acyl-ACP synthesis and increased lipid biosynthesis protein
A Cyanobacterium containing: - A first modification introducing a polynucleotide encoding an acyl carrier protein (ACP), thereby increasing acyl-ACP synthesis in the organism. - A second modification introducing a polynucleotide encoding a lipid biosynthesis protein such as acyl-ACP thioesterase (TES), diacylglycerol acyltransferase (DGAT), acetyl coenzyme A carboxylase (ACCase), phosphatidic acid phosphatase (PAP), triacylglycerol hydrolase, fatty acyl-CoA synthetase, or phospholipase, including combinations thereof. - The Cyanobacterium produces an increased amount of lipid compared to a corresponding wild-type or singly-modified Cyanobacterium.
Cyanobacterium with reduced glycogen accumulation through genetic modification
A Cyanobacterium further comprising a third modification that reduces glycogen accumulation, achieved by either: - A full or partial deletion of a gene in the glycogen biosynthesis or storage pathway or - Reduced expression of such a gene (including, for example, glucose-1-phosphate adenyltransferase (glgC), phosphoglucomutase (pgm)), - Or by introduction or overexpression of a gene encoding a protein involved in glycogen breakdown. This results in further enhancement of lipid production.
Methods of producing genetically modified Cyanobacteria with increased lipid production
Methods for making a Cyanobacterium comprising: 1. Introducing a modification to increase acyl-ACP synthesis by incorporating a polynucleotide encoding an acyl carrier protein (ACP). 2. Introducing a polynucleotide encoding a lipid biosynthesis protein (as detailed above). 3. Further optional modifications include introducing a polynucleotide encoding an acyl-ACP synthetase (Aas), or altering glycogen metabolic genes as described above.
Method for lipid production by culturing the modified Cyanobacterium
Culturing the described genetically modified Cyanobacterium to produce or accumulate increased amounts of lipids, which include triglycerides and/or free fatty acids.
The claims cover modified Cyanobacteria with engineered acyl-ACP and lipid biosynthesis pathways, optionally combined with altered glycogen metabolism, as well as methods for making these organisms and using them for increased lipid and triglyceride production.
Stated Advantages
Photosynthetic microorganisms engineered as described produce increased amounts of lipids, such as fatty acids and triglycerides, compared to unmodified strains.
Genetically modified Cyanobacteria can be used as efficient sources of feedstock for biofuel and specialty chemical production, utilizing minimal inputs like sunlight, water, and basic nutrients.
Engineered strains with overexpressed ACP and lipid biosynthesis proteins show no significant growth defects and continual fatty acid production, making them attractive for continuous lipid production.
Documented Applications
Production of biofuels using lipids (such as fatty acids and triglycerides) produced by genetically modified photosynthetic microorganisms.
Use of the modified microorganisms as feedstocks for the production of specialty chemicals, including production of glycerin as a byproduct.
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