Modified photosynthetic microorganisms for producing triglycerides
Inventors
Roberts, James • Cross, Fred • Warrener, Paul • Munoz, Ernesto Javier • Lee, Martin Henry • Romari, Khadidja • Kotovic, Kimberly Marie • Hickman, Jason W.
Assignees
Publication Number
US-8394614-B2
Publication Date
2013-03-12
Expiration Date
2029-10-23
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Abstract
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.
Core Innovation
This invention demonstrates that photosynthetic microorganisms, such as Cyanobacteria which do not naturally produce triglycerides, can be genetically modified to synthesize triglycerides by introducing polynucleotide sequences encoding enzymes associated with triglyceride biosynthesis. Enzymes of particular focus include diacylglycerol acyltransferase (DGAT) and phosphatidate phosphatase. The expression of such enzymes enables Cyanobacteria to convert naturally occurring fatty acids into triglycerides, serving as energy storage molecules.
The invention also shows that genetically modifying Cyanobacteria to increase the biosynthesis of fatty acids, notably by introducing exogenous genes encoding acetyl-CoA carboxylase (ACCase), results in an increased fatty acid supply. Since fatty acids are required for triglyceride formation, this strategy enhances the overall production of triglycerides. Codon-optimization and expression regulation, such as inducible promoters, are among the methods used to improve the expression of exogenous genes in the host microorganism.
The problem addressed by this invention arises from the limited ability of traditional photosynthetic organisms, such as algae, to be genetically manipulated for increased triglyceride yield under culture conditions, combined with the lack of endogenous enzymatic pathways for triglyceride formation in Cyanobacteria. Through genetic engineering, the present invention overcomes these limitations, enabling the use of Cyanobacteria as a controllable and potentially high-yielding source for the biosynthesis of triglycerides and fatty acids.
Claims Coverage
The patent contains two independent claims, each covering a distinct inventive feature relating to the production of triglycerides and/or wax esters in photosynthetic microorganisms using specific genetic modifications.
Culturing a modified Cyanobacterium comprising an exogenous polynucleotide encoding a prokaryotic diacylglycerol acyltransferase (DGAT)
A method is provided in which a photosynthetic microorganism, specifically a modified Cyanobacterium, is cultured where it contains an exogenous polynucleotide that encodes a prokaryotic diacylglycerol acyltransferase (DGAT). This genetic modification enables the microorganism to produce a triglyceride, a wax ester, or both. Key aspects include: - The modified Cyanobacterium contains a DGAT from prokaryotic sources such as Acinetobacter, Streptomyces, or Alcanivorax. - The method may further include introducing exogenous polynucleotides encoding a phosphatidate phosphatase and/or acetyl-CoA carboxylase (ACCase). - Expression constructs, stable genome integration, inducible promoters, and codon-optimization for Cyanobacterium expression are additional features. - The modified Cyanobacterium may be S. elongatus PCC 7942, a salt-tolerant variant thereof, Synechococcus PCC 7002, or Synechocystis PCC 6803.
Culturing a modified Cyanobacterium comprising an exogenous polynucleotide encoding a prokaryotic diacylglycerol acyltransferase (DGAT) that uses acyl-ACP as a substrate
A method is provided in which a photosynthetic microorganism, specifically a genetically modified Cyanobacterium, is cultured wherein the microorganism contains an exogenous polynucleotide encoding a prokaryotic DGAT that uses acyl-ACP as a substrate. This enables the production of a triglyceride, a wax ester, or both.
In summary, the claims cover methods of producing triglycerides and wax esters using photosynthetic Cyanobacteria genetically engineered with exogenous prokaryotic diacylglycerol acyltransferase genes, with some embodiments emphasizing DGATs that utilize acyl-ACP as substrates and including additional features such as specific enzyme sources, supporting biosynthetic enzymes (phosphatidate phosphatase, ACCase), and optimized expression strategies.
Stated Advantages
Enables the production of large amounts of triglycerides from minimal energy and nutrient input using sunlight, water, air, and minimal nutrients.
Provides a manageable and efficient source of feedstock for the production of biofuels and specialty chemicals.
Allows genetic manipulation of Cyanobacteria, which are more tractable than algae, resulting in increased fatty acid and triglyceride yields.
Documented Applications
Production of triglycerides to be used as feedstock for biofuels, such as biodiesel.
Production of specialty chemicals, such as glycerin for the pharmaceutical and cosmetic industries.
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