Production of beta-phellandrene using genetically engineered photosynthetic microorganisms

Abstract

The present invention provides methods and compositions for producing β-phellandrene hydrocarbons from a photosynthetic microorganism such as cyanobacteria.

What is claimed is: 
     
         1 . A method of obtaining β-phellandrene from photosynthetic microorganisms, the method comprising:
 culturing a strain of a photosynthetic microorganism that has been genetically modified to express a heterologous β-phellandrene synthase under conditions in which the β-phellandrene synthase is expressed; and 
 collecting from the surface of the culture medium β-phellandrene hydrocarbons that have spontaneously diffused into the medium from the photosynthetic microorganisms across the cell wall, wherein the culture is in a continuous growth phase and the β-phellandrene hydrocarbons are continuously generated. 
 
     
     
         2 . The method of  claim 1 , wherein the strain of the photosynthetic microorganism is a cyanobacteria strain or a microalgae strain. 
     
     
         3 . The method of  claim 1 , wherein the strain of the photosynthetic microorganism is a cyanobacteria strain. 
     
     
         4 . The method of  claim 3 , wherein the cyanobacteria strain is from a genus selected from the group consisting of  Synechocystis, Synechococcus, Arthrospira, Nostoc,  and  Anabaena.    
     
     
         5 . The method of  claim 3 , where the β-phellandrene synthase is encoded by a nucleic acid having at least 80% nucleic acid sequence identity to SEQ ID NO:3. 
     
     
         6 . The method of  claim 1 , wherein the strain of the photosynthetic microorganism is a microalgae strain. 
     
     
         7 . The method of  claim 1 , wherein the β-phellandrene synthase has at least 70% identity to SEQ ID NO:1. 
     
     
         8 . The method of  claim 7 , wherein the β-phellandrene synthase has at least 90% identity to SEQ ID NO:1. 
     
     
         9 . The method of  claim 1 , wherein collecting β-phellandrene hydrocarbons comprises siphoning or skimming the β-phellandrene hydrocarbons from the surface of the culture medium. 
     
     
         10 . The method of  claim 1 , wherein collecting β-phellandrene hydrocarbons comprises overlaying a solvent onto the surface of the culture medium. 
     
     
         11 . The method of  claim 10 , wherein the solvent is selected from the group consisting of heptane, decane, and dodecane. 
     
     
         12 . A cell culture comprising a photosynthetic microorganism, wherein the photosynthetic microorganism is genetically modified to express a heterologous β-phellandrene synthase; and cell culture media comprises β-phellandrene produced by the photosynthetic microorganism that has diffused into the cell culture media from the photosynthetic microorganism and floats on the surface of the culture medium, wherein the cell culture is in a continuous growth phase and the β-phellandrene hydrocarbons are continuously generated. 
     
     
         13 . The cell culture of  claim 12 , wherein the photosynthetic microorganism strain is a cyanobacteria strain or a microalgae strain. 
     
     
         14 . The cell culture of  claim 12 , wherein the photosynthetic microorganism strain is a cyanobacteria strain. 
     
     
         15 . The cell culture of  claim 14 , wherein the cyanobacteria strain is of a genus selected from the group consisting of the genera  Synechocystis, Synechococcus, Arthrospira, Nostoc,  and  Anabaena.