Trophic Conversion of Obligate Phototrophic Algae Through Metabolic Engineering

Abstract

Most microalgae are obligate photoautotrophs and their growth is strictly dependent on the generation of photosynthetically-derived energy. In this study it is shown that the microalga Phaeodaclylurn tricornutum can be engineered to import glucose and grow in the dark through the introduction of genes encoding glucose transporters. Both the human and Chlorella kessleri glucose transporters facilitated the uptake of glucose by P. tricornutum , allowing the cells to metabolize exogenous organic carbon and thrive, independent of light. This is the first successful trophic conversion of an obligate photoautotroph through metabolic engineering, and it demonstrates that methods of cell nourishment can be fundamentally altered with the introduction of a single gene. Since strains transformed with the glucose transport genes are able to grow non-photosynthetically, they can be exploited for the analysis of photosynthetic processes through mutant generation and characterization. Finally, this work also represents critical progress toward large-scale commercial exploitation of obligate phototrophic algae through the use of microbial fermentation technology, eliminating significant limitations resulting from light-dependent growth.

1 - 22 . (canceled) 
     
     
         23 . A method of producing an algal biomass comprising culturing Bacillariophyta alga cells capable of growing in the absence of light to produce a biomass, wherein Bacillariophyta alga cells in the culture comprise an exogenous transgene, wherein the transgene comprises a nucleic acid construct encoding a glucose transporter selected from the group consisting of Glut1 (human erythrocyte glucose transporter 1) and Hup1 ( Chlorella  HUP1 Monosaccharide-H+ Symporter) under the control of a functionally linked promoter, wherein upon expression of the glucose transporter in an amount sufficient to transport glucose into the Bacillariophyta alga cells, the Bacillariophyta alga cells grow on glucose in the absence of light as compared to untransformed wild-type Bacillariophyta alga cells, and wherein the untransformed wild-type Bacillariophyta alga cells are obligate photoautotrophs. 
     
     
         24 . The method of  claim 23 , wherein the Bacillariophyta alga cells are selected from the group consisting of  Nitzschia, Navicula, Thalassiosira , and  Phaeodactylum  cells. 
     
     
         25 . The method of  claim 24 , wherein the  Phaeodactylum  cells are  Phaeodactylum tricornutum  cells. 
     
     
         26 . The method of  claim 23 , wherein the promoter is a light harvesting promoter. 
     
     
         27 . The method of  claim 26 , wherein the light harvesting promoter is a fucoxanthin chlorophyll binding protein (fcp) promoter. 
     
     
         28 . The method of  claim 27 , wherein the fop promoter is fcpA, fcpB fcpC, or fcpE. 
     
     
         29 . The method of  claim 23 , wherein the method further comprises harvesting the biomass. 
     
     
         30 . A biomass produced by the method of  claim 23 . 
     
     
         31 . An animal feed comprising the biomass of  claim 30 . 
     
     
         32 . A method of producing an oil, comprising extracting an oil from the biomass of  claim 30 . 
     
     
         33 . A method of producing a pigment, comprising extracting a pigment from the biomass of  claim 30 . 
     
     
         34 . The method of  claim 33 , wherein the pigment is selected from the group consisting of β-carotene, a phycobiliprotein, a carotenoid, and a xanthophyll. 
     
     
         35 . A method for the heterotrophic conversion of Bacillariophyta alga cells, comprising:
 (a) transforming Bacillariophyta alga cells with an exogenous transgene, wherein the transgene comprises a nucleic acid construct encoding a glucose transporter selected from the group consisting of Glut1 (human erythrocyte glucose transporter 1) and Hup1 ( Chlorella  HUP1 Monosaccharide-H+ Symporter) tinder the control of a functionally linked promoter, wherein upon expression of the glucose transporter in an amount sufficient to transport glucose into the Bacillariophyta alga cells, the Bacillariophyta alga cells grow on glucose in the absence of light as compared to untransformed wild-type Bacillariophyta alga cells, and wherein the untransformed wild-type Bacillariophyta alga cells are obligate photoautotrophs, and   (b) selecting transformed alga cells by growing the cells on glucose in the absence of light.   
     
     
         36 . The method of  claim 35 , wherein the Bacillariophyta alga cells are selected from the group consisting of  Nitzschia, Navicula, Thalassiosira , and  Phaeodactylum  cells. 
     
     
         37 . The method of  claim 36 , wherein the  Phaeodactylum  cells are  Phaeodactylum tricornutum  cells. 
     
     
         38 . The method of  claim 35 , wherein the promoter is a light harvesting promoter. 
     
     
         39 . The method of  claim 38 , wherein the light harvesting promoter is a fucoxanthin chlorophyll binding protein (fcp) promoter. 
     
     
         40 . The method of  claim 39 , wherein the fcp promoter is fcpA, fcpC, or fcpE. 
     
     
         41 . The method of  claim 35 , wherein the method further comprises transforming the Bacillariophyta alga cells with a gene of interest. 
     
     
         42 . The method of  claim 41 , wherein the gene of interest encodes a recombinant protein. 
     
     
         43 . A transformed alga cell produced by the method of  claim 41 . 
     
     
         44 . A method of producing a recombinant protein comprising culturing the transformed alga cell of  claim 43  under conditions allowing the expression of the gene of interest, wherein the gene of interest encodes a recombinant protein, to produce the recombinant protein.