Recombinant DNA techniques are used to produce oleaginous recombinant cells that produce triglyceride oils having desired fatty acid profiles and regiospecific or stereospecific profiles. Genes manipulated include those encoding stearoyl-ACP desturase, delta 12 fatty acid desaturase, acyl-ACP thioesterase, ketoacyl-ACP synthase, and lysophosphatidic acid acyltransferase. The oil produced can have enhanced oxidative or thermal stability, can be useful as a frying oil, shortening, roll-in shortening, tempering fat, cocoa butter replacement, as a lubricant, or as a feedstock for various chemical processes. The fatty acid profile can be enriched in midchain profiles or the oil can be enriched in triglycerides of the saturated-unsaturated-saturated type.
1. A method for producing an oil comprising:
providing a recombinant oleaginous microalga of the genus Prototheca comprising an exogenous polynucleotide encoding a palmitoyl-ACP desaturase, wherein said exogenous polynucleotide is targeted by homologous recombination to a locus on the genome of said recombinant oleaginous microalga; and an endogenous FATA gene encoding an acyl-ACP thioesterase that has been disrupted by insertion of an exogenous polynucleotide into said endogenous FATA gene via homologous recombination and/or an exogenous FATA polynucleotide encoding an acyl-ACP thioesterase;
cultivating said recombinant oleaginous microalga, thereby producing said oil.
2. The method of claim 1 , wherein said recombinant oleaginous microalga comprises an endogenous FATA gene encoding an acyl-ACP thioesterase that has been disrupted by insertion of an exogenous polynucleotide into said endogenous FATA gene via homologous recombination.
3. The method of claim 1 , wherein said recombinant oleaginous microalga further comprises an exogenous polynucleotide encoding a KASII enzyme.
4. The method of claim 1 , wherein said recombinant oleaginous microalga further comprises an endogenous Δ12 fatty acid desaturase gene that has been disrupted by insertion of an exogenous polynucleotide into said endogenous Δ12 desaturase gene via homologous recombination.
5. The method of claim 1 , wherein said recombinant oleaginous microalga further comprises an exogenous polynucleotide encoding an inhibitory RNA targeted against FATA transcripts to reduce production of an acyl-ACP thioesterase, relative to an oleaginous microalga not comprising said exogenous polynucleotide.
6. The method of claim 1 , wherein said recombinant oleaginous microalga further comprises an exogenous nucleic acid encoding a Δ12 fatty acid desaturase operably linked to a pH-regulatable promoter.
7. The method of claim 1 , wherein said recombinant oleaginous microalga is Prototheca moriformis, Prototheca krugani, Prototheca stagnora , or Prototheca zopfii.
8. The method of claim 7 , wherein said recombinant oleaginous microalga is Prototheca moriformis.
9. The method of claim 5 , wherein said inhibitory RNA is a hairpin RNA.
10. The method of claim 5 , wherein said inhibitory RNA is a micro RNA, short interfering RNA, double stranded RNA or antisense RNA.
11. The method of claim 1 , wherein said recombinant oleaginous microalga further comprises an exogenous polynucleotide encoding an inhibitory RNA targeted against desaturase transcripts to reduce production of a desaturase, relative to an oleaginous microalga not comprising said exogenous polynucleotide.
12. The method of claim 11 , wherein said desaturase is a Δ12 fatty acid desaturase.
13. The method of claim 11 , wherein said desaturase is a stearoyl-ACP desaturase.
14. The method of claim 11 , wherein said inhibitory RNA is a hairpin RNA.
15. The method of claim 11 , wherein said inhibitory RNA is a microRNA, short interfering RNA, double stranded RNA or antisense RNA.
16. The method of claim 1 , wherein said oleaginous microalga comprises an exogenous FATA polynucleotide encoding an acyl-ACP thioesterase.