Acyl-acp thioesterases and mutants thereof

Abstract

Novel plant acyl-ACP thioesterase genes of the FatB and FatA classes and proteins encoded by these genes are disclosed. The genes are useful for constructing recombinant host cells having altered fatty acid profiles. Expression of the novel and/or mutated FATB and FATA genes is demonstrated in oleaginous microalga host cells. Furthermore, a method for producing an oil elevated in one or more of C12:0, C14:0, C16:0, C18:0 and/or C18:1 fatty acids includes transforming a cell with novel and/or mutated FATB and/or FATA genes, e.g., having an N-terminal deletion. The cells produce triglycerides with altered and useful fatty acid profiles.

1 . A cDNA or nucleic acid construct comprising a polynucleotide sequence encoding a heterologous regulatory element and a FatB acyl-ACP thioesterase gene operable to produce an altered fatty acid profile in an oil produced by a cell expressing the nucleic acid construct, wherein the FatB gene expresses a protein having an amino acid sequence having at least 65% identity to SEQ ID NO: 40 or SEQ ID NO: 42. 
     
     
         2 . The cDNA or nucleic acid construct of  claim 1 , wherein the acyl-ACP thioesterase coding sequence comprises at least 65% identity to SEQ ID NO: 44 or any equivalent sequences by virtue of the degeneracy of the genetic code. 
     
     
         3 . (canceled) 
     
     
         4 . The nucleic acid construct of  claim 1 , further comprising a polynucleotide encoding a plastid targeting peptide with at least 65% identity to SEQ ID NO: 37. 
     
     
         5 . (canceled) 
     
     
         6 . A host cell capable of expressing the cDNA or nucleic acid construct of  claim 1  so as to produce a triglyceride oil having an altered composition relative to a control cell without the construct, the oil optionally having an increase in C8-C12 fatty acids. 
     
     
         7 . A host cell capable of expressing the cDNA or nucleic acid construct of  claim 1  so as to produce a triglyceride oil having an altered composition relative to a control cell without the construct, the oil optionally having an increase in C10 and C12 fatty acids. 
     
     
         8 . The host cell of  claim 6 , wherein the host cell is selected from the group consisting of a plant cell, a microbial cell, and a microalgal cell. 
     
     
         9 . A method of producing a recombinant cell that produces an altered fatty acid profile, the method comprising transforming a cell with a cDNA or nucleic acid construct of  claim 1 . 
     
     
         10 . A host cell produced by the method of  claim 9 . 
     
     
         11 . The host cell of  claim 10 , wherein the host cell is selected from the group consisting of a plant cell, a microbial cell, and a microalgal cell. 
     
     
         12 . A method for producing an oil or oil-derived product, the method comprising cultivating a host cell of  claim 6  and extracting oil produced thereby, optionally wherein the cultivation is by heterotrophic growth on sugar. 
     
     
         13 . The method of  claim 12 , further comprising producing a fatty acid, fuel, chemical, food, or other oil-derived product from the oil. 
     
     
         14 . (canceled) 
     
     
         15 . An oil produced by the method of  claim 12 , optionally having a fatty acid profile comprising at least 20% C8, C10, C12, C14, C16 or C18 fatty acids. 
     
     
         16 . The oil of  claim 15 , wherein the oil is produced by a microalgae, has a microalgal sterol profile, and optionally, lacks C24-alpha sterols. 
     
     
         17 . (canceled) 
     
     
         18 . A method for producing an oil, the method comprising:
 (a) providing a plastidic, oleaginous cell, optionally a microbial cell, the cell expressing a functional, acyl-ACP thioesterase gene encoded by a FATB gene having a deletion mutation in a region corresponding to the region coding for amino acids 66-98 of SEQ ID NO: 8;   (b) cultivating the cell to produce a cell-oil; and   (c) isolating the cell-oil from the cell.   
     
     
         19 . The method of  claim 17 , wherein the cell-oil is enriched in C12 due to the deletion. 
     
     
         20 . The method of  claim 18  wherein the FATB gene encodes a protein with at least 65% sequence identity to SEQ ID NO: 42. 
     
     
         21 . (canceled) 
     
     
         22 .- 38 . (canceled) 
     
     
         39 . The oil produced by the method of  claim 19 , wherein the fatty acid profile of the cell-oil comprises at least 5% C12 fatty acids. 
     
     
         40 . The oil produced by the method of  claim 39 , wherein the fatty acid profile of the cell-oil comprises at least 10% C12 fatty acids. 
     
     
         41 . The oil produced by the method of  claim 40 , wherein the fatty acid profile of the cell-oil comprises at least 15% C12 fatty acids.