Novel ketoacyl acp synthase genes and uses thereof

Abstract

The present invention relates to beta-ketoacyl ACP synthase genes of the KASI/KASIV type and proteins encoded by these genes. The genes can be included in nucleic acid constructs, vectors or host cells. Expression of the gene products can alter the fatty acid profile of host cells. The KAS genes can be combined with a FATA or FATB thioesterase gene to create a cell that produces an increased amount of C8-C16 fatty acids. Suitable host cells include plastidic cells of plants or microalgae. Oleaginous microalga host cells with the new genes are disclosed.

1 . A non-natural, isolated polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to any one of SEQ ID NOs: 21-37, or 39-55, or encoding a KASI-like protein having at least 80, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% amino acid sequence identity to any one of SEQ ID NOs: 2-18, 62-72, or a mature protein produced therefrom, or the complement of the polynucleotide. 
     
     
         2 . A transformation vector comprising the cDNA of  claim 1 . 
     
     
         3 . The vector of  claim 2 , comprising promoter and 3′UTR sequences in operable linkage to the cDNA, and optionally a flanking sequence for homologous recombination. 
     
     
         4 . A host cell comprising the polynucleotide of  claim 1  and/or the vector of  claim 2  or  3 . 
     
     
         5 . The host cell of  claim 4 , wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway. 
     
     
         6 . The host cell of  claim 5 , wherein the host cell is a microalga. 
     
     
         7 . The host cell of  claim 6 , wherein the host cell is of Trebouxiophyceae, and optionally of the genus  Chlorella  or  Prototheca.    
     
     
         8 . The host cell of  claim 7 , wherein the microalga is of the species  Prototheca moriformis.    
     
     
         9 . A method for making a cell-oil, the method comprising cultivating a host cell of  claim 1 , so as produce the cell-oil, wherein the oil comprises triglycerides and microalgal sterols. 
     
     
         10 . The method of  claim 9 , wherein the cell oil comprises sterols characterized by a sterol profile and the sterol profile has an excess of ergosterol over β-sitosterol and/or the presence of 22, 23-dihydrobrassicasterol, poriferasterol or clionasterol. 
     
     
         11 . A host cell comprising
 a) a non-natural, isolated polynucleotide having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity or equivalent sequence by virtue of the degeneracy of the genetic code to any one of SEQ ID NOs: 21-37, or 39-55, or encoding a KASI-like protein having at least 80, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% amino acid sequence identity to any one of SEQ ID NOs: 2-18, 62-72, or a mature protein produced therefrom, or the complement of the polynucleotide; and   b) a non-natural, isolated polynucleotide encoding a FATA acyl-ACP thioesterase or FATB acyl-ACP thioesterase.   
     
     
         12 . The host cell of  claim 11 , wherein the FATB acyl-ACP thioesterase has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 57. 
     
     
         13 . The host cell of  claim 12 , wherein the host cell produces a cell oil characterized by a fatty acid profile with (i) at least 30, 40, 50, or 55% C14:0, (ii) at least 7, 8, 9, 10, 11, 12, 13, or 14% C8:0, (iii) at least 10, 15, 20, 25, 30, or 35 area % for the sum of C8:0 and C10:0, or (iv) a C8/C10 ratio in the range of 2.2-2.5, 2.5-3.0, or 3.0-3.4. 
     
     
         14 . The host cell of  claim 11 , wherein the host cell is a plastidic oleaginous cell having a type II fatty acid biosynthesis pathway. 
     
     
         15 . The host cell of  claim 14 , wherein the host cell is a microalga. 
     
     
         16 . The host cell of  claim 15 , wherein the host cell is of Trebouxiophyceae, and optionally of the genus  Chlorella  or  Prototheca.    
     
     
         17 . The host cell of  claim 16 , wherein the microalga is of the species  Prototheca moriformis.    
     
     
         18 . The host cell of  claim 11 , wherein one or more of the polynucleotides is codon-optimized for expression in the host cell such that the polynucleotide's coding sequence contains the most or second most preferred codon for at least 60% of the codons of the coding sequence such that the codon-optimized sequence is more efficiently translated in the host cell relative to a non-optimized sequence. 
     
     
         19 . The host cell of  claim 18 , wherein the coding sequence contains the most preferred codon for at least 80% of the codons of the coding sequence. 
     
     
         20 . A method for making a cell-oil, the method comprising cultivating a host cell of  claim 11 , so as produce the cell-oil, wherein the oil comprises triglycerides and microalgal sterols.