Recombinant microalgae including keto-acyl acp synthase

Abstract

Methods and compositions for the production of food compositions, oils, fuels, oleochemicals, and other compounds in recombinant microorganisms are provided, including oil-bearing microorganisms and methods of low cost cultivation of such microorganisms. Microalgal cells containing exogenous genes encoding, for example, a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, a polysaccharide-degrading enzyme, a keto acyl-ACP synthase enzyme, a fatty acyl-ACP thioesterase, a fatty acyl-CoA/aldehyde reductase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty aldehyde decarbonylase, and/or an acyl carrier protein are useful in manufacturing food compositions, and transportation fuels such as renewable diesel, biodiesel, and renewable jet fuel, as well as oleochemicals such as functional fluids, surfactants, soaps and lubricants.

1 .- 14 . (canceled) 
     
     
         15 . A recombinant microalgal host cell comprising an exogenous nucleic acid sequence encoding a keto-acyl ACP synthase (KAS), wherein said host cell expresses the nucleic acids and the fatty acid profile of the host cell is altered. 
     
     
         16 . The recombinant microalgal host cell of  claim 15 , wherein said exogenous nucleic acid sequence encodes a KAS selected from the group consisting of KASI, KASII, KASIII or KASIV. 
     
     
         17 . The recombinant microalgal host cell of  claim 16 , wherein said KAS is KASI or KASIV. 
     
     
         18 . The recombinant microalgal host cell of  claim 16 , wherein said KAS is KASII. 
     
     
         19 . The recombinant microalgal host cell of  claim 15 , wherein said host cell is a cell of the genus  Prototheca  or  Chlorella.    
     
     
         20 . The recombinant microalgal host cell of  claim 19 , wherein said host cell is  Prototheca moriformis.    
     
     
         21 . The recombinant microalgal host cell of  claim 15  further comprising a second exogenous nucleic acid sequence encoding a polypeptide selected from the group consisting of sucrose invertase, fatty acyl-ACP thioesterase, desaturase, or a selectable marker. 
     
     
         22 . The recombinant microalgal host cell of  claim 21 , wherein the second exogenous nucleic acid sequence encodes a fatty acyl-ACP thioesterase selected from the group consisting of  Umbellularia californica  fatty acyl-ACP thioesterase,  Cinnamomum camphora  fatty acyl-ACP thioesterase,  Umbellularia californica  fatty acyl-ACP thioesterase,  Myristica fragrans  fatty acyl-ACP thioesterase,  Elaeis guineensis  fatty acyl-ACP thioesterase,  Populus tomentosa  fatty acyl-ACP thioesterase,  Arabidopsis thaliana  fatty acyl-ACP thioesterase,  Gossypium hirsutum  fatty acyl-ACP thioesterase,  Cuphea lanceolata  fatty acyl-ACP thioesterase,  Cuphea hookeriana  fatty acyl-ACP thioesterase,  Cuphea calophylla  subsp.  mesostemon  fatty acyl-ACP thioesterase,  Cuphea lanceolata  fatty acyl-ACP thioesterase,  Elaeis guineensis  fatty acyl-ACP thioesterase,  Gossypium hirsutum  fatty acyl-ACP thioesterase,  Vitis vinifera  fatty acyl-ACP thioesterase,  Garcinia mangostana  fatty acyl-ACP thioesterase,  Brassica juncea  fatty acyl-ACP thioesterase,  Madhuca longifolia  fatty acyl-ACP thioesterase,  Brassica napus  fatty acyl-ACP thioesterase,  Oryza sativa  fatty acyl-ACP thioesterase,  Cuphea hookeriana  fatty acyl-ACP thioesterase,  Ulmus Americana  fatty acyl-ACP thioesterase,  Cuphea lanceolata  fatty acyl-ACP thioesterase,  Cuphea palustris  fatty acyl-ACP thioesterase,  Iris germanica  fatty acyl-ACP thioesterase,  Cuphea palustris  fatty acyl-ACP thioesterase,  Myristica fragrans  fatty acyl-ACP thioesterase,  Cuphea wrightii  fatty acyl-ACOP thioesterase,  Ricinus communis  fatty acyl-ACP thioesterase,  Carthamus tinctorus  fatty acyl-ACP thioesterase gene,  Elaeis guiniensis  fatty acyl-ACP thioesterase gene,  Cuphea hookeriana  fatty acyl-ACP thioesterase gene,  Brassica napus  fatty acyl-ACP thioesterase, and  Prototheca moriformis  fatty acyl-ACP thioesterase. 
     
     
         23 . A recombinant microalgal host cell comprising an exogenous nucleic acid sequence, wherein said exogenous nucleic acid disrupts an endogenous nucleic acid sequence that encodes a keto-acyl ACP synthase and the fatty acid profile of the host cell is altered. 
     
     
         24 . The recombinant microalgal host cell of  claim 23 , wherein said endogenous nucleic acid sequence encodes a KAS selected from the group consisting of KASI, KASII, KASIII or KASIV. 
     
     
         25 . The recombinant microalgal host cell of  claim 23 , wherein said host cell is a cell of the genus  Prototheca  or  Chlorella.    
     
     
         26 . The recombinant microalgal host cell of  claim 25 , wherein said host cell is  Prototheca moriformis.    
     
     
         27 . The recombinant microalgal host cell of  claim 23 , wherein said recombinant host cell further comprises one or more exogenous nucleic acid sequences encoding a polypeptide selected from the group consisting of sucrose invertase, fatty acyl-ACP thioesterase, desaturase, or a selectable marker. 
     
     
         28 . The recombinant microalgal host cell of  claim 27 , wherein the second exogenous nucleic acid sequence encodes a fatty acyl-ACP thioesterase selected from the group consisting of  Umbellularia californica  fatty acyl-ACP thioesterase,  Cinnamomum camphora  fatty acyl-ACP thioesterase,  Umbellularia californica  fatty acyl-ACP thioesterase,  Myristica fragrans  fatty acyl-ACP thioesterase,  Elaeis guineensis  fatty acyl-ACP thioesterase,  Populus tomentosa  fatty acyl-ACP thioesterase,  Arabidopsis thaliana  fatty acyl-ACP thioesterase,  Gossypium hirsutum  fatty acyl-ACP thioesterase,  Cuphea lanceolata  fatty acyl-ACP thioesterase,  Cuphea hookeriana  fatty acyl-ACP thioesterase,  Cuphea calophylla  subsp. mesostemon fatty acyl-ACP thioesterase,  Cuphea lanceolata  fatty acyl-ACP thioesterase,  Elaeis guineensis  fatty acyl-ACP thioesterase,  Gossypium hirsutum  fatty acyl-ACP thioesterase,  Vitis vinifera  fatty acyl-ACP thioesterase,  Garcinia mangostana  fatty acyl-ACP thioesterase,  Brassica juncea  fatty acyl-ACP thioesterase,  Madhuca longifolia  fatty acyl-ACP thioesterase,  Brassica napus  fatty acyl-ACP thioesterase,  Oryza sativa  fatty acyl-ACP thioesterase,  Cuphea hookeriana  fatty acyl-ACP thioesterase,  Ulmus Americana  fatty acyl-ACP thioesterase,  Cuphea lanceolata  fatty acyl-ACP thioesterase,  Cuphea palustris  fatty acyl-ACP thioesterase,  Iris germanica  fatty acyl-ACP thioesterase,  Cuphea palustris  fatty acyl-ACP thioesterase,  Myristica fragrans  fatty acyl-ACP thioesterase,  Cuphea wrightii  fatty acyl-ACOP thioesterase,  Ricinus communis  fatty acyl-ACP thioesterase,  Carthamus tinctorus  fatty acyl-ACP thioesterase gene,  Elaeis guiniensis  fatty acyl-ACP thioesterase gene,  Cuphea hookeriana  fatty acyl-ACP thioesterase gene,  Brassica napus  fatty acyl-ACP thioesterase, and  Prototheca moriformis  fatty acyl-ACP thioesterase. 
     
     
         29 . A recombinant polynucleotide encoding a keto-acyl ACP synthase optimized for expression in a host cell selected from the group consisting of  Prototheca, Chlorella , or  Dunaliella , wherein said recombinant polynucleotide consists of codons comprising the most preferred codons of Table 2, 28, 29 or 30 for at least 80% of the codons. 
     
     
         30 . The recombinant polynucleotide encoding the keto-acyl ACP synthase of  claim 29 , wherein said host cell is  Prototheca  and said codons are disclosed in Table 2. 
     
     
         31 . The recombinant polynucleotide encoding the keto-acyl ACP synthase of  claim 30 , wherein said recombinant polynucleotide consist of codons comprising the most preferred codon for at least 85% of the codons. 
     
     
         32 . The recombinant polynucleotide encoding the keto-acyl ACP synthase of  claim 30 , wherein said recombinant polynucleotide consist of codons comprising the most preferred codon for at least 90% of the codons. 
     
     
         33 . The recombinant polynucleotide encoding the keto-acyl ACP synthase of  claim 30 , wherein said recombinant polynucleotide consist of codons comprising the most preferred codon for at least 95% of the codons. 
     
     
         34 . A method of producing a triglyceride oil, the method comprising the steps of:
 heterotrophically cultivating the recombinant microalgal host cell of  claim 15 ; and   isolating triglyceride oil from said recombinant microalgal host cell.   
     
     
         35 . A method of producing a triglyceride oil, the method comprising the steps of:
 heterotrophically cultivating the recombinant microalgal host cell of  claim 23 ; and   isolating triglyceride oil from said recombinant microalgal host cell.