VARIANT THIOESTERASES AND METHODS OF USE
The present invention relates to variant thioesterases and their use in plants, e.g., to increase enzymatic activity and to promote increased production of mid-chain length fatty acids (e.g., 8 to 14 carbons) and at desired ratios. Further disclosed herein are methods of manufacturing renewable chemicals through the manufacture of novel triglyceride oils followed by chemical modification of the oils. Oils containing fatty acid chain lengths of C8, C10, C12 or C14 are also disclosed and are useful as feedstocks in the methods described herein.
1 . A nucleic acid molecule encoding a variant acyl-ACP thioesterase comprising a C-terminal catalytic domain, and an N-terminal hydrophobic domain and specificity domain, wherein one or more of the hydrophobic domain and/or the specificity domain are heterologous to the catalytic domain, wherein the hydrophobic domain comprises:
a) amino acid residues of an acyl-ACP-TE corresponding to an amino acid sequence selected from the group consisting of amino acid residues 61-77 of SEQ ID NO:43; amino acid residues 61-77 of SEQ ID NO:44; amino acid residues 85-101 of SEQ ID NO:45; amino acid residues 78-95 of SEQ ID NO:46; amino acid residues 50-66 of SEQ ID NO:47; amino acid residues 91-107 of SEQ ID NO:60; and amino acid residues 90-106 of SEQ ID NO:61;
b) a motif comprising the amino acid sequence (P/H)(G/D/V)(W/L)(S/N)(M/R/V)(P/L/S)(L/F)(E/A/T/S)(L/A/K)(I/V)TT(I/V)F(S/L/V/G)(A/K/V)(A/P);
c) at least 60% sequence identity to an amino acid sequence selected from the group consisting of amino acid residues 61-77 of SEQ ID NO:43; amino acid residues 61-77 of SEQ ID NO:44; amino acid residues 85-101 of SEQ ID NO:45; amino acid residues 78-95 of SEQ ID NO:46; amino acid residues 50-66 of SEQ ID NO:47; amino acid residues 91-107 of SEQ ID NO:60; and amino acid residues 90-106 of SEQ ID NO:61; and/or
d) amino acid residues 61-77 of SEQ ID NO:43; amino acid residues 61-77 of SEQ ID NO:44; amino acid residues 85-101 of SEQ ID NO:45; amino acid residues 78-95 of SEQ ID NO:46; amino acid residues 50-66 of SEQ ID NO:47; amino acid residues 91-107 of SEQ ID NO:60; or amino acid residues 90-106 of SEQ ID NO:61.
2 . The nucleic acid of claim 1 , wherein the nucleic acid encodes a variant acyl-acyl carrier protein (ACP) thioesterase (TE) comprising:
i) the specificity domain from a C10:0 acyl-ACP preferring TE and a catalytic domain from a C12:0 acyl-ACP preferring TE;
ii) the specificity domain from a C12:0 acyl-ACP preferring TE and a catalytic domain from a C14:0 acyl-ACP preferring TE;
iii) the specificity domain from a C14:0 acyl-ACP preferring TE and a catalytic domain from a C12:0 acyl-ACP preferring TE;
iv) the specificity domain from a C12:0 acyl-ACP preferring TE and a catalytic domain from a C10:0 acyl-ACP preferring TE; or
v) the specificity domain from a C10:0 acyl-ACP preferring TE and a catalytic domain from a C8:0 acyl-ACP preferring TE.
3 - 11 . (canceled)
12 . The nucleic acid of claim 1 , wherein the nucleic acid encodes a hydrophobic domain that comprises an N-terminal Leucine residue.
13 . A nucleic acid of claim 1 , wherein the nucleic acid further encodes an N-terminal sequence encoding a plastid transit peptide.
14 - 15 . (canceled)
16 . The nucleic acid of claim 1 , wherein the nucleic acid further encodes a linker domain positioned N-terminal to the hydrophobic domain.
17 - 18 . (canceled)
19 . A nucleic acid of claim 1 , comprising codon bias for improved expression in an algal host cell.
20 . An expression cassette comprising the nucleic acid of claim 1 .
21 . A vector comprising the nucleic acid of claim 1 .
22 . A variant acyl-acyl carrier protein (ACP) thioesterase (TE) encoded by the nucleic acid of claim 1 .
23 . A host cell comprising the nucleic acid of claim 1 .
24 - 32 . (canceled)
33 . A plant, algae or microalgae comprising the nucleic acid of claim 1 .
34 - 35 . (canceled)
36 . An oil product produced by the plant, algae or microalgae of claim 33 , or a chemical, material, or food product produced from that oil.
37 . A method of producing a plant, an algae or a microalgae that produces an oil having a desired fatty acid profile, comprising transforming the plant, algae or microalgae with a nucleic acid sequence of claim 1 , and cultivating the plant, algae or microalgae so as to produce the oil.
38 - 41 . (canceled)
42 . A method of producing an oil, comprising transforming the plant, algae or microalgae with a nucleic acid molecule of claim 1 , expressing the variant acyl-ACP TE to produce fatty acids, and recovering the oil produced by the plant, algae or microalgae comprising the fatty acids.
43 . A method of producing an oil, comprising culturing the plant, algae or microalgae of claim 1 , expressing the variant acyl-ACP TE to produce fatty acids, and recovering the oil produced by the plant, algae or microalgae comprising the fatty acids.