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. An oleaginous microalgal cell expressing a non-natural FATA enzyme, wherein the non-natural FATA enzyme has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and compared to a parent Garcinia mangostana FATA enzyme comprises the amino acid substitutions A at the position corresponding to position 171 of SEQ ID NO: 45 (corresponding to V193 of wild-type Garcinia mangostana FATA enzyme), and A or V at the position corresponding to position 86 of SEQ ID NO: 45 (corresponding to G108 of wild-type Garcinia mangostana FATA enzyme), and wherein expression of the non-natural FATA enzyme increases C18:0 fatty acids in the fatty acid profile of the oil produced by the cell relative to a corresponding cell expressing the parent FATA enzyme.
2. The oleaginous microalgal cell expressing a non-natural enzyme according to claim 1 , further wherein the non-natural enzyme comprises A or V at the position corresponding to position 89 of SEQ ID NO: 45 (corresponding to S111 of wild-type Garcinia mangostana FATA enzyme).
3. The oleaginous microalgal cell expressing a non-natural enzyme according to claim 1 , further wherein the non-natural enzyme comprises A at the position corresponding to position 89 of SEQ ID NO: 45 (corresponding to S111 of wild-type Garcinia mangostana FATA enzyme).
4. An oleaginous microalgal cell expressing a non-natural FATA enzyme, wherein the non-natural FATA enzyme has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 45 and compared to a parent Garcinia mangostana FATA enzyme comprises the amino acid substitutions A at the position corresponding to position 171 of SEQ ID NO: 45 (corresponding to V193 of wild-type Garcinia mangostana FATA enzyme), and A at the position corresponding to position 86 of SEQ ID NO: 45 (corresponding to G108 of wild-type Garcinia mangostana FATA enzyme), and wherein expression of the non-natural FATA enzyme increases C18:0 fatty acids in the fatty acid profile of the oil produced by the cell relative to a corresponding cell expressing the parent FATA enzyme.
5. The oleaginous microalgal cell expressing a non-natural enzyme according to claim 4 , further wherein the non-natural enzyme comprises A or V at the position corresponding to position 89 of SEQ ID NO: 45 (corresponding to S111 of wild-type Garcinia mangostana FATA enzyme).
6. The oleaginous microalgal cell expressing a non-natural enzyme according to claim 5 , wherein the non-natural enzyme comprises A at the position corresponding to position 89 of SEQ ID NO: 45 (corresponding to S111 of wild-type Garcinia mangostana FATA enzyme).
7. The oleaginous microalgal cell according to claim 1 , wherein the oleaginous microalgal cell is a Prototheca cell.
8. The oleaginous microalgal cell according to claim 7 , wherein the cell is a Prototheca moriformis cell.
9. A method for producing a triglyceride oil, the method comprising cultivating an oleaginous microalgal cell according to claim 1 ; and isolating the oil.
10. A method for producing a triglyceride oil, the method comprising cultivating an oleaginous microalgal cell according to claim 2 ; and isolating the oil.
11. A method for producing a triglyceride oil, the method comprising cultivating an oleaginous microalgal cell according to claim 3 ; and isolating the oil.
12. A method for producing a triglyceride oil, the method comprising cultivating an oleaginous microalgal cell according to claim 4 ; and isolating the oil.
13. A method for producing a triglyceride oil, the method comprising cultivating an oleaginous microalgal cell according to claim 5 ; and isolating the oil.
14. A method for producing a triglyceride oil, the method comprising cultivating an oleaginous microalgal cell according to claim 6 ; and isolating the oil.