Variant lipid acyltransferases and methods of making
View Patent ↗The present invention relates to a method for the production of a variant lipid acyltransferase comprising the steps of: (i) selecting a parent enzyme which is a lipid acyltranserase enzyme; (ii) modifying one or more amino acids to produce a variant lipid acyltransferase; (iii) testing the activity of the variant lipid acyltransferase on a galactolipid and/or phospholipid and/or triglyceride substrate; (iv) selecting a variant enzyme with enhanced activity towards galactolipids compared with the parent enzyme; (v) providing a Bacillus licheniformis cell; (vi) transforming the Bacillus licheniformis cell with a heterologous nucleotide sequence encoding said variant lipid acyltransferase; and (iii) expressing said variant lipid acyltransferase in the cell under the control of a promoter sequence. The variant lipid acyltransferase can undergo post-translations modification, truncation and/or clipping, i.e., to remove a signal peptide. In addition, the present invention further relates to the use of Bacillus licheniformis to express a lipid acyltransferase, a Bacillus licheniformis host cell comprising a heterologous lipid acyltransferase and a vector comprising a nucleotide sequence encoding a lipid acyltransferase operably linked to a promoter sequence homologous to B. licheniformis.
1. An isolated lipid acyltransferase comprising: the amino acid sequence of SEQ ID No. 16.
2. The isolated lipid acyltransferase of claim 1 , wherein the amino acid sequence undergoes post-translational modification.
3. The isolated lipid acyltransferase of claim 2 , wherein the post-translational modification is a truncation.
4. The isolated lipid acyltransferase of claim 2 , wherein the post-translational modification comprises removal of amino acids from position 235 to position 273 of SEQ ID No. 16.
5. An isolated lipid acyltransferase comprising:
(i) the amino acid sequence of SEQ ID No. 70; or
(ii) an amino acid sequence having at least 90% identity to SEQ ID No. 70 along its full length;
wherein the amino acid sequence possesses acyltransferase activity, and
wherein the amino acid sequence contains the motif GDSX, wherein X is one of amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S.
6. The isolated lipid acyltransferase of claim 5 , wherein the amino acid sequence has at least 95% identity to SEQ ID No. 70.
7. The isolated lipid acyltransferase of claim 5 , wherein the amino acid sequence has at least 98% identity to SEQ ID No. 70.
8. The isolated lipid acyltransferase of claim 5 , wherein the amino acid sequence is SEQ ID No. 70.
9. The isolated lipid acyltransferase of claim 5 , wherein X is L or Y.
10. The isolated lipid acyltransferase of claim 5 , wherein X is L.
11. A method of preparing a lyso-glycolipid comprising treating a foodstuff or an oil comprising a glycolipid with the lipid acyltransferase of claim 1 , thereby preparing a lyso-glycolipid.
12. The method of claim 11 , wherein the lyso-glycolipid is digalactosyl monoglyceride (DGMG) and the glycolipid is digalactosyl diglyceride (DGDG).
13. The method of claim 11 , wherein the lyso-glycolipid is monogalactosyl monoglyceride (MGMG) and the glycolipid is monogalactosyl diglyceride (MGDG).
14. The method of claim 11 , wherein the foodstuff is a dough.
15. The method of claim 11 , wherein the foodstuff is or comprises an egg.
16. The method of claim 11 , wherein an oil is treated with the lipid acyltransferase.
17. The method of claim 16 , wherein the method is a degumming process.
18. A method of preparing a lyso-glycolipid comprising treating a foodstuff or an oil comprising a glycolipid with the lipid acyltransferase of claim 5 , thereby preparing a lyso-glycolipid.
19. The method of claim 18 , wherein the lyso-glycolipid is digalactosyl monoglyceride (DGMG) and the glycolipid is digalactosyl diglyceride (DGDG).
20. The method of claim 18 , wherein the lyso-glycolipid is monogalactosyl monoglyceride (MGMG) and the glycolipid is monogalactosyl diglyceride (MGDG).
21. The method of claim 18 , wherein the foodstuff is a dough.
22. The method of claim 18 , wherein the foodstuff is or comprises an egg.
23. The method of claim 18 , wherein an oil is treated with the lipid acyltransferase.
24. The method of claim 23 , wherein the method is a degumming process.