US 11,987,819 B2
Method of increasing omega-3 polyunsaturated fatty acids production in microalgae
Anne-Cecile V. Bayne, Ellicott City, MD (US); and Ross E. Zirkle, Mount Airy, MD (US)
Assigned to DSM IP Assets B.V., Heerlen (NL)
Appl. No. 16/300,212
Filed by DSM IP Assets B.V., Heerlen (NL)
PCT Filed May 11, 2017, PCT No. PCT/EP2017/061347
§ 371(c)(1), (2) Date Nov. 9, 2018,
PCT Pub. No. WO2017/194683, PCT Pub. Date Nov. 16, 2017.
Claims priority of provisional application 62/335,498, filed on May 12, 2016.
Prior Publication US 2019/0144838 A1, May 16, 2019
Int. Cl. A01H 5/00 (2018.01); C12N 9/10 (2006.01); C12N 15/00 (2006.01); C12N 15/82 (2006.01); C12P 7/6432 (2022.01); C12P 7/6472 (2022.01)
CPC C12N 9/1029 (2013.01) [A01H 5/00 (2013.01); C12N 15/00 (2013.01); C12N 15/8247 (2013.01); C12P 7/6432 (2022.01); C12P 7/6472 (2013.01); C12Y 203/01085 (2013.01)] 13 Claims
 
1. A recombinant microalga that has been modified to express increased levels of polyunsaturated fatty acid synthase, comprising:
(a) a first gene encoding a polyunsaturated fatty acid synthase subunit 1 (PFA1) which comprises a first amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, wherein the first amino acid sequence comprises a β-ketoacyl-ACP synthase (KS) domain having at least 95% sequence identity to SEQ ID NO: 8, a malonyl-CoA:ACP acyltransferase (MAT) domain having at least 95% sequence identity to SEQ ID NO: 10, an acyl carrier protein (ACP) domain having at least 95% sequence identity to SEQ ID NO: 12, 14, 16, 18, 20, 22, or 24, a β-ketoacyl-ACP reductase (KR) domain of SEQ ID NO: 26 and a FabA-like β-hydroxyacyl-ACP dehydrase (DH) domain having at least 95% sequence identity to SEQ ID NO: 28;
(b) a second gene encoding a polyunsaturated fatty acid synthase subunit 2 (PFA2) which comprises a second amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4, wherein the second amino acid sequence comprises a KS domain having at least 95% sequence identity to SEQ ID NO: 30, a chain length factor (CLF) domain having at least 95% sequence identity to SEQ ID NO: 32, an acyltransferase (AT) domain having at least 95% sequence identity to SEQ ID NO: 34 and an enoyl-ACP reductase (ER) domain having at least 95% sequence identity to SEQ ID NO: 36; and
(c) a third gene encoding a polyunsaturated fatty acid synthase subunit 3 (PFA3) which comprises a third amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6, wherein the third amino acid sequence comprises a DH domain having at least 95% sequence identity to SEQ ID NO: 38 or 40 and an ER domain having at least 95% sequence identity to SEQ ID NO: 42,
wherein the recombinant microalga is a recombinant Schizochytrium,
wherein the recombinant microalga comprises multiple copies of the first and/or third gene but only a single copy of the second gene, and
wherein the recombinant microalga produces lipids with an increased level of eicosapentaenoic acid (EPA) compared to an unmodified microalga.