期刊
PLANT BIOTECHNOLOGY JOURNAL
卷 16, 期 1, 页码 298-309出版社
WILEY
DOI: 10.1111/pbi.12772
关键词
Nannochloropsis; LC-PUFA; eicosapentaenoic acid; multigene expression; gene stacking; genetic engineering toolkit; 2A peptides; bidirectional promoters
资金
- National Science Foundation [IOS-1354721]
- United States Department of Energy (Division of Chemical Sciences, Geosciences and Biosciences, Office) [DE-FG02-91ER20021]
- United States Department of Energy (Great Lakes Bioenergy Research Center) [DE-FC02-07ER64494]
- AgBioResearch, Michigan State University
- People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme [627266]
Nannochloropsis oceanica is an oleaginous microalga rich in omega 3 long-chain polyunsaturated fatty acids (LC-PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC-PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC-PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC-OX and pNOC-stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase-encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC-PUFAs, with the overexpression of Delta 12 and Delta 5 FAD encoding sequences leading to an increase in the final omega 3 product, EPA.
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