A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production
Abstract
Nannochloropsis oceanica is an oleaginous microalga rich in ω3 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 Δ12 and Δ5 FAD encoding sequences leading to an increasemore »
- Authors:
-
- MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing MI USA, Cell and Molecular Biology Program, Michigan State University, East Lansing MI USA
- Department of Plant Biology, Michigan State University, East Lansing MI USA
- MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing MI USA, Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Gottingen Germany
- MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing MI USA, Department of Plant Biology, Michigan State University, East Lansing MI USA, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing MI USA
- Publication Date:
- Research Org.:
- Univ. of Wisconsin, Madison, WI (United States); Michigan State Univ., East Lansing, MI (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division
- OSTI Identifier:
- 1369495
- Alternate Identifier(s):
- OSTI ID: 1369496; OSTI ID: 1545799
- Grant/Contract Number:
- FG02-91ER20021; FC02-07ER64494
- Resource Type:
- Published Article
- Journal Name:
- Plant Biotechnology Journal
- Additional Journal Information:
- Journal Name: Plant Biotechnology Journal; Journal ID: ISSN 1467-7644
- Publisher:
- Society for Experimental Biology; Association of Applied Biology
- Country of Publication:
- United Kingdom
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; Nannochloropsis; LC‐PUFA; eicosapentaenoic acid; multigene expression; gene stacking; genetic engineering toolkit; 2A peptides; bidirectional promoters
Citation Formats
Poliner, Eric, Pulman, Jane A., Zienkiewicz, Krzysztof, Childs, Kevin, Benning, Christoph, and Farré, Eva M. A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production. United Kingdom: N. p., 2017.
Web. doi:10.1111/pbi.12772.
Poliner, Eric, Pulman, Jane A., Zienkiewicz, Krzysztof, Childs, Kevin, Benning, Christoph, & Farré, Eva M. A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production. United Kingdom. https://doi.org/10.1111/pbi.12772
Poliner, Eric, Pulman, Jane A., Zienkiewicz, Krzysztof, Childs, Kevin, Benning, Christoph, and Farré, Eva M. Mon .
"A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production". United Kingdom. https://doi.org/10.1111/pbi.12772.
@article{osti_1369495,
title = {A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production},
author = {Poliner, Eric and Pulman, Jane A. and Zienkiewicz, Krzysztof and Childs, Kevin and Benning, Christoph and Farré, Eva M.},
abstractNote = {Nannochloropsis oceanica is an oleaginous microalga rich in ω3 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 Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.},
doi = {10.1111/pbi.12772},
journal = {Plant Biotechnology Journal},
number = ,
volume = ,
place = {United Kingdom},
year = {Mon Jun 12 00:00:00 EDT 2017},
month = {Mon Jun 12 00:00:00 EDT 2017}
}
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