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

Poliner, Eric; Pulman, Jane A.; Zienkiewicz, Krzysztof; Childs, Kevin; Benning, Christoph; Farré, Eva M.

doi:10.1111/pbi.12772

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

Journal Article · Mon Jun 12 00:00:00 EDT 2017 · Plant Biotechnology Journal

DOI:https://doi.org/10.1111/pbi.12772· OSTI ID:1369495

Poliner, Eric ^[1]; Pulman, Jane A. ^[2]; Zienkiewicz, Krzysztof ^[3]; Childs, Kevin ^[2]; Benning, Christoph ^[4]; Farré, Eva M. ^[2]

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

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.

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Research Organization:: Univ. of Wisconsin, Madison, WI (United States); Michigan State Univ., East Lansing, MI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division

Grant/Contract Number:: FG02-91ER20021; FC02-07ER64494

OSTI ID:: 1369495

Alternate ID(s):: OSTI ID: 1369496; OSTI ID: 1545799

Journal Information:: Plant Biotechnology Journal, Journal Name: Plant Biotechnology Journal; ISSN 1467-7644

Publisher:: Society for Experimental Biology; Association of Applied BiologyCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Nannochloropsis
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eicosapentaenoic acid
multigene expression
gene stacking
genetic engineering toolkit
2A peptides
bidirectional promoters

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

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