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 increase in the final ω3 product, EPA.

Keywords: Nannochloropsis; 2A peptides; LC-PUFA; bidirectional promoters; eicosapentaenoic acid; gene stacking; genetic engineering toolkit; multigene expression.

Figure 1

EPA biosynthetic pathway identification in N. oceanica CCMP1779. (a) Identification of the fatty acid species by comparison with canola oil and 20C LC‐PUFAs standards (20:3 ^{Δ8,Δ11,Δ14} , di‐homo gamma linolenic acid; 20:4 ^{Δ5,Δ8,Δ11,Δ14} , ω6 eicosatetraenoic acid; 20:4 ^{Δ8,Δ11,Δ14,Δ17} , ω3 eicosatetraenoic acid; and 20:5 ^{Δ5,Δ8,Δ11,Δ14,Δ17} , eicosapentaenoic acid). (b) The EPA biosynthetic pathway includes five desaturases (red ovals, FADs) and an elongase (blue oval, FAE). Gene IDs are shortened from the NannoCCMP1779_# format. (c) Expression of the EPA biosynthetic pathway encoding genes during a light:dark cycle. The arrow indicates the time of cell harvest for cDNA isolation of EPA biosynthetic pathway genes. Gene expression was calculated using a previous experiment (Poliner et al., 2015) and the corrected gene annotation. Values are average ± range of two independent cultures.

Figure 2

Computational annotation of protein sequences of isolated EPA biosynthetic genes. Protein sequences were generated based upon isolated cDNA sequences. Active‐site amino acids and conserved domains identified using conserved domain BLAST. ER localization signals identified with HECTAR. Transmembrane domains predicted using TMHMM2.

Figure 3

Galactose‐inducible expression of the EPA pathway genes in S. cerevisiae. (a) The stacking strategy for EPA production in S. cerevisiae used three plasmids, containing different auxotrophic markers. Dual expression of FADs (pESC‐D12 + D6, and pESC‐D5 + W3) was achieved by a bidirectional Gal10 promoter, and expression of the FAE was under control of the Gal1 promoter (pYES‐E6). (Co)transformation of vectors yielded S. cerevisiae strains Sc‐Δ12 + Δ6, Sc‐Δ12 + Δ6 + E6 and Sc‐Δ12 + Δ6 + E6 + Δ5 + ω3. (b) Representative GC‐FID fatty acid profiles of Sc‐LacZ‐negative control and FAD expressing yeast strains 48 h postgalactose induction.

Figure 4

Assembly of native promoters, terminators and a range of reporters to generate a transgenic expression toolkit for N. oceanica CCMP1779. (a) The pNOC‐OX vector series contains a series of reporters under the control the EF promoter with the LDSP terminator, and a hygromycin resistance gene under the control of the LDSP promoter and 35S terminator. Epitopes include the cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP) and NanoLuciferase (Nlux), and hemagglutinin peptide (HA) encoding sequences. P2A(60) is an extended 2A peptide coding sequence placed 3′ of the zeocin resistance gene (BleR) or hygromycin resistance gene (HygR) for bicistronic expression by ribosomal skipping. The pNOC‐stacked vectors utilize a bidirectional promoter (Ribi) for coexpression of a reporter and resistance gene with 2A peptide coding sequence and multicloning site followed by a heat‐shock terminator. (b) RNA expression of endogenous genes corresponding to the promoters, NannoCCMP1779_10181 (EFpro), and the gene pair NannoCCMP1779_9669 and NannoCCMP1779_9669 (Ribi promoter) under light:dark cycles (data from (Poliner et al., 2015)). (c) Transgenic protein confirmation by immunoblot of pNOC‐OX‐CFP transformants detected with α‐GFP. Total protein was stained using the dye DB71.

Figure 5

Optimization of 2A peptide ribosomal skipping efficiency in N. oceanica CCMP1779. (a) Schematic of pNOC‐2A vector series containing a zeocin resistance coding sequence (BleR) followed by a 2A peptide coding sequence and a multicloning site. Numbers in parentheses correspond to numbers of amino acids of the 2A peptide incorporated, and the encoded amino acid sequence is above (arrow indicates skipping site). For assessing function in N. oceanica CCMP1779, a coding sequence for firefly luciferase (Flux) with a C‐terminal HA tag was inserted downstream of the 2A peptide. (b‐c) Immunoblotting with α‐HA antibody of lines transformed with the pNOC‐2A vector series expressing full‐length BleR‐2A‐Flux (FL) and Flux (Flux*). Total protein stained using DB71.

Figure 6

Vectors for FAD overexpression in N. oceanica CCMP1779 FADs. (a) Schematics of pNOC‐DOX vectors for overexpression of N. oceanica FADs. The Δ9 FAD coding sequence was expressed 3′ of the BleR‐P2A(30) coding cassette in the vector pNOC‐DOX9. Expression of Δ5 or Δ12 FAD coding sequences by a pNOC‐OX vector with a CFP or HA epitope tag. The stacking vector (pNOC‐stacked‐DOX) uses the Ribi promoter to coexpress the coding sequences for BleR‐P2A(30)‐Δ9 FAD, and Δ12 FAD. (b) Immunoblotting N. oceanica single desaturase DOX lines using α‐HA or α‐GFP antibodies. Total protein was stained using DB71. (c) Gene stacking strategy for N. oceanica by use of bicistronic pNOC‐stacked‐DOX and sequential introduction of vectors with different selection markers. (d) Immunoblotting of stacked DOX9 + 12 and DOX5 + 9 + 12 lines using α‐HA or α‐GFP antibodies. Total protein was stained using the dye DB71.

Figure 7

Desaturase overproduction alters the fatty acid profile of N. oceanica CCMP1779. (a) Gene expression of FADs measured by qPCR using the ACTR gene as control (average ± SEM of 3 independent cultures). (b) Cell growth of DOX lines during 5 days under constant light (average ± SEM of four independent cultures). (c) Cell diameter of DOX lines (average ± SEM of four independent cultures). WT, wild type; EV, empty vector control.