EPA+DHA canola

The canola (Brassica napus) was modified for long chain polyunsaturated fatty acid synthesis and accumulation in the seed tissues through the insertion of a mutli-enzyme biochemical pathway to convert oleic acid into polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The enzymes were sourced from Phytophthora sojae, Ostreococcus tauri, Thalassiosira pseudonana, Physcomitrella patens, Thraustochytrium sp., Pythium irregulare, Phytophthora infestans and Pavlova lutheri. For a summary of the biochemical pathway, kindly refer to the "Additional Information" section. The canola-based system allows for scalable production without sourcing these polyunsaturated fatty acids from marine animals. The modified canola additionally contains Arabidopsis thaliana acetohydroxy acid synthase, which confers tolerance to the imidazolinone herbicides, allowing for selection during transformation and post-emergence weed control.

Brassica napus cultivar Kumily

LTM593

Gene expression
- The genetic cassettes are present in the clockwise (sense) orientation.
- Transcription of all of the coding sequence, except  Arabidopsis thaliana acetohydroxy acid synthase large-subunit (Atahas), are under control of seed-specific promoters.
- Atahas is under control of Petroselinum crispum ubiquitin 4-2 promoter, which promotes strong and constitutive expression in all vegetative tissues.
- Some gene cassetes contain introns to enhance the expression of the coding sequence.

Inserted elements
- Agrobacterium rhizogenes-mediated transformation was used instead of the common Agrobacterium tumefaciens-mediated transformation.
- Conlinin promoter was not specified (whether Conlinin-1 or -2). Thus, Conlinin-1 promoter was chosen as a placeholder.
- Two Thraustochytrium sp. delta-6 desaturase and two P. irregulare omega-3 desaturase sequences are present per insert.
- Arabidopsis thaliana acetohydroxy acid synthase large-subunit contains S653N and A122T substitutions.
- The following coding sequences were codon optimized for expression in plants:
   -- Thraustochytrium sp. delta-5 desaturase;
   -- Ostreococcus tauri delta-6 desaturase;
   -- Thalassiosira pseudonana delta-6 elongase;
   -- Phytophthora sojae delta-12 desaturase;
   -- Pythium irregulare omega-3 desaturase;
   -- Phytophthora infestans omega-3 desaturase;
   -- Thraustochytrium sp. delta-4 desaturase;
   -- Pavlova lutheri delta-4 desaturase; and
   -- Ostreococcus tauri delta-5 elongase.

Vector backbone
- The backbone contains: Escherichia coli neomycin phosphotransferase II; E. coli origin of replication (F plasmid); E. coli sopA and sopB; Agrobacterium tumefaciens repB and repC; E. coli Tn5 sequence; and A. tunmefaciens oriT.

Genomic analysis by next generation sequencing (Illumina)
- Next generation sequencing confirmed two separate T-DNA insertions are present, without any rearrangement of the genetic cassettes.
- Insert 1 contained a 184-basepair truncation of the 5΄ end of the RB and a 72-basepair truncation of the 3΄ end of the LB. The first 64 basepairs in the RB of Insert 1 was determined to be a rearrangement of short T-DNA RB-derived repeats. Insert 2 contained 184-basepair truncation of the 5΄ end of the RB with a 53-basepair truncation of the 3΄ end of the LB.
- Insert 1 and Insert 2 contained all 13 intended gene expression cassettes.
- Insert 1 had one cytosine to adenine nucleotide change in the coding sequence of the delta-12 desaturase gene, resulting in a phenylalanine to leucine amino acid substitution (F83L) in the protein, and a nucleotide change in the L. usitatissimum peroxiredoxin-like protein promoter of the P. irregulare omega-3 desaturase.
- Insert 1 was inserted into the the Cnn-random chromosome and resulted in a 8-basepair deletion in the canola genome.
- Insert 2 contains a guanine to thymine nucleotide change in the coding sequences of P. lutheri delta-4 desaturase, resulting in an alanine to serine amino acid substitution (A102S) in the protein.
- Insert 2 was inserted into the C03 chromosome and resulted in a 31-basepair deletion in the canola genome.
- The amino acid changes in the proteins did not show altered activity.
- No backbone sequences were detected.

The fatty acid desaturase and elongase proteins can be detected in mature seeds by ELISA. Due to the seed-specific nature of the promoters, the proteins cannot be detected elsewhere in the plant tissues.

Acetohydroxy acid synthase protein can be detected in all tissues of the plants, except the mature seeds, by ELISA and quantitative Western blot.

Polyunsaturated fatty acid synthesis pathway
1) Oleic acid (C18:1n-9) → Linoleic acid (C18:2n-6) catalyzed by P. sojae delta-12 desaturase
2) Linoleic acid (C18:2n-6) → Gamma-linolenic acid (C18:3-6) catalyzed O. tauri delta-6 desaturase
3) Gamma-linolenic acid (C18:3-6) → Dihomo-gamma-linolenic acid (C20:3n-6) catalyzed by T. pseudonana and P. patens delta-6 elongase
4) Dihomo-gamma-linolenic acid (C20:3n-6) → Arachidonic acid (C20:4n-6) catalyzed by Thraustochytrium sp. delta-5 desaturase
5) Arachidonic acid (C20:4n-6) → Eicosapentaenoic acid (EPA; C20:5n-3) catalyzed by P. irregulare and P. infestans omega-3 desaturase
6) Eicosapentaenoic acid (C20:5n-3) → Docosapentaenoic acid (C22:5n-3) catalyzed by O. tauri delta-5 elongase
7) Docosapentaenoic acid (C22:5n-3) → Docosahexaenoic acid (DHA; C22:6n-3) catalyzed by Thraustochytrium sp. and P. lutheri delta-4 desaturase.