Abstract
Eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17) and Docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) are nutritionally beneficial to human health. Transgenic production of EPA and DHA in oilseed crops by transferring genes originating from lower eukaryotes, such as microalgae and fungi, has been attempted in recent years. However, the low yield of EPA and DHA produced in these transgenic crops is a major hurdle for the commercialization of these transgenics. Many factors can negatively affect transgene expression, leading to a low level of converted fatty acid products. Among these the codon bias between the transgene donor and the host crop is one of the major contributing factors. Therefore, we carried out codon optimization of a fatty acid delta-6 desaturase gene PinD6 from the fungus Phytophthora infestans, and a delta-9 elongase gene, IgASE1 from the microalga Isochrysis galbana for expression in Saccharomyces cerevisiae and Arabidopsis respectively. These are the two key genes encoding enzymes for driving the first catalytic steps in the Δ6 desaturation/Δ6 elongation and the Δ9 elongation/Δ8 desaturation pathways for EPA/DHA biosynthesis. Hence expression levels of these two genes are important in determining the final yield of EPA/DHA. Via PCR-based mutagenesis we optimized the least preferred codons within the first 16 codons at their N-termini, as well as the most biased CGC codons (coding for arginine) within the entire sequences of both genes. An expression study showed that transgenic Arabidopsis plants harbouring the codon-optimized IgASE1 contained 64% more elongated fatty acid products than plants expressing the native IgASE1 sequence, whilst Saccharomyces cerevisiae expressing the codon optimized PinD6 yielded 20 times more desaturated products than yeast expressing wild-type (WT) PinD6. Thus the codon optimization strategy we developed here offers a simple, effective and low-cost alternative to whole gene synthesis for high expression of foreign genes in yeast and Arabidopsis.
Highlights
Very long chain polyunsaturated fatty acids (VCL-PUFAs), such as EPA and DHA have a wide range of physiological and biological functions in cells
Our results show that codon optimization of both genes resulted in marked increases in the production of VLC-PUFAs in transgenic hosts compared to the expression of the native genes
We found that the three arginine-encoding CGCArg codons at positions 10, 35 and 84 are the least preferred codons used by Arabidopsis having a predicted codon usage efficiency of only 20% (S1 Fig)
Summary
Very long chain polyunsaturated fatty acids (VCL-PUFAs), such as EPA and DHA have a wide range of physiological and biological functions in cells. In the Δ6 desaturation/Δ6 elongation pathway, the first step is catalyzed by the Δ6 desaturase that converts linolenic acid (LA, 18:2Δ9,12) and α-linolenic acid (ALA, 18:3Δ9,12,15) to γ-linolenic acid (GLA, 18:3Δ6,9,12) and stearidonic acid (SDA, 18:4Δ6,9,12,15), respectively This is followed by a Δ6 elongation step and a Δ5 desaturation step to achieve the production of arachidonic acid EPA and (AA, 20:4Δ5,8,11,14). In the alternative Δ9 elongation/Δ8 desaturation pathway, LA and ALA are first elongated by a Δ9 elongase to yield eicosadienoic acid (EDA, 20:2Δ11,14) and eicosatrienoic acid (EtrA, 20:2Δ11,14,17), respectively. These products are desaturated by Δ8 and Δ5 desaturation to produce EPA and AA. DHA is produced by Δ5 elongation and Δ4 desaturation of EPA
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