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Eicosapentaenoic acid (EPA, 20:5Δ 5,8,11,14,17 ) and Docosahexaenoic acid (DHA, 22:6Δ 4,7,10,13,16,19 ) 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 .

Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes