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

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

Molecular characterisation of bacterial resistance to heavy metals: present understanding and future directions

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Effect of Excess Li on the Structural and Electrical Properties of Garnet-Type Li6La3Ta1.5Y0.5O12

Volatility of lithium during preparation of lithium-stuffed garnet-type metal oxide solid Li ion electrolytes is a common problem, which affects phase formation, ionic conductivity, mechanical strength and density. Synthesis of Li-stuffed garnets has been performed generally using the conventional solid-state reactions at elevated temperature in air. The present study describes the effect of excess LiNO3 (2.5 to 15 wt.%) addition during the ceramic synthesis on the structural and electrical properties of garnet-type Li6La3Ta1.5Y0.5O12. Powder X-ray diffraction (PXRD) confirmed that cubic phase was formed in all tested cases, and there is no significant variation in lattice parameter with amount of excess LiNO3 used. However, increasing amounts of excess lithium decreased inter-particle contact and increased grain growth during sintering, producing sharply varied microstructures. PXRD showed no secondary phase and scanning electron microscopy (SEM) analysis showed rather uniform morphology and absence of "glassy" materials at the grain-boundaries. The bulk Li ion conductivity was found to increase with amount of excess lithium, reaching a maximum room temperature conductivity of 1.62 × 10−4 Scm−1 for the sample prepared using 10 wt.% excess LiNO3. Raman microscopy study indicated the presence of Li2CO3 in all aged Li6La3Ta1.5Y0.5O12 samples prepared using excess LiNO3.Ye