Codon Optimization Of The Key Enzymes Involved In The Biosynthesis Of Very Long Chain Polyunsaturated Fatty Acids And Cloning Of The Key Enzymes Involved In Lipid Biosynthesis

Very long chain (≥C18) polyunsaturated fatty acids (VLCPUFA), such as arachidonicacid (AA;20:4ω6), eicosapentaenoic acid (EPA;20:5ω3) and docosapentaenoic acid (DHA;22:6ω3) are essential for human health and nutrition. EPA and DHA are the main activeingredients of fish oil. The oily fish, such as salmon and mackerel are good sources for thesefatty acids. However, the natural fish resources have declined rapidly in recent years. Inaddition, the recent findings of toxic chemicals in fish oil have raised the safety concerns onthe consumption of fish and fish products, this again reduced the intake of these beneficialfatty acids. Therefore, alternative source of these VLCPUFAs are desirable. To reconstructEPA/DHA metabolic pathway in crop plants through genetic engineering will be a sustainableand safe alternative to supplement these fatty acids. But the yield of EPA especially DHA intransgenic plants is relatively low. Research showed that there are many causes for the lowyield of EPA/DHA，including the host species, promoters used for driving the expression ofthe transgenes, metabolic pathways, combination of transgenes, the codon bias between thetransgenes and the host species, etc.In this study, we focused our research on the codon bias between different species. Wecarried out various codon optimization methods to modify the key enzymes involved in thebiosynthesis of EPA/DHA, followed by the analysis of their enzyme activities in eitherArabidopsis or yeast. By comparing these different strategies, we aimed at finding a particularcodon optimization method for increasing gene expression levels that leads to higher yields ofEPA/DHA in plants.In addition, we also cloned two important genes involved in the commited steps of lipidbiosynthesis that encoded the glycerol-3-phosphate dehydrogenase (GPDH) and acetyl-CoAcarboxylase (ACCase) from the microalgae Amphidinium carterae. These genes may be usedto increase EPA/DHA levels by increasing the overall lipid content in transgenic plants.Below are the main findings:(1) The codon optimization of the Δ9elongase from Isochrysis galbana The first step of the Δ8desaturation pathway is catalysed by the Δ9elongase which has asignificant impact on the subsequent steps in this pathway. We compared the codon usage ofthe Δ9elongase in Isochrysis galbana and Arabidopsis thaliana. We took two strategies tooptimize the Δ9elongase for its expression in Arabidopsis. The first one was to optimize themost bias codon, the Arg encoding CGC codon that has a frequence of codon usage of only20%in Arabidopsis. Through PCR-based site-directed mutagenesis we changed CGC codonsto AGA codons, maintaining the coded amino acid the same. The second strategy was tooptimization the first16codons in the N terminus of the enzyme. All of these codons werechanged to the codons that are used most frequently in Arabidopsis thaliana. These variantswere used to transform Arabidopsis thaliana and their enzyme activities were analysed. Thewild type Δ9elongase with no codon change were used as a control. We found that the Δ9elongase gene variants with single CGC and mutilple CGC codon optimization had enzymeactivities comparable to the wild type control. However, the enzyme activities of the variantswith three CGC and the N terminal16-codon optimization showed significantly highercompared to the wild type and the latter was slightly higher than that of the former.(2) The codon optimization of Δ6desaturase from Phytophthora infestansIn order to test the usefulness of the result obtained from the codon optimization strategyfor the Isochrysis galbana Δ9elongase, we further made codon optimization for a Δ6desaturase isolated from Phytophthora infestans. The functionality of the mutated genes wereanalysed by expression in Saccharomyces cerevisiae. Δ6desaturase catalyses the first step ofthe Δ6pathway that desaturates LA and ALA to GLA and STA, respectively. Similar to the Δ9elongase in the Δ8desaturation pathway, it has a profound impact on the entire pathway. Wemade a comparasion on codon usage of the Δ6desaturase in Phytophthora infestans andSaccharomyces cerevisiae. According to the result of codon optimization for the Δ9elongase,we took two strategies to optimize the Δ6desaturase for its expression in Saccharomycescerevisiae. The first one was to optimize the most bias codon, the CGC codon that has afrequence of codon usage of only13%in Saccharomyces cerevisiae. Through PCR-basedsite-directed mutagenesis we changed all three CGC codons to AGA codons, maintaining thecoded amino acid the same. The second strategy was to optimization the first16codons in theN terminus of the enzyme. All of these codons were changed to the codons that are used most frequently in Saccharomyces cerevisiae. These two variants were used to transformSaccharomyces cerevisiae and their enzyme activities were analysed. The wild type Δ6desaturase with no codon change were used as a control. We found that the enzyme activitiesof both variants were significantly higher compared to the wild type, and Δ6desaturase withoptimization in its N terminus yielded slightly higher enzyme activity than that with threeCGC codons being optimized. This was consistent with the result obtained from codonoptimization for the Δ9elongase, hence further confirming the usefulness of these codonoptimization strategies.(3) Isolation of genes involved in lipid biosynthesis from the microalgae AmphidiniumcarteraeWe sequenced the transcripts of microalgae A.carterae. Bioinformatics analysis enabledus to identify many expressed sequence tags (ESTs) of the key enzymes involved in lipidbiosynthesis.5′and3′RACE strategies were employed to amplify these sequences from firststrand cDNAs prepared from total RNAs isolated from this microalgae. Four partialsequences at the3′end of the glycerol-3-phosphate dehydrogenase genes (GPDH) and twopartial sequences of the accD subunits of acetyl-CoA carboxylase genes (ACCase) wereobtained. This laid a foundation for the further cloning of the full length coding regions ofthese genes that could be used to increase the total lipid content in transgenic oilseed crops.