title: Elevation of the Yields of Very Long Chain Polyunsaturated Fatty Acids via Minimal Codon Optimization of Two Key Biosynthetic Enzymes.
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.
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Xia
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Xueying
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Xinzheng
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Desong
authors.initials: D
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Quanxi
authors.initials: Q
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Yaxiao
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Plant Genetics & Breeding, College of Agronomy and Biotechnology, China Agricultural University, No 2, Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
authors.forename: Jinping
authors.initials: J
authors.lastname: Hua
authors.affiliation: State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
authors.forename: Baoxiu
authors.identifier: http://orcid.org/0000-0002-9425-935X
authors.initials: B
authors.lastname: Qi
mesh_terms: D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003062:Codon; D004281:Docosahexaenoic Acids / Q000096:biosynthesis* / Q000235:genetics; D015118:Eicosapentaenoic Acid / Q000096:biosynthesis* / Q000235:genetics; D000081031:Fatty Acid Elongases; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D058087:Haptophyta / Q000201:enzymology / Q000235:genetics; D050602:Linoleoyl-CoA Desaturase / Q000235:genetics* / Q000378:metabolism; D055750:Phytophthora infestans / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D019076:Transgenes
chemical_list: D003062:Codon; D004281:Docosahexaenoic Acids; D015118:Eicosapentaenoic Acid; D050602:Linoleoyl-CoA Desaturase; D000123:Acetyltransferases; D000081031:Fatty Acid Elongases