Xia Fei, Li Xueying, Li Xinzheng, Zheng Desong, Sun Quanxi, Liu Jiang, Li Yaxiao, Hua Jinping, Qi Baoxiu
State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, 271000, China.
Department of Plant Genetics & Breeding, College of Agronomy and Biotechnology, China Agricultural University, No 2, Yuanmingyuan West Road, Haidian District, Beijing, 100193, China.
PLoS One. 2016 Jul 19;11(7):e0158103. doi: 10.1371/journal.pone.0158103. eCollection 2016.
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.
二十碳五烯酸（EPA，20:5Δ5,8,11,14,17）和二十二碳六烯酸（DHA，22:6Δ4,7,10,13,16,19）对人体健康具有营养益处。近年来，人们尝试通过转移源自低等真核生物（如微藻和真菌）的基因，在油料作物中进行EPA和DHA的转基因生产。然而，这些转基因作物中EPA和DHA的低产量是这些转基因商业化的主要障碍。许多因素会对转基因表达产生负面影响，导致转化脂肪酸产物水平较低。其中，转基因供体与宿主作物之间的密码子偏好性是主要影响因素之一。因此，我们分别对来自致病疫霉的脂肪酸δ-6去饱和酶基因PinD6和来自等鞭金藻的δ-9延长酶基因IgASE1进行了密码子优化，使其分别在酿酒酵母和拟南芥中表达。这两个基因是编码酶的关键基因，驱动着EPA/DHA生物合成的Δ6去饱和/Δ6延长以及Δ9延长/Δ8去饱和途径中的第一步催化反应。因此，这两个基因的表达水平对于确定EPA/DHA的最终产量很重要。通过基于PCR的诱变，我们优化了这两个基因N端前16个密码子中最不常用的密码子，以及整个序列中偏好性最高的CGC密码子（编码精氨酸）。一项表达研究表明，携带密码子优化后的IgASE1的转基因拟南芥植株中，延长脂肪酸产物比表达天然IgASE1序列的植株多64%，而表达密码子优化后的PinD6的酿酒酵母产生的去饱和产物比表达野生型（WT）PinD6的酵母多20倍。因此，我们在此开发的密码子优化策略为在酵母和拟南芥中高效表达外源基因提供了一种简单、有效且低成本的全基因合成替代方法。
