Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica
Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica
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来自微绿球藻的酰基-酰基载体蛋白α9去饱和酶的鉴定和功能分析
DOI：
10.1007/s12275-022-00001-9
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发表时间：
2023-01-31
影响因子：
3
通讯作者：
Zhang，Dongyi
中科院分区：
文献类型：
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作者：
Yang，Ruigang;Wang，Hui;Zhang，Dongyi
产油海洋微藻Nannochloropsis oceanica菌株IMET1由于其独特的积累大量三酰甘油和二十碳五烯酸的优异能力，作为一种有前途的光合细胞工厂而受到越来越多的关注。为了完成大洋藻脂肪酸生物合成途径基因的基因组注释，我们进行了本研究，以确定一个新的候选基因编码典型的叶绿体基质酰基酰基载体蛋白Δ9desaturase。利用cDNA末端的快速扩增得到全长cDNA，并确定了被4个内含子中断的编码区结构。RT-qPCR结果显示，在氮饥饿条件下，该基因的转录丰度上调。利用egfp融合蛋白进行荧光定位研究，发现翻译蛋白定位在叶绿体基质中。翻译蛋白的催化活性在大肠杆菌和酵母突变株BY4389中被诱导表达，表明其对棕榈酰acp （C16:0-ACP）和硬脂酰acp （C18:0-ACP）具有潜在的去饱和能力。进一步用BY4839进行板上功能互补实验表明，表达的酶恢复了油酸的生物合成。这些结果支持了海洋新菌株IMET1叶绿体基质中表达蛋白的去饱和活性，以满足单不饱和脂肪酸的生物合成和积累。
The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.