Plant Biochemistry and Infection Biology, Institute of Plant Science and Microbiology, Universität Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany.
Zybio Inc, Chongqing Municipality, 400084, China.
Appl Microbiol Biotechnol. 2022 Oct;106(19-20):6535-6549. doi: 10.1007/s00253-022-12106-7. Epub 2022 Sep 7.
Nannochloropsis oceanica is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish N. oceanica as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of N. oceanica strain CCMP1779 with the codon-optimized VP2 gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that N. oceanica was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet. KEY POINTS: • We engineered N. oceanica for recombinant protein production. • The antigenic surface protein 2 of IPN virus could indeed be expressed in the host. • A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.
海洋盐藻是一种具有新兴生物技术应用前景的单细胞产油微藻，其基因组已被测序、注释，转录组和蛋白质组数据可用，并且具有成熟的遗传工程基本分子工具。为了将海洋盐藻建立为真核宿主，用于重组蛋白合成，并开发疫苗生产的分子技术，我们选择了一种引起传染性胰腺坏死的致病性鱼类病毒的病毒表面蛋白 2 (VP2) 作为模型疫苗。通过对 N. oceanica 菌株 CCMP1779 的稳定核转化，用密码子优化的 VP2 基因，利用 Venus 报告融合物，通过 qPCR 和流式细胞术评估转化体群体中不同内源性启动子的强度。在伸长因子启动子下，VP2 产量最高，其 N 端前导序列具有增强作用。个别转化体在报告基因-free VP2 的产生能力方面差异达数量级。当将最佳候选物进行在光生物反应器中生长和 VP2 生产的动力学分析时，重组蛋白完整性一直保持到早期稳定生长阶段，并实现了总可溶性蛋白 4.4%的 VP2 高产量。最大产量与表达载体在核基因组中的多次整合相关。结果表明，海洋盐藻已成功构建为用于生产模型亚单位疫苗的高产量的稳健平台。此处建立的分子方法很可能可以直接适用于同一宿主中进一步疫苗的生产，从而可以通过含有微藻的饮食将其分发给鱼类、脊椎动物或人类。关键点：
我们对 N. oceanica 进行了基因工程改造，用于重组蛋白生产。
病毒的抗原表面蛋白 2 确实可以在宿主中表达。
在海洋盐藻中实现了总可溶性蛋白 4.4%的 VP2 高产量。
