de Grahl Imke, Rout Sweta Suman, Maple-Grødem Jodi, Reumann Sigrun
Plant Biochemistry and Infection Biology, Institute of Plant Science and Microbiology, Universität Hamburg, Ohnhorststr. 18, D-22609, Hamburg, Germany.
The Norwegian Centre for Movement Disorders, Stavanger University Hospital, N-4021, Stavanger, Norway.
Appl Microbiol Biotechnol. 2020 Oct;104(20):8747-8760. doi: 10.1007/s00253-020-10789-4. Epub 2020 Sep 9.
Photoautotrophic microalgae offer a great potential as novel hosts for efficient recombinant protein production. Nannochloropsis oceanica produces an extraordinarily high content of polyunsaturated fatty acids, and its robust growth characteristics, published genome sequence and efficient nuclear transformation make N. oceanica a promising candidate for biotechnological applications. To establish a robust and flexible system for recombinant protein production, we cloned six endogenous, potentially constitutive or inducible promoters from N. oceanica strain CCMP1779 and investigated their strength using monomeric Venus as reporter gene. Microscopic pre-screening of individual transformants revealed that the promoters of elongation factor (EF), tubulin (TUB) and nitrate reductase (NR) enabled high reporter gene expression. Comparative quantitative analyses of transformant populations by flow cytometry and qRT-PCR demonstrated the highest Venus expression from the EF promoter and the NR promoter if extended by an N-terminal 14-amino acid leader sequence. The kinetics of reporter gene expression were analysed during photobioreactor cultivation, achieving Venus yields of 0.3% (for EF) and 4.9% (for NR::LS) of total soluble protein. Since inducible expression systems enable the production of toxic proteins, we developed an auto-induction medium for the NR promoter transformants. By switching the N source from ammonium to nitrate in the presence of low ammonium concentrations, the starting point of Venus induction could be fine-tuned and shifted towards exponential growth phase while maintaining high recombinant protein yields. Taken together, we demonstrate that a model recombinant protein can be produced robustly and at very high levels in N. oceanica not only under constitutive but also under auto-inducible cultivation conditions. KEY POINTS: • Nannochloropsis oceanica might serve as host for recombinant protein production. • Comparative promoter strength analyses were conducted for twelve different constructs. • Robust high-yield recombinant protein production was achieved under constitutive conditions. • The nitrate reductase promoter enabled protein production under auto-induction conditions.
光合自养微藻作为高效重组蛋白生产的新型宿主具有巨大潜力。海洋微拟球藻产生的多不饱和脂肪酸含量极高，其强大的生长特性、已公布的基因组序列以及高效的核转化能力，使海洋微拟球藻成为生物技术应用的一个有前途的候选者。为建立一个强大且灵活的重组蛋白生产系统，我们从海洋微拟球藻CCMP1779菌株中克隆了六个内源性、可能组成型或诱导型启动子，并使用单体维纳斯作为报告基因研究了它们的强度。对单个转化体进行显微镜预筛选发现，延伸因子（EF）、微管蛋白（TUB）和硝酸还原酶（NR）的启动子能够使报告基因高表达。通过流式细胞术和qRT-PCR对转化体群体进行比较定量分析表明，如果EF启动子和NR启动子延伸一个N端14个氨基酸的前导序列，维纳斯的表达量最高。在光生物反应器培养过程中分析了报告基因表达的动力学，维纳斯产量分别达到总可溶性蛋白的0.3%（EF启动子）和4.9%（NR::LS启动子）。由于诱导表达系统能够生产有毒蛋白，我们为NR启动子转化体开发了一种自动诱导培养基。通过在低铵浓度下将氮源从铵切换为硝酸盐，可以微调维纳斯诱导的起始点，并将其转移到指数生长期，同时保持高重组蛋白产量。综上所述，我们证明了一种模型重组蛋白不仅可以在组成型培养条件下，而且可以在自动诱导培养条件下在海洋微拟球藻中大量稳健地生产。要点：• 海洋微拟球藻可能作为重组蛋白生产的宿主。• 对十二种不同构建体进行了启动子强度比较分析。• 在组成型条件下实现了稳健的高产重组蛋白生产。• 硝酸还原酶启动子能够在自动诱导条件下生产蛋白。
