Abstract

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.

中文翻译：

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开发用于微藻Nannochloropsis oceanica中重组蛋白生产的组成型和自动诱导的高产量表达系统。

摘要

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。拟南芥产生异常高含量的多不饱和脂肪酸，并且其强大的生长特性，已公布的基因组序列和有效的核转化使大肠念珠菌成为生物技术应用的有前途的候选者。为了建立一个强大而灵活的重组蛋白生产系统，我们从大洋念珠菌菌株CCMP1779克隆了六个内源性，潜在组成型或诱导型启动子，并使用单体金星研究了它们的强度。作为报告基因。单个转化体的显微镜预筛选显示，延伸因子（EF），微管蛋白（TUB）和硝酸还原酶（NR）的启动子能够实现较高的报告基因表达。通过流式细胞仪和qRT-PCR进行的转化子群体比较定量分析表明，金星最高从EF启动子和NR启动子的表达，如果延伸了N-末端的14个氨基酸的前导序列。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，实现了总可溶性蛋白的金星产量为0.3％（对于EF）和4.9％（对于NR :: LS）。由于可诱导表达系统能够产生有毒蛋白质，因此我们开发了用于NR启动子转化子的自动诱导培养基。通过在低铵盐浓度下将氮源从铵盐转换为硝酸盐，可以微调金星诱导的起点，并向指数生长期转移，同时保持高重组蛋白产量。综上所述，我们证明了在海洋猪笼草中可以稳健地产生很高水平的模型重组蛋白。 不仅在本构条件下，而且在自诱导培养条件下。

关键点

•Nannochloropsis oceanica可能是重组蛋白生产的宿主。

•对十二种不同的构建体进行了比较启动子强度分析。

•在组成条件下实现了稳健的高产量重组蛋白生产。

•硝酸还原酶启动子能够在自动诱导条件下生产蛋白质。