The nucleolus as a genomic safe harbor for strong gene expression in Nannochloropsis oceanica

Abstract

Microalgae are used in food and feed, and they are considered a potential feedstock for sustainably produced chemicals and biofuel. However, production of microalgal-derived chemicals is not yet economically feasible. Genetic engineering could bridge the gap to industrial application and facilitate the production of novel products from microalgae. Here, we report the discovery of a novel gene expression system in the oleaginous microalga Nannochloropsis that exploits the highly efficient transcriptional activity of RNA polymerase I and an internal ribosome entry site for translation. We identified the nucleolus as a genomic safe harbor for Pol I transcription and used it to construct transformant strains with consistently strong transgene expression. The new expression system provides an outstanding tool for genetic and metabolic engineering of microalgae and thus will probably make substantial contributions to microalgal research.

Biochemistry and Biotechnology of Lipid Accumulation in the Microalga Nannochloropsis oceanica

“…oceanica lines at best. The recent development of effective genome editing and transgene expression systems may contribute to the future metabolic engineering efforts. ,,,− For example, a novel chimeric gene expression system using the nucleolus as a genomic safe harbor has been recently established for strong and consistent transgene expression in N. oceanica .…”

“…The recent development of effective genome editing and transgene expression systems may contribute to the future metabolic engineering efforts. ,,,− For example, a novel chimeric gene expression system using the nucleolus as a genomic safe harbor has been recently established for strong and consistent transgene expression in N. oceanica . Moreover, improving enzyme performance (e.g., activity and substrate specificity) by synthetic protein design may be promising in developing algal strains for tailor-made lipid production.…”

“…N. oceanica can accumulate lipids up to 60% of the dry cell weight, including the high-value ω-3 LC-PUFA EPA, and has long been used as feeds in the aquacultural industry. , In addition, N. oceanica is a good source of protein, accounting for 35–44% of the dry cell weight, and has potential to be used as a single-cell protein for novel food and feed .…”

“…oceanica strains have been sequenced, including CCMP1779 and IMET1, and additional transcriptomic and proteomic resources for many Nannochloropsis species are available (e.g., Nannochloropsis Design and Synthesis, NanDeSyn, , accessed August 2022). − Moreover, genetic toolboxes for manipulating gene expression in N. oceanica have been successfully developed, including gene stacking for multiple gene overexpression and clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques for precise genome editing ,− (for a review, see ref ). The high lipid and LC-PUFA content along with recent advances in genetic engineering, therefore, promote strong interests in developing N.…”

“…2 Therefore, microalgae hold the promise of a sustainable resource for lipidbased biofuel production as a result of their high lipid accumulation ability and no competition with agricultural crops for arable land. 2 Some microalgal species also produce health-promoting long-chain polyunsaturated fatty acids (LC-PUFAs), such as eicosapentaenoic acid (EPA, C20:5Δ 5,8,11,14,17 ) and docosahexaenoic acid (DHA, C22:6Δ 4,7,10,13,16,19 ). 3,4 EPA and DHA are essential ω-3 fatty acids that have important roles in neuronal and retinal development, immune system, cardiovascular health, and cognitive function and have been widely used in nutritional supplements.…”