Nannochloropsis is a genus of fast-growing microalgae that have a high lipid content. Nannochloropsis species have a high triacylglycerol (TAG) content and contain a large amount of the omega-3 long-chain polyunsaturated fatty acid, eicosapentaenoic acid (EPA). There is a growing interest in Nannochloropsis species as models for the study of microalga lipid metabolism and as a platform for synthetic biology. Genome sequences are available for several species, and genetic engineering... Show moreNannochloropsis is a genus of fast-growing microalgae that have a high lipid content. Nannochloropsis species have a high triacylglycerol (TAG) content and contain a large amount of the omega-3 long-chain polyunsaturated fatty acid, eicosapentaenoic acid (EPA). There is a growing interest in Nannochloropsis species as models for the study of microalga lipid metabolism and as a platform for synthetic biology. Genome sequences are available for several species, and genetic engineering techniques are being introduced. In this study, I developed a new generation of transgenic vectors for gene stacking and marker-free gene disruption in Nannochloropsis oceanica CCMP1779. These tools enable gene specific studies and were applied to investigate a lipid biosynthetic pathway that is co-expressed under different light conditions. As for all photosynthetic organisms, light plays an important role in driving metabolism and regulation by photosensing in Nannochloropsis species. Each day photosynthetic organisms must maximize their energy capture during the day and be able to sustain themselves during the night. Nannochloropsis cultures synchronize cell division during a light:dark cycle, with cell division occurring at night, along with the usage of stored metabolites that are accumulated during the day. RNA-sequencing measures global transcript abundance, that ultimately might lead to changes in enzymatic activity, metabolism and physiology. I investigated the role of transcriptional regulation on metabolite levels and cell physiology using RNA-sequencing. In the study I found coordination between cell growth, triacylglycerol and hexose content, and transcript abundance of the genes in relevant pathways. Briefly anabolic processes were phased to the light period and catabolic processes phased to the dark period. Furthermore, promoters for transgenic expression were chosen based on transcriptomic measurements gathered in this study. Eicosapentaenoic acid is a high-value fatty acid that is a necessary nutrient for humans, with a biosynthetic pathway consisting of 5 fatty acids desaturases (FADs) and a fatty acid elongase (FAE). Interestingly, the genes of this biosynthetic pathway were strongly co-expressed during light:dark cycles, and I set out to characterize the pathway. Expression of isolated cDNAs in S. cerevisiae resulted in the production of the expected long-chain polyunsaturated fatty acids (LC-PUFAs), and ultimately EPA when all 4 LC-PUFA FADs and an FAE were co-expressed. Selected FADs were overexpressed in N. oceanica and resulted in increased LC-PUFA and EPA content. CRISPR/Cas9 is a potent tool for gene editing. The RNA-guided nuclease, Cas9, was tested as a fusion with green fluorescent protein (GFP) and NanoLuciferase (Nlux) reporters, and the Cas9-Nlux fusion was readily detectable for efficient screening of transformants for recombinant protein production. Single-guide RNAs (sgRNAs) when fused to 5’ and 3’ self-cleaving ribozymes efficiently targeted genes. The two components of the system were expressed from a bidirectional promoter. N. oceanica is capable of expressing transgenes from circular episomal DNA, and an episomal CRISPR construct was generated. The nitrate reductase gene was targeted and the mutants generated with frame-shifts in the coding sequence were unable to grow on nitrate. When antibiotic selection was removed, the episome was lost, and a mutant line that was “cured” of the episome was isolated. These tools are being utilized for gene specific studies in N. oceanica. Show less
