Lipids and more : advancing Nannochloropsis as a biotechnological multipurpose chassis

Microalgae hold promise for sustainable production of commodities including food, feed, and green chemicals. Nannochloropsis oceanica is a particularly interesting candidate for industrial purposes and an emerging model organism for oleaginous. Although production of bulk commodities from N. oceanica is not yet economically feasible, strain improvement strategies could transform this microalga into a cell factory for cost-effective production of designer food, feed and bioproducts. This will require a comprehensive framework for metabolic engineering and synthetic biology as cornerstones to maximization of yields and productivities. These practices rely on a proper genome annotation, on an in-depth understanding of metabolic fluxes, and on a highly developed genetic manipulation toolset. Therefore, advancing our understanding of the metabolic network and expanding the toolbox for genetic engineering are of the utmost importance to establish N. oceanica as a commodity production platform.

To improve the available tools for conducting forward genetic screens with N. oceanica, we developed a high-throughput screening for cellular neutral lipid content in chapter 2. Fluorescence activated cell sorting (FACS) was employed as a powerful single cell methodology. A robust and efficient neutral lipid staining procedure for N. oceanica was devised, utilizing the fluorophore BODIPY505/515. It was found that 6% DMSO, 1.2 µg ml-1 and 15 min staining duration are ideal for staining neutral lipids in exponentially growing N. oceanica cultures. For cultures that were stressed by nutrient deprivation, 10% DMSO, 1.2-1.6 µg ml-1 and 36 min duration were required. The combination of this staining method with FACS allowed quantitative prediction of cellular neutral lipid contents.

Chapters 3-4 focused on developing novel tools for genetic manipulation of N. oceanica using CRISPR-Cas technology. Cas12a was employed as a significant asset in a CRISPR toolbox due to several differences and advantages compared with Cas9. First, we established a CRISPR-Cas12a-based genome editing procedure for N. oceanica using a ribonucleoprotein (RNP) transformation strategy. Efficiency strongly depended on the chosen Cas protein, with Cas12a from Francisella novicida (FnCas12a) being the most efficient, and Cas12a from Acidaminococcus sp. being the least. We further demonstrated that the Cas12a RNP-based CRISPR strategy can be used to generate scarless and markerless deletion mutants of N. oceanica when combining it with FACS. Moreover, we established an episomal CRISPR-Cas12a genome editing strategy, and we showed that Cas12a is able to efficiently process pre-crRNA into mature crRNAs in N. oceanica. We demonstrated that this ability can be exploited for multiplexed CRISPR gene editing, by expressing a CRISPR array from the episomal plasmid. Multiplexing efficiencies were as high as 37% for inducing double-strand breaks at two target sites simultaneously, and 14% for three loci. Lastly, we developed a CRISPR interference system for gene silencing in N. oceanica, employing episomal expression of catalytically inactive versions of SpCas9 and FnCas12a as genetic fusions with a sequence encoding a human KRAB transcription repressor domain. Highest repression efficiencies were observed when crRNA sequences were targeting the beginning of the silenced gene, with up to 85% reduction in transcript levels for dCas9-KRAB.

In addition to strategies for targeted gene knockouts and knockdowns, a sophisticated genetic manipulation toolbox requires means for gene overexpression. One of the most important factors for gene expression is the choice of transcriptional promoter as a key regulator of expression efficiency. To identify novel transcriptional promoters suitable for metabolic engineering, we constructed a promoter-trapped random insertional mutant library using a promoterless GFP cassette, and screened the library for GFP levels in chapter 5. The insertion site in a mutant with particularly strong GFP fluorescence was traced to a 25S rRNA gene. It was discovered that transgene expression at the rDNA locus was rendered possible by transcription through RNA polymerase I (Pol I), in conjunction with activity of an artificial internal ribosome entry site (IRES), which lifted the translational block of the uncapped chimeric rRNA-GFP transcript. We defined the minimum genetic elements required for gene expression using Pol I and this IRES, and it was revealed that the nucleolar organizer region acts as a genomic safe harbor for gene expression with these elements. Based on these insights a pipeline for constructing transformant strains with consistent and exceptionally strong transgene expression was devised. Finally, we demonstrated the potential of the new gene expression system for high-efficiency production of recombinant protein in N. oceanica, by constructing strains that produce a VHH antibody at up to 8.5% of extract protein.

In chapter 6, a forward genetic screen was conducted to identify genes related to lipid metabolism in N. oceanica. We implemented an efficient and robust procedure for genotyping insertional mutants, and screened an insertional mutant library of the microalga for increased neutral lipid contents using FACS. Through this, we identified a transcription factor (NO06G03670) with similarity to APETALA2-like proteins of higher plants, which functions as a negative regulator of neutral lipid accumulation in N. oceanica. Transcriptomic analysis revealed that NO06G03670 knockout causes transcriptional upregulation of genes related to chloroplastic fatty acid biosynthesis and the Calvin-Benson-Bassham cycle.

Medium chain fatty acids (MCFAs) are desirable compounds for food and feed, but they are produced at marginal levels in most microalgae. To improve the potential of N. oceanica for food and feed applications, we generated transformant strains with increased MCFA contents by heterologous expression of an MCFA-biased acyl-CoA thioesterase from the eudicot Cuphea palustris in chapter 7.

To improve lipid productivity, N. oceanica was genetically engineered to express a glycerol-3-phosphate-acyltransferase (GPAT) in chapter 8. Overexpression of an endogenous and heterologous GPAT both increased neutral lipid accumulation in transformants by up to 51% under favourable growth conditions. Expression of the endogenous GPAT further resulted in increased production of polyunsaturated fatty acids.

In the final chapter, key advancements of the genetic manipulation toolbox for Nannochloropsis were highlighted and suitable targets for additional improvements were suggested. Moreover, strategies were proposed that could enhance our understanding of this microalga's metabolism. We painted a vision of N. oceanica as a multifunctional chassis with diverse industrial applications, inspired by this microalga's innate industrial potential and the recent improvements in genetic manipulation techniques.