Genetic engineering of microalgae for enhanced lipid production.

Microalgae have the potential to become microbial cell factories for lipid production. Their ability to convert sunlight and CO2 into valuable lipid compounds has attracted interest from cosmetic, biofuel, food and feed industries. In order to make microalgae-derived products cost-effective and commercially competitive, enhanced growth rates and lipid productivities are needed, which require optimization of cultivation systems and strain improvement. Advances in genetic tool development and omics technologies have increased our understanding of lipid metabolism, which has opened up possibilities for targeted metabolic engineering. In this review we provide a comprehensive overview on the developments made to genetically engineer microalgal strains over the last 30 years. We focus on the strategies that lead to an increased lipid content and altered fatty acid profile. These include the genetic engineering of the fatty acid synthesis pathway, Kennedy pathway, polyunsaturated fatty acid and triacylglycerol metabolisms and fatty acid catabolism. Moreover, genetic engineering of specific transcription factors, NADPH generation and central carbon metabolism, which lead to increase of lipid accumulation are also reviewed.

High-throughput insertional mutagenesis reveals novel targets for enhancing lipid accumulation in Nannochloropsis oceanica.

The microalga Nannochloropsis oceanica is considered a promising platform for the sustainable production of high-value lipids and biofuel feedstocks. However, current lipid yields of N. oceanica are too low for economic feasibility. Gaining fundamental insights into the lipid metabolism of N. oceanica could open up various possibilities for the optimization of this species through genetic engineering. Therefore, the aim of this study was to discover novel genes associated with an elevated neutral lipid content. We constructed an insertional mutagenesis library of N. oceanica, selected high lipid mutants by five rounds of fluorescence-activated cell sorting, and identified disrupted genes using a novel implementation of a rapid genotyping procedure. One particularly promising mutant (HLM23) was disrupted in a putative APETALA2-like transcription factor gene. HLM23 showed a 40%-increased neutral lipid content, increased photosynthetic performance, and no growth impairment. Furthermore, transcriptome analysis revealed an upregulation of genes related to plastidial fatty acid biosynthesis, glycolysis and the Calvin-Benson-Bassham cycle in HLM23. Insights gained in this work can be used in future genetic engineering strategies for increased lipid productivity of Nannochloropsis.

Characterizing a thermostable Cas9 for bacterial genome editing and silencing.

CRISPR-Cas9-based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here we identify and characterize ThermoCas9 from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that in vitro ThermoCas9 is active between 20 and 70 °C, has stringent PAM-preference at lower temperatures, tolerates fewer spacer-protospacer mismatches than SpCas9 and its activity at elevated temperatures depends on the sgRNA-structure. We develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55 °C in Bacillus smithii and for gene deletion at 37 °C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish a Cas9-based bacterial genome editing and silencing tool with a broad temperature range.