John Verruto

Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator

Lipid production in the industrial microalga Nannochloropsis gaditana exceeds that of model algal species and can be maximized by nutrient starvation in batch culture. However, starvation halts growth, thereby decreasing productivity. Efforts to engineer N. gaditana strains that can accumulate biomass and overproduce lipids have previously met with little success. We identified 20 transcription factors as putative negative regulators of lipid production by using RNA-seq analysis of N. gaditana during nitrogen deprivation. Application of a CRISPR–Cas9 reverse-genetics pipeline enabled insertional mutagenesis of 18 of these 20 transcription factors. Knocking out a homolog of fungal Zn(II)2Cys6-encoding genes improved partitioning of total carbon to lipids from 20% (wild type) to 40–55% (mutant) in nutrient-replete conditions. Knockout mutants grew poorly, but attenuation of Zn(II)2Cys6 expression yielded strains producing twice as much lipid (∼5.0 g m−2 d−1) as that in the wild type (∼2.5 g m−2 d−1) under semicontinuous growth conditions and had little effect on growth.

Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies

Significance Stacking traits in microalgae is limited by a lack of robust genome modification tools and selectable marker availability. This presents a key hurdle in developing strains for renewable products including biofuels. Here, we overcome these limitations by combining inducible Cre recombinase with constitutive Cas9 nuclease expression in the industrial strain, Nannochloropsis gaditana. With this system, we demonstrate marker- and reporter-free recapitulation of an important lipid productivity trait. In addition, we generate a strain harboring seven-gene knockouts within the photosystem antennae encoding genes. The combined use of relatively mature (Cre) and emerging (CAS9) genome modification technologies can thus accelerate the pace of industrial strain development and facilitate basic research into functionally redundant gene families. Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+. Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5′-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family.