The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas Reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing

Abstract

Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas9 allows directed exploration of new endogenous selectable markers. Since editing frequencies with CRISPR/Cas9 techniques are often low, the Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, increasing editing frequencies from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was targeted to either the cytosol or the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

Teaching an old ‘doc’ new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection

“…Previous reports have demonstrated that multiple fluorescent proteins can be combined in the alga cell simultaneously by transformation and mating using micro-plate readers and confocal microscopy imaging techniques (Rasala et al, 2013(Rasala et al, , 2014. FP expression can also screened at the agar-plate level with whole-plant or stereo-microscopy imaging apparatus, a technique which assists selection of robust expressing cell lines quickly from a population (Lauersen et al, 2016(Lauersen et al, , 2018Wichmann et al, 2018;Freudenberg et al, 2021Freudenberg et al, , 2022aFreudenberg et al, , 2022b. Fluorescent proteins are not the only bio-molecules which emit some amount of fluorescence signal in host cells and tissues.…”

“…Genetic fusion of target recombinant proteins with FPs has become a common strategy to identify high transgene expressing transformants in a population and facilitate genetic/metabolic engineering concepts with C. reinhardtii (Lauersen et al, 2016(Lauersen et al, , 2018Wichmann et al, 2018;Perozeni, 2020;Einhaus et al, 2022;Freudenberg et al, 2021;Freudenberg et al, 2022a). Target protein-FP fusions allow rapid identification of cell lines exhibiting robust transgene expression and translation as stronger fluorescence signal correlates to higher abundance of target-fusion protein products.…”