Integration of a Galdieria plasma membrane sugar transporter enables heterotrophic growth of the obligate photoautotrophic red alga Cynanidioschyzon merolae

Abstract

The unicellular thermoacidophilic red alga Cyanidioschyzon merolae is an emerging model organism of photosynthetic eukaryotes. Its relatively simple genome (16.5 Mbp) with very low‐genetic redundancy and its cellular structure possessing one chloroplast, mitochondrion, peroxisome, and other organelles have facilitated studies. In addition, this alga is genetically tractable, and the nuclear and chloroplast genomes can be modified by integration of transgenes via homologous recombination. Recent studies have attempted to clarify the structure and function of the photosystems of this alga. However, it is difficult to obtain photosynthesis‐defective mutants for molecular genetic studies because this organism is an obligate autotroph. To overcome this issue in C. merolae , we expressed a plasma membrane sugar transporter, Gs SPT 1, from Galdieria sulphuraria , which is an evolutionary relative of C. merolae and capable of heterotrophic growth. The heterologously expressed GsSPT1 localized at the plasma membrane. GsSPT1 enabled C. merolae to grow mixotrophically and heterotrophically, in which cells grew in the dark with glucose or in the light with a photosynthetic inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea ( DCMU ) and glucose. When the GsSPT1 transgene multiplied on the C. merolae chromosome via the URA Cm‐Gs selection marker, which can multiply itself and its flanking transgene, GsSPT1 protein level increased and the heterotrophic and mixotrophic growth of the transformant accelerated. We also found that Gs SPT 1 overexpressing C. merolae efficiently formed colonies on solidified medium under light with glucose and DCMU . Thus, Gs SPT 1 overexpresser will facilitate single colony isolation and analyses of photosynthesis‐deficient mutants produced either by random or site‐directed mutagenesis. In addition, our results yielded evidence supporting that the presence or absence of plasma membrane sugar transporters is a major cause of difference in trophic properties between C. merolae and G. sulphuraria .

Exploiting the potential of Cyanidiales as a valuable resource for biotechnological applications

“…Heterotrophic growth rates of C. merolae are substantially lower than those of Galdieria (Moriyama et al, 2015). However, the genomic integration of a sugar transporter gene from G. sulphuraria resulted in higher growth rates of C. merolae (Fujiwara et al, 2019). Furthermore, C. merolae lacks a cell wall and thus allows for a simple and effective cell extraction by osmotic shock as demonstrated by Rahman and co-workers (Rahman et al, 2017).…”

“…The future for exploitation of Cyanidiales Both G. sulphuraria and C. merolae have unique advantages for biotechnological applicationson one hand an efficient generation of biomass for Galdieria and on the other an advanced understanding of the genome and options for genetic manipulation in C. merolae (Table 1). It is evident that knowledge of these organisms could be combined, as shown by generating modified C. merolae strains with metabolite transporters from G. sulphuraria (Fujiwara et al, 2019). Lately, the potential of Cyanidiales as a biotechnological resource has increasingly been recognized.…”

The Unicellular Red AlgaCyanidioschyzon merolae—The Simplest Model of a Photosynthetic Eukaryote

“…However, it later turned out that the selection of transformants by the URA Cm-Gs marker resulted in the multicopy insertion of the marker and transgene at high frequencies accompanied by recombination events at the targeted locus. The number of copies varies among transformed clones ( Fujiwara et al 2013 , Fujiwara et al 2019 ). By contrast, the selection of transformants by the URA marker ( C. merolae wild-type gene) resulted in the efficient single-copy insertion of the marker and transgene ( Fujiwara et al 2013 , Fujiwara et al 2019 ).…”

“…The number of copies varies among transformed clones ( Fujiwara et al 2013 , Fujiwara et al 2019 ). By contrast, the selection of transformants by the URA marker ( C. merolae wild-type gene) resulted in the efficient single-copy insertion of the marker and transgene ( Fujiwara et al 2013 , Fujiwara et al 2019 ). Although the reason for the multiple insertions of URA Cm-Gs into the genome is unclear at this point, the activity of URA Cm-Gs is likely lower than that of normal URA; hence, only clones in which URA Cm-Gs and a transgene are multiplicated can grow and form colonies on a uracil-depleted medium.…”

Grand Challenges in Microalgae Domestication

“…In Red Algae, Galdieria sulphuraria is considered for its cultivation at high temperature and acidity. Its small genome has been known for nearly two decades (Barbier et al, 2005 ), and genetic engineering is possible (Fujiwara et al, 2019 ).…”