Abstract
Key messageWe have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism.Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae—an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi:10.1007/s00709-015-0936-9). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae—a new model organism.
Highlights
Cyanidioschyzon merolae is an extremophilic red microalga that dwells in moderately high temperatures (40– 56 °C) and highly acidic environments (Ciniglia et al 2004)
The ability to express exogenous proteins from the plastid genome has attracted a considerable attention (Maliga 2004; Daniell et al 2009; Bock 2007) due to its capacity to accumulate large quantities of foreign proteins. This propensity is advantageous for biotechnological applications (Meyers et al 2010), as plastid protein expression may reach up to 70% of leaf or cell protein content (Daniell et al 2009)
C. merolae is known to grow in high ambient temperature of 42 °C and higher but it tolerates, reasonably well, temperature as low as 37 °C, it might be well suited for human proteins production, requiring the precise human body temperature for maturation
Summary
Cyanidioschyzon merolae is an extremophilic red microalga that dwells in moderately high temperatures (40– 56 °C) and highly acidic (between pH 0.2–4) environments (Ciniglia et al 2004). Genomes of all three of these organelles were fully sequenced (Matsuzaki et al 2004; Ohta et al 1998, 2003). This simplicity can be utilized for genetic engineering and production of transgenic organisms, especially via site-directed mutagenesis, interesting in physiological and structural studies of whole cells as well as intracellular organelles i.e. chloroplasts. All of the nuclear transformation protocols relayed on introducing the wild-type URA5.3 (CMK046C) gene into C. merolae a spontaneous mutant (M4 strain), deficient in the UMP synthase gene (due to a frame-shift mutation) exhibiting uracil-dependent growth. The combination of the wild-type URA5.3 with uracil-deficient mutant sets up a limitation
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