The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal β-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.
Bibliographical noteFunding Information:
KJL acknowledges baseline research funding provided by King Abdullah University of Science & Technology. KAUST team is grateful to Paulo C. Aurelio of KAUST Core Labs Lab Equipment Maintenance (LEM) team for install and maintenance of the Algem photobioreactors and flow cytometer. PJL acknowledges financial support from Xylem, Inc. and ASU Lightworks; and would also like to acknowledge Keirsten Allen for her technical support. The authors wish to express their gratitude to Dr. Martha Stark for her invaluable assistance with the transformation protocol. The authors thank Dr. Sebastian Overmans for creating Algem bioreactor profiles from provided data. The authors thank Dr. Thomas Baier for invaluable discussions around the use of CHYB in C. reinhardtii and sharing these insights with us.
© 2023 The Author(s)
- Microalgae. red algae. ketocarotenoids. Cyanidiophyceae. polyextremophiles. astaxanthin
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Biomedical Engineering