Engineering the green algae Chlamydomonas incerta for recombinant protein production

Kang, Kalisa; Santo, Évellin do Espirito; Diaz, Crisandra Jade; Oliver, Aaron; Saxton, Lisa; May, Lauren; Mayfield, Stephen; Molino, João Vitor Dutra

doi:10.1371/journal.pone.0321071

Engineering the green algae Chlamydomonas incerta for recombinant protein production

Journal Article · Wed Apr 16 00:00:00 EDT 2025 · PLoS ONE

DOI:https://doi.org/10.1371/journal.pone.0321071· OSTI ID:2569324

^[1]; Santo, Évellin do Espirito ^[2]; Diaz, Crisandra Jade ^[1]; ^[1]; Saxton, Lisa ^[1]; May, Lauren ^[3]; Mayfield, Stephen ^[4]; ^[1]

Univ. of California, San Diego, CA (United States)
Univ. of California, San Diego, CA (United States); Univ. of Sao Paulo (Brazil)
California Polytechnic State Univ. (CalPoly), San Luis Obispo, CA (United States)
Univ. of California, San Diego, CA (United States); Algenesis Materials, San Diego, CA (United States)

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of California, San Diego, CA (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Grant/Contract Number:: EE0009671; EE0009671

OSTI ID:: 2569324

Journal Information:: PLoS ONE, Journal Name: PLoS ONE Journal Issue: 4 Vol. 20; ISSN 1932-6203

Publisher:: Public Library of ScienceCopyright Statement

Country of Publication:: United States

Language:: English

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