Kang Kalisa, Santo Évellin do Espirito, Diaz Crisandra Jade, Oliver Aaron, Saxton Lisa, May Lauren, Mayfield Stephen, Molino João Vitor Dutra
Department of Molecular Biology, School of Biological Sciences, University of California San Diego, La Jolla, California, United States of America.
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil.
PLoS One. 2025 Apr 16;20(4):e0321071. doi: 10.1371/journal.pone.0321071. eCollection 2025.
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.
不确定衣藻（Chlamydomonas incerta）是模式绿藻莱茵衣藻（Chlamydomonas reinhardtii）的亲缘关系很近的遗传近亲，作为重组蛋白表达的宿主具有显著潜力。由于不确定衣藻和莱茵衣藻之间存在密切的遗传关系，该物种为增进我们对光合生物的理解提供了一个额外的参考点，也为生物技术应用提供了一个潜在的新候选者。本研究调查了不确定衣藻表达三种重组蛋白的能力：荧光蛋白mCherry、半纤维素降解酶木聚糖酶和塑料降解酶PHL7。我们还研究了将蛋白表达靶向该藻类各种细胞区室的能力，包括细胞质、分泌途径、细胞质膜和细胞壁。与莱茵衣藻直接比较时，不确定衣藻表现出独特但显著的重组蛋白生产能力。用编码mCherry的载体进行细胞转化显示，不确定衣藻产生的荧光水平比莱茵衣藻高约3.5倍，免疫印迹强度增加了3.7倍。对于木聚糖酶的表达和分泌，不确定衣藻和莱茵衣藻都表现出相似的分泌能力和酶活性，木聚糖降解率相当，突出了木聚糖酶在微藻中表达的工业适用性。最后，通过聚酯聚氨酯悬浮液Impranil® DLN的体外降解证明，不确定衣藻显示出与莱茵衣藻相当的PHL7活性水平。最后，我们还探索了细胞融合在不确定衣藻和莱茵衣藻之间产生遗传杂种的潜力，以此作为增强表型多样性和增加遗传变异的一种手段。我们能够产生遗传融合体，它可以将重组蛋白基因以及相关的选择标记基因交换到重组后代中。这些发现强调了不确定衣藻作为重组蛋白生产的强大平台以及作为深入了解微藻生物学的有力工具的潜力。
