Biology subjects

do Espirito Santo, E.

Publications and source records attributed to do Espirito Santo, E..

4 recordsLinked to original sources

bioRxiv2025

Sustainable production of plastic-degrading enzymes in Chlamydomonas pacifica

The discovery of a new extremophile alga, Chlamydomonas pacifica, provides an opportunity to expand on heterologous protein expression beyond the traditional Chlamydomonas reinhardtii. C. pacifica is a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown that C. reinhardtii can secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in the C. pacifica algae biomass. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/654053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@10065a3org.highwire.dtl.DTLVardef@110c456org.highwire.dtl.DTLVardef@1c9efc6org.highwire.dtl.DTLVardef@9ceea1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Low-Cost Screening of Algae for Extreme Tolerance to pH, Temperature, Salinity, and Light

Bioprospecting algae strains with tolerance to extreme conditions such as pH, temperature, salinity, and light is crucial for advancing biotechnology and environmental applications. However, traditional screening methods often involve significant costs and labor, restricting their accessibility and practical use. In this study, we developed and validated low-cost, high-throughput screening techniques, predominantly employing agar plates and liquid culture assays, to effectively differentiate tolerance levels among various algae strains. The methodologies were optimized using the model microalga Chlamydomonas reinhardtii and its closely related species Chlamydomonas incerta and the recently discovered extremophilic Chlamydomonas pacifica. We systematically evaluated the algae for tolerance to extremes by establishing precise gradients of pH (acidic to alkaline conditions), salinity (0 to 5 M NaCl), temperature (34-42{degrees}C), and light intensity (40 to 2977 E{middle dot}m-{superscript 2}{middle dot}s-{superscript 1}). Our results demonstrated that these cost-effective, agar plate-based methods effectively distinguished algae strains exhibiting superior tolerance to extreme environmental conditions. These screening techniques not only provided clear differentiation among the closely related strains but also delivered reproducible outcomes suitable for scaling up to larger bioprospecting efforts. Furthermore, the affordability and simplicity of these methods facilitate their implementation in resource-limited laboratories, thereby broadening participation in algae bioprospecting endeavors. This study highlights the potential of low-cost, accessible screening techniques to significantly enhance the discovery and characterization of algal strains with extreme traits. Ultimately, these methods support the development of robust algae-based resources, driving innovation in diverse industrial processes and environmental solutions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/653396v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@6b179borg.highwire.dtl.DTLVardef@dadbfforg.highwire.dtl.DTLVardef@a1750dorg.highwire.dtl.DTLVardef@baffc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Engineering microalgal cell wall-anchored proteins using GP1 PPSPX motifs and releasing with intein-mediated fusion

Harnessing and controlling the localization of recombinant proteins is essential for advancing synthetic biology, industrial biotechnology, and drug delivery. This study presents a dual system for protein anchoring and controlled release in Chlamydomonas reinhardtii. Using truncated variants of the GP1 glycoprotein fused to the plastic-degrading enzyme PHL7, we identified the PPSPX motif as critical for anchoring proteins to the cell wall. Constructs with increased PPSPX content exhibited reduced secretion but enhanced anchoring, revealing key anchor-signal sites within GP1. To enable controlled release, we incorporated a pH-sensitive intein from derived from Mycobacterium tuberculosis RecA. Under acidic conditions (pH 5.5), this intein efficiently cleaved and released mCherry and PHL7 from the GP1 anchor. Fluorescence kinetics demonstrated significant mCherry release from the mCherry-intein-GP1 construct within 4 hours at pH 5.5, while release was minimal at pH 8.0 and negligible for the mCherry-GP1 control. Western blot analysis confirmed efficient cleavage, showing a lower band corresponding to free mCherry at pH 5.5 and no release at pH 8.0. This anchor-release strategy integrates glycomodules with pH-sensitive inteins to enable precise spatial and temporal protein control. The system offers broad utility for targeted drug delivery, environmental biosensing, and biocatalyst deployment. Overall, we establish a versatile framework for optimizing protein localization and environmentally triggered release in C. reinhardtii, with broad implications for proteomics, biofilm engineering, and scalable therapeutic delivery systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/634604v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@8bdc2org.highwire.dtl.DTLVardef@1e213c7org.highwire.dtl.DTLVardef@1808559org.highwire.dtl.DTLVardef@b2c1e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production

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. incertas 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(R) 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. incertas potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/618925v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@173d398org.highwire.dtl.DTLVardef@148ad36org.highwire.dtl.DTLVardef@63b5e9org.highwire.dtl.DTLVardef@3be081_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗