Biology subjects

Diaz, C. J.

Publications and source records attributed to Diaz, C. J..

7 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

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↗

bioRxiv2024

Efficient secretion of a plastic degrading enzyme from the green algae Chlamydomonas reinhardtii

Plastic pollution has become a global crisis, with microplastics contaminating every environment on the planet, including our food, water, and even our bodies. In response, there is a growing interest in developing plastics that biodegrade naturally, thus avoiding the creation of persistent microplastics. As a mechanism to increase the rate of polyester plastic degradation, we examined the potential of using the green microalga Chlamydomonas reinhardtii for the expression and secretion of PHL7, an enzyme that breaks down post-consumer polyethylene terephthalate (PET) plastics. We engineered C. reinhardtii to secrete active PHL7 enzyme and selected strains showing robust expression, by using agar plates containing a polyester polyurethane (PU) dispersion as an efficient screening tool. This method demonstrated the enzymes efficacy in degrading ester bond-containing plastics, such as PET and bio-based polyurethanes, and highlights the potential for microalgae to be implemented in environmental biotechnology. The effectiveness of algal-expressed PHL7 in degrading plastics was shown by incubating PET with the supernatant from engineered strains, resulting in substantial plastic degradation, confirmed by mass spectrometry analysis of terephthalic acid (TPA) formation from PET. Our findings demonstrate the feasibility of polyester plastic recycling using microalgae to produce plastic-degrading enzymes. This eco-friendly approach can support global efforts toward eliminating plastic in our environment, and aligns with the pursuit of low-carbon materials, as these engineered algae can also produce plastic monomer precursors. Finally, this data demonstrates C. reinhardtii capabilities for recombinant enzyme production and secretion, offering a "green" alternative to traditional industrial enzyme production methods. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/619606v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@108e29dorg.highwire.dtl.DTLVardef@55ceaborg.highwire.dtl.DTLVardef@173793eorg.highwire.dtl.DTLVardef@1d14c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Description of a novel extremophile green algae, Chlamydomonas pacifica, and its potential as a biotechnology host

We present the comprehensive characterization of a newly identified microalga, Chlamydomonas pacifica, originally isolated from a soil sample in San Diego, CA, USA. This species showcases remarkable biological versatility, including a broad pH range tolerance (6-11.5), high thermal tolerance (up to 42{degrees}C), and salinity resilience (up to 2% NaCl). Its amenability to genetic manipulation and sexual reproduction via mating, particularly between the two opposing strains CC-5697 & CC-5699, now publicly available through the Chlamydomonas Resource Center, underscores its potential as a biotechnological chassis. The biological assessment of C. pacifica revealed versatile metabolic capabilities, including diverse nitrogen assimilation capability, motility and phototaxis. Genomic and transcriptomic analyses identified 17,829 genes within a 121 Mb genome, featuring a GC content of 61%. The codon usage of C. pacifica closely mirrors that of C. reinhardtii, indicating a conserved genetic architecture that supports a trend in codon preference with minor variations. Phylogenetic analyses position C. pacifica within the core-Reinhardtinia clade yet distinct from known Volvocales species. The lipidomic data revealed an abundance of triacylglycerols (TAGs), promising for biofuel applications and lipids for health-related benefits. Our investigation lays the groundwork for exploiting C. pacifica in biotechnological applications, from biofuel generation to synthesizing biodegradable plastics, positioning it as a versatile host for future bioengineering endeavors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/611117v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1ead226org.highwire.dtl.DTLVardef@18af631org.highwire.dtl.DTLVardef@291c2borg.highwire.dtl.DTLVardef@f860ac_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

molecular biology↗

bioRxiv2024

Engineering the novel extremophile alga Chlamydomonas pacifica for high lipid and high starch production as a path to developing commercially relevant strains

Microalgae offer a compelling platform for the production of commodity products, due to their superior photosynthetic efficiency, adaptability to non-arable lands and non-potable water, and their capacity to produce a versatile array of bioproducts, including biofuels and biomaterials. However, the scalability of microalgae as a bioresource has been hindered by challenges such as costly biomass production related to vulnerability to pond crashes during large-scale cultivation. This study presents a pipeline for the genetic engineering and pilot-scale production of biodiesel and thermoplastic polyurethane precursors in the extremophile species Chlamydomonas pacifica. This extremophile microalga exhibits exceptional resilience to high pH, high salinity, and elevated temperatures. Initially, we evolved this strain to also have a high tolerance to high light intensity through mutagenesis, breeding, and selection. Subsequently, we genetically engineered C. pacifica to produce high levels of lipids and starch without compromising growth. We demonstrated the scalability of these engineered strains by cultivating them in pilot-scale raceway ponds and converting the resulting biomass into biodiesel and thermoplastic polyurethanes. This study showcases the complete cycle of transforming a newly discovered species into a commercially relevant commodity production strain. This research underscores the potential of extremophile algae, including C. pacifica, as a key species for the burgeoning sustainable bioeconomy, offering a viable path forward in mitigating environmental challenges and supporting global bioproduct demands. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/604193v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@84948eorg.highwire.dtl.DTLVardef@352616org.highwire.dtl.DTLVardef@1577082org.highwire.dtl.DTLVardef@19b8644_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae

Heritable variation in a genes expression arises from mutations impacting cis- and trans-acting components of its regulatory network, with expression variation often derived from trans-regulatory mutations within species. Here, we investigate how trans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans-regulatory effects on expression of the same focal gene in Saccharomyces cerevisiae. Relative to 1766 mutations without effects on expression of this focal gene, we found that these trans-regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of the trans-regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism and signal transduction. Finally, we find that the genomic distribution of these new regulatory mutations significantly overlaps with the genomic distribution of trans-regulatory variants segregating within S. cerevisiae.

genetics↗