Biology subjects

Molino, J. V. D.

Publications and source records attributed to Molino, J. V. D..

4 recordsLinked to original sources

bioRxiv2025

Identification and Overexpression of Endogenous Transcription Factors to Enhance Lipid Accumulation in the Commercially Relevant Species Chlamydomonas pacifica

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wildtype. However, CpaPSR1 increased lipid accumulation compared to wildtype in normal media despite causing no effect under nitrogen depravation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/651737v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@8ab995org.highwire.dtl.DTLVardef@19dff1forg.highwire.dtl.DTLVardef@1d865aborg.highwire.dtl.DTLVardef@1fafd12_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com.

systems biology↗

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↗

bioRxiv2021

Biohybrid Microswimmers Against Bacterial Infections

Biohybrid microswimmers exploit the natural abilities of motile microorganisms e.g. in releasing cargo on-demand with high spatial and temporal control. However, using such engineered swarms to deliver antibiotics addressing bacterial infections has not yet been realized. In the present study, a design strategy for biohybrid microswimmers is reported, which features the covalent attachment of antibiotics to the motile green algae Chlamydomonas reinhardtii via a photo-cleavable linker. The surface engineering of the algae does not rely on genetic manipulations, proceeds with high efficiency, does not impair the viability or phototactic ability of microalgae, and allows for caging of the antibiotic on the surface for subsequent release via external stimuli. Two different antibiotic classes have been separately utilized, which result in activity against both gram-positive and gram-negative strains. Guiding the biohybrid microswimmers by an external beacon, and on-demand delivery of the drugs by light with high spatial and temporal control, allowed for strong inhibition of bacterial growth in vivo. This efficient strategy could potentially allow for the selective treatment of bacterial infections by engineered algal microrobots with high precision in space and time. Overall, this work presents an operationally simple production of biohybrid microswimmers loaded with antibiotic cargo to combat bacterial infections precisely delivered in three-dimensional space.

synthetic biology↗

bioRxiv2021

Development of a Cell Surface Display System in Chlamydomonas reinhardtii

Cell-surface display systems are biotechnological techniques used to express heterologous proteins on the cell surface. Their application depends directly on the cell system used, as well as on the anchoring point for the surface displayed protein. To meet most application demands an inexpensive, safe, and scalable production platform, that reduces the economic barriers for large scale use is needed. Towards this goal, we screened three possible cell surface anchoring points in the green algae Chlamydomonas by fusing mVenus to prospective anchors moieties. The vectors harboring mVenus:anchor were screened for mVenus fluorescence and tested for cellular localization by confocal laser scanning microscopy. This strategy allowed the identification of two functional anchors, one for the cytoplasmic membrane using the MAW8 GPI-anchor signal, and one for the cell wall using the GP1 protein. We also exploited GP1 chemical and biological traits to release the fused proteins efficiently during cell wall shedding. Our work provides a foundation for surface engineering of C reinhardtii supporting both cell biology studies and biotechnology applications.

synthetic biology↗