13 research outputs found
Systems Thinking in Science Education and Outreach toward a Sustainable Future
Systems thinking, interdisciplinary research projects, and creative problem solving are ways to frame modern chemistry curricula to inspire the next generation of scientists, engineers, teachers, and citizens to use their skills and education to create a sustainable future. By integrating planetary boundaries, green chemistry, and the UN sustainable development goals, we use a systems thinking approach in undergraduate education and outreach to a range of diverse populations to drive discussion, exploration of scientific principles, and teach students how they can use chemistry to solve the distinctive challenges of the anthropocene. Interdisciplinary research projects employ critical thinking, problem solving, and creativity as part of the scientific method. Translating undergraduate research in nanotechnology, renewable energy, and sustainability into lesson plans and engaging in outreach to diverse populations promotes equity in science education and encourages underrepresented groups to seek careers in a scientific field. Community college students act as role models in outreach as they teach chemistry using a systems thinking approach, connect sustainability to STEM careers that can make a positive impact on local communities, and show underrepresented groups that they are needed in these disciplines. Engaging, interdisciplinary laboratories used in outreach, such as the synthesis of algae biodiesel, making paints from natural resources, sustainable agriculture and engineering, and DNA origami, access all aspects of systems thinking. Using systems thinking as a framework in science education and outreach teaches students the significance and relevance of chemistry while creating a platform for women and underrepresented groups to learn how important their representation is to contribute to a sustainable, equitable future
Investigating fatty acid biosynthesis within the algal chloroplast using Chlamydomonas reinhardtii as a model
As finite petroleum reserves run their course and combustion-related COβ emissions rise concerns about global warming, humanity is faced with the challenge of finding new sources of energy that are carbon-neutral, renewable and sustainable to meet the growing demand. Photosynthetic organisms convert solar energy and COβ directly into metabolic products that can serve as fungible biofuels. Microalgae are particularly attractive as a biodiesel feedstock, as they produce oil in high yields, grow at fast rates in habitats not suitable for conventional agriculture, and do not compete with the food supply. However, oil accumulation occurs under environmental stress, which compromises biomass productivity, and algal fatty acids are not ideal for biodiesel quality. The ability to manipulate algal fatty acid biosynthesis would thus be a significant stride towards developing algae as a biodiesel feedstock. In fatty acid biosynthesis within an algal chloroplast, an acyl carrier protein (ACP) tethers the growing fatty acid as it undergoes iterative cycles of elongation, and a thioesterase (TE) domain catalyzes the release of a mature fatty acid from the ACP. As plant TEs specific for certain chain length fatty acids have altered the fatty acid profile of transgenic plants and bacteria, this has emerged as a promising strategy to modify algal fatty acid content to fashion an optimized biodiesel feedstock. The work outlined in this thesis aims to investigate intermolecular interactions in algal fatty acid biosynthesis to facilitate engineering. A novel strategy was employed in which a chemical probe inspired by the enzymatic activity of the algal TE was synthesized, attached to the algal ACP chemoenzymatically, and used to trap algal ACP-TE interactions in vitro. No protein- protein interactions were detected between plant TEs and the algal ACP in vitro, and thus plant TEs did not elicit the desired phenotype when engineered into the algal chloroplast. Using protein-protein interactions as a means to control product identity may shift the paradigm towards rationally designed engineering approaches to optimize algae as a bioenergy source. Renewable energy outreach and education has been an indispensable facet of this work to generate awareness and instill passion for sustainable energy in our future scientist
Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions
Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions
<div><p>Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga <em>Chlamydomonas reinhardtii</em> (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar <em>in silico</em> binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate <em>in vitro</em> that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated <em>in vivo</em>, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the <em>C. reinhardtii</em> chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.</p> </div
Thioesterase modeling, docking of ACP-TE protein-protein interactions, and blind substrate docking of fatty acid substrate to <i>C. reinhardtii</i> TE.
<p>(<i>A</i>) Docking of CrTE (grey) with Cr-cACP (blue) showing a <10 Γ
distance between Cr-cACP Ser<sub>43</sub> (orange) and the active site Cys<sub>306</sub>His<sub>270</sub>Asn<sub>268</sub> triad (magenta) of CrTE. (<i>B</i>) Docked complex of CrTE (grey) and ChTE (yellow) showing similar binding modes of Cr-cACP to both plant and algal thioesterases. (<i>C</i>) Surface representation of blind docking of stearyl-4β²-phosphopantetheine to CrTE showing the thioester bond of the substrate in close proximity to the TE active site and stearate in the binding tunnel of CrTE.</p
Activity-based crosslinking as a determinant of functional interaction with <i>C. reinhardtii</i> cACP.
<p>(<i>A</i>) Activity-based substrate mimics used in crosslinking assay; (<i>B</i>) SDS-PAGE gel showing Cr-cACP/CrTE interaction. <i>Apo</i>-Cr-cACP was modified with pantetheine analogue <b>2</b> or <b>3</b> to generate the corresponding <i>crypto</i>-Cr-cACPs (<b>4</b> and <b>5</b>). <i>Crypto</i>-Cr-cACPs were incubated with CrTE and crosslinking was visualized by SDS-PAGE analysis following FLAG affinity purification of the CrACP/TE complex. The band observed at 50 kDa is the FLAG-tagged CrTE and the band detected at βΌ75 kDa is the ACP/TE complex. During overnight incubation at 37Β°C, reduced CrTE shows spontaneous oxidation (bands at βΌ100 kDa). Pantetheine analogues used in crosslinking reactions are noted under the gel. A red arrow illustrates the ACP-TE complex in (<i>B</i>) and (<i>C</i>). (<i>C</i>) <i>Apo</i>-Cr-cACP was reacted with either Ξ±-bromopalmitic pantetheine probe <b>3</b> or Ξ±-bromohexyl pantetheine probe <b>6</b> to generate <i>crypto</i>-Cr-cACPs with C16 and C6 acyl chains attached, respectively. Each <i>crypto</i>- Cr-cACP was incubated with CrTE and monitored for extent of crosslinking by SDS-PAGE analysis of Ni-NTA-purified reactions. (<i>D</i>) <i>Apo</i>-Cr-cACP was modified with Ξ±-bromopalmitic pantetheine analogue <b>3</b> to form <i>crypto</i>-Cr-cACP, which was incubated with UcTE (left 2 lanes) or ChTE (right 2 lanes). Crosslinking was measured by SDS-PAGE analysis following FLAG affinity purification. The bands at βΌ50 kDa are plant TEs.</p
Thioesterase activity assay.
<p>Activity of plant and algal thioesterases and porcine pancreas type II lipase were determined by monitoring the hydrolysis of para-nitrophenylhexanoate for 16 hours at 30Β°C. (<i>A</i>) pH 7, TEs expressed in <i>E. coli</i>; (<i>B</i>) pH 8, TEs expressed in <i>E. coli</i>; (<i>C</i>) pH 7, TEs expressed in <i>C. reinhardtii</i>; (<i>D</i>) pH 8, TEs expressed in <i>C. reinhardtii</i>.</p
Schematic of activity-based crosslinking between CrACP and TEs.
<p>(<i>A</i>) <i>Apo</i>-CrACP is formed by treating <i>holo</i>-CrACP with ACP hydrolase from <i>P. aeruginosa </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042949#pone.0042949-Murugan1" target="_blank">[36]</a>, removing the pantetheine moiety from the conserved serine of CrACP. Presence of <i>apo</i>-CrACP is confirmed using a one-pot fluorescent labeling method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042949#pone.0042949-Worthington3" target="_blank">[37]</a>, detected by visualization of a resulting SDS-PAGE gel at 365 nm. (+) formation of fluorescent <i>crypto</i>-CrACP; (β) control reaction in which fluorescent pantetheine analogue <b>1</b> was omitted. (<i>B</i>) Activity-based crosslinking scheme. <i>Apo</i>-CrACP is incubated with <b>2</b> or <b>3</b>, Sfp, ATP, CoA-A, CoA-D, and CoA-E to generate the corresponding <i>crypto</i>-CrACPs <b>4</b> and <b>5</b>. Upon incubation of <i>crypto</i>-CrACP with TE, protein-protein interactions trigger a site-specific covalent crosslinking reaction with the chloroacrylamide in <b>4</b> or the Ξ±-bromoamide in <b>5</b>, forming an ACP-TE crosslinked complex. (<i>C</i>) Predicted enzymatic mechanism of the hydrolytic release of a fatty acid by CrTE using a Cys-Asn-His catalytic triad. (<i>D</i>) Mechanism of irreversible crosslink between TE and <i>crypto</i>-CrACP containing a reactive bromide on the carbon Ξ± to the site of nucleophilic attack by the TE.</p
Fatty acid analysis of <i>C. reinhardtii</i> strains expressing thioesterases.
<p>Fatty acid composition of Cr strains was determined by GC/MS analysis and comparison to authentic standards. Peak areas were integrated and compared to an external standard for quantification. Bar graphs denote abundances of (<i>A</i>) Myristic acid (14:0), (<i>B</i>) Palmitic acid (16:0), and (<i>C</i>) Oleic acid (18:1), and labels on the Y-axis correspond to the percentages of these fatty acids of the total fatty acid content. (<i>D</i>) Full GC/MS chromatograms of Cr strains expressing CrTE (red), UcTE (Blue) and wildtype CrTE (black). Three separate cultures of each strain were analyzed for fatty acid content and composition, and data were recorded and averaged with a mean deviation of 7% in each experiment. Statistical analyses were performed using SPSS (v13.0), and for all data analysis, a <i>p</i>-value<0.5 was considered statistically significant.</p