Plants and algae require light for photosynthesis, but absorption of too much light can lead to photo‐oxidative damage to the photosynthetic apparatus and sustained decreases in the efficiency and ...rate of photosynthesis (photoinhibition). Light stress can adversely affect growth and viability, necessitating that photosynthetic organisms acclimate to different environmental conditions in order to alleviate the detrimental effects of excess light. The model unicellular green alga, Chlamydomonas reinhardtii, employs diverse strategies of regulation and photoprotection to avoid, minimize, and repair photo‐oxidative damage in stressful light conditions, allowing for acclimation to different and changing environments.
Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis ...was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.
Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in ...nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ
C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF,
and
, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy.
Esterases have emerged as important biocatalysts for enzyme-based polyester recycling of poly(ethylene terephthalate) (PET) to terephthalic acid (TPA) and ethylene glycol (EG). Here, we present ...process modeling, techno-economic, life-cycle, and socioeconomic impact analyses for an enzymatic PET depolymerization-based recycling process, which we compare with virgin TPA manufacturing. We predict that enzymatically recycled TPA (rTPA) can be cost-competitive and highlight key areas to achieve this. In addition to favorable long-term socioeconomic benefits, rTPA can reduce total supply chain energy use by 69%–83% and greenhouse gas emissions by 17%–43% per kg of TPA. An economy-wide assessment for the US estimates that the TPA recycling process can reduce environmental impacts by up to 95% while generating up to 45% more socioeconomic benefits, also relative to virgin TPA production. Sensitivity analyses highlight impactful research opportunities to pursue toward realizing biological PET recycling and upcycling.
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•Enzymes expand PET-recycling options beyond chemo-catalytic and thermal methods•Process analyses reveal key economic drivers for enzymatic PET recycling•Enzymatic PET recycling can be cost-competitive with virgin PET manufacturing costs•Circularity of the PET economy offers sustainability and socioeconomic benefits
Chemical recycling and upcycling of plastics will be critical technologies to address the plastic pollution challenge. Given multiple process options for recycling plastics, rigorous process analysis is necessary to identify challenges that must be overcome for a technology to reach an industrial scale. For PET recycling, several chemical recycling strategies have been proposed and, in some cases—chemo-catalytic and thermal approaches—are being scaled up. Given that PET exhibits labile ester bonds that are also common in natural biological systems, the research community is vigorously pursuing the engineering of esterase enzymes to depolymerize PET. This study applies process analysis to highlight drivers that the community can focus on to accelerate the development of a biological PET depolymerization process and also provides a basis to compare current and future enzyme-based approaches for PET-recycling to chemo-catalytic and thermal methods.
This study presents a comprehensive process, economic, environmental, and socioeconomic analysis of the enzymatic recycling of poly(ethylene terephthalate), which is the most widely used synthetic polyester. The analyses predict that PET deconstruction using enzymes can achieve cost parity with terephthalic acid manufacturing as well as substantial reductions in both supply chain energy use and greenhouse gas emissions relative to virgin polyester manufacturing. This study also highlights key research areas for further impactful development of biocatalysis-enabled plastics recycling.
Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer ...glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
Light harvesting complex stress-related 3 (LHCSR3) is the protein essential for photoprotective excess energy dissipation (non-photochemical quenching, NPQ) in the model green alga Chlamydomonas ...reinhardtii. Activation of NPQ requires low pH in the thylakoid lumen, which is induced in excess light conditions and sensed by lumen-exposed acidic residues. In this work we have used site-specific mutagenesis in vivo and in vitro for identification of the residues in LHCSR3 that are responsible for sensing lumen pH. Lumen-exposed protonatable residues, aspartate and glutamate, were mutated to asparagine and glutamine, respectively. By expression in a mutant lacking all LHCSR isoforms, residues Asp117, Glu221, and Glu224 were shown to be essential for LHCSR3-dependent NPQ induction in C. reinhardtii. Analysis of recombinant proteins carrying the same mutations refolded in vitro with pigments showed that the capacity of responding to low pH by decreasing the fluorescence lifetime, present in the wild-type protein, was lost. Consistent with a role in pH sensing, the mutations led to a substantial reduction in binding the NPQ inhibitor dicyclohexylcarbodiimide.
Plants respond to shade-modulated light signals via phytochrome (phy)-induced adaptive changes, termed shade avoidance. To examine the roles of Phytochrome-lnteracting basic helix-loop-helix Factors, ...PIF1, 3, 4, and 5, in relaying such signals to the transcriptional network, we compared the shade-responsive transcriptome profiles of wild-type and quadruple pif (pifq) mutants. We identify a subset of genes, enriched in transcription factor-encoding loci, that respond rapidly to shade, in a PIF-dependent manner, and contain promoter G-box motifs, known to bind PIFs. These genes are potential direct targets of phy-PIF signaling that regulate the primary downstream transcriptional circuitry. A second subset of PIF-dependent, early response genes, lacking G-box motifs, are enriched for auxin-responsive loci, and are thus potentially indirect targets of phy-PIF signaling, mediating the rapid cell expansion induced by shade. Comparing deetiolation-and shade-responsive transcriptomes identifies another subset of G-box-containing genes that reciprocally display rapid repression and induction in response to light and shade signals. These data define a core set of transcriptional and hormonal processes that appear to be dynamically poised to react rapidly to light-environment changes via perturbations in the mutually antagonistic actions of the phys and PIFs. Comparing the responsiveness of the pifq and triple pif mutants to light and shade confirms that the PIFs act with overlapping redundancy on seedling morphogenesis and transcriptional regulation but that each PIF contributes differentially to these responses.
Summary
Plants must switch rapidly between light harvesting and photoprotection in response to environmental fluctuations in light intensity. This switch can lead to losses in absorbed energy usage, ...as photoprotective energy dissipation mechanisms can take minutes to hours to fully relax. One possible way to improve photosynthesis is to engineer these energy dissipation mechanisms (measured as non‐photochemical quenching of chlorophyll a fluorescence, NPQ) to induce and relax more quickly, resulting in smaller losses under dynamic light conditions. Previous studies aimed at understanding the enzymes involved in the regulation of NPQ have relied primarily on labor‐intensive and time‐consuming generation of stable transgenic lines and mutant populations – approaches limited to organisms amenable to genetic manipulation and mapping. To enable rapid functional testing of NPQ‐related genes from diverse organisms, we performed Agrobacterium tumefaciens‐mediated transient expression assays in Nicotiana benthamiana to test if NPQ kinetics could be modified in fully expanded leaves. By expressing Arabidopsis thaliana genes known to be involved in NPQ, we confirmed the viability of this method for studying dynamic photosynthetic processes. Subsequently, we used naturally occurring variation in photosystem II subunit S, a modulator of NPQ in plants, to explore how differences in amino acid sequence affect NPQ capacity and kinetics. Finally, we functionally characterized four predicted carotenoid biosynthesis genes from the marine algae Nannochloropsis oceanica and Thalassiosira pseudonana and examined the effect of their expression on NPQ in N. benthamiana. This method offers a powerful alternative to traditional gene characterization methods by providing a fast and easy platform for assessing gene function in planta.
Significance Statement
Transient expression of genes in Nicotiana benthamiana has become a method of choice to quickly assess gene function in planta. Here we illustrate the potential of transient assays in N. benthamiana to provide a medium‐throughput platform for assessing gene function with regards to photosynthetic processes. A promising system is described for studying the regulation of NPQ dynamics toward minimizing energy losses and improving photosynthetic yields.
Abstract As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a ...generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.
Numerous mutations and covalent modifications of the highly abundant, long‐lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The ...nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native‐state polymers are commonly found in opaque lenses. The βγ‐crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV‐B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD‐crystallin. Such substitutions may represent a model of UV‐induced photodamage—introduction of a charged group into the hydrophobic core generating “denaturation from within.” The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold—W42E and W130E—yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB‐crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N‐terminal vs C‐terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β‐sheets. These features are most consistent with domain‐swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.
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