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Aliphatic polyamides, or nylons, are typically highly crystalline and thermally robust polymers used in high-performance applications. Nylon 6, a high-ceiling-temperature (HCT) polyamide from ε-caprolactam, lacks expedient chemical recyclability, while low-ceiling temperature (LCT) nylon 4 from pyrrolidone exhibits complete chemical recyclability, but it is thermally unstable and not melt-processable. Here, we introduce a hybrid nylon, nylon 4/6, based on a bicyclic lactam composed of both HCT ε-caprolactam and LCT pyrrolidone motifs in a hybridized offspring structure. Hybrid nylon 4/6 overcomes trade-offs in (de)polymerizability and performance properties of the parent nylons, exhibiting both excellent polymerization and facile depolymerization characteristics. This stereoregular polyamide forms nanocrystalline domains, allowing optical clarity and high thermal stability, however, without displaying a melting transition before decomposition. Of a series of statistical copolymers comprising nylon 4/6 and nylon 4, a 50/50 copolymer achieves the greatest synergy in both reactivity and polymer properties of each homopolymer, offering an amorphous nylon with favorable properties, including optical clarity, a high glass transition temperature, melt processability, and full chemical recyclability.

Pd/C and Pt/C show high activity for hydrogen-free reductive catalytic fractionation compared to Ru/C and Ni/C.

Less than 10% of the plastics generated globally are recycled, while the rest are incinerated, accumulated in landfills, or leach into the environment. New technologies are emerging to chemically recycle waste plastics that are receiving tremendous interest from academia and industry. Chemists and chemical engineers need to understand the fundamentals of these technologies to design improved systems for chemical recycling and upcycling of waste plastics. In this paper, we review the entire life cycle of plastics and options for the management of plastic waste to address barriers to industrial chemical recycling and further provide perceptions on possible opportunities with such materials. Knowledge and insights to enhance plastic recycling beyond its current scale are provided. Outstanding research problems and where researchers in the field should focus their efforts in the future are also discussed.

When excess base is required to drive desired reactions, such as in lignin alkaline oxidation, Sr(OH)2 can offer a reversibly-soluble alternative to NaOH that allows simple recycle of the excess base with concomitant cost and environmental benefits.

Life cycle assessment of enzymatic poly(ethylene terephthalate) (PET) recycling highlights key challenges and opportunities for improving environmental impacts.

We combine metabolic engineering and polymer chemistry to convert cresols, a main component of biomass-derived catalytic fast pyrolysis wastewater, to methyl muconic acids that have performance-advantaged properties in nylons and plasticizers.

Hybrid processes, featuring biological conversion of lignocellulose to small molecules followed by chemo-catalytic conversion to larger molecules suitable for difficult-to-electrify transport modes, are a promising route to biomass-derived fuels in demand for climate stabilization.

Summary This perspective discusses a set of design principles for next-generation kinetically trapped, intrinsically circular polymers (iCPs) that are inherently, selectively, and expediently depolymerizable to their monomer state once their kinetic barriers of deconstruction are overcome, thereby enabling not only the ideal shortest chemical circularity but also tunable performance properties. After describing four elements of the design principles—thermodynamics and kinetics, strategies to overcome trade-offs and unify conflicting properties, predictive modeling, and supply-chain life-cycle assessment and techno-economic analysis, which are illustrated with state-of-the-art examples—it concludes with presenting key challenges and opportunities for sustainable development of iCPs.

Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium . Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

Lignin is the largest source of bio-based aromatic compounds in nature, and its valorization is essential to the sustainability of lignocellulosic biorefining. Characterizing lignin-derived compounds remains challenging due to the heterogeneity of this biopolymer. Tandem mass spectrometry is a promising tool for lignin structural analytics, as fragmentation patterns of model compounds can be extrapolated to identify characteristic moieties in complex samples. This work extended previous resonance excitation-type collision-induced dissociation (CID) methods that identified lignin oligomers containing β-O-4, β-5, and β-β bonds, to also identify characteristics of 5-5, β-1, and 4-O-5 dimers, enabled by quadrupole time-of-flight (QTOF) CID with energy-resolved mass spectrometry (ERMS). Overall, QTOF-ERMS offers in-depth structural information and could ultimately contribute to tools for high-throughput lignin dimer identification.

Bio-based compounds with unique chemical functionality can be obtained through selective transformations of plant and other non-fossil, biogenic feedstocks for the development of new polymers to displace those produced from fossil carbon feedstocks. Although substantial efforts have been invested to produce bio-based polymers that are chemically identical to and directly replace those from petroleum, a long-pursued goal is to synthesize new, sustainable, bio-based polymers that either functionally replace or exhibit performance advantages relative to incumbent polymers. Owing to anthropogenic climate change and the environmental consequences of global plastics pollution, the need to realize a bio-based materials economy at scale is critical. To that end, in this Review we describe the concept of performance-advantaged, bio-based polymers (PBPs), highlighting examples wherein superior performance is facilitated by the inherent chemical functionality of bio-based feedstocks. We focus on PBPs with C–O and C–N inter-unit chemical bonds, as these are often readily accessible from bio-based feedstocks, which are heteroatom-rich relative to petroleum-derived feedstocks. Finally, we outline guiding principles and challenges to aid progress in the development of PBPs. Bio-based polymers that exhibit superior performance relative to petroleum-based incumbents can encourage industry adoption and offset fossil carbon use. This Review introduces performance-advantaged, bio-based polymers and highlights examples wherein superior performance is facilitated by the inherent chemical functionality of bio-based feedstocks.

The development of technologies to recycle polyethylene (PE) and polypropylene (PP), globally the two most produced polymers, is critical to increase plastic circularity. Here, we show that 5 wt % cobalt supported on ZSM-5 zeolite catalyzes the solvent-free hydrogenolysis of PE and PP into propane with weight-based selectivity in the gas phase over 80 wt % after 20 h at 523 K and 40 bar H

Randomly barcoded transposon insertion sequencing (RB-TnSeq) is an efficient, multiplexed method to determine microbial gene function during growth under a selection condition of interest. This technique applies to growth, tolerance, and persistence studies in a variety of hosts, but the wealth of data generated can complicate the identification of the most critical gene targets. Experimental and analytical methods for improving the resolution of RB-TnSeq are proposed, using

Summary Reducing the carbon intensity of plastics production by sourcing sustainable feedstocks while simultaneously enabling effective polymer recycling represents a potential transformation of 21st century manufacturing. To evaluate technologies that could enable such changes, it is imperative to compare the sustainability of bio-based and/or circular plastic flows to those of incumbent manufacturing paradigms. To that end, we estimate the supply chain energy requirements and greenhouse gas (GHG) emissions associated with US-based plastics consumption. Major commodity polymers, each of which has a global consumption of at least 1 MMT per year, account for an estimated annual 3.2 quadrillion Btutus (quads) of energy and 104 MMTCO2e of GHG emissions in the US alone. This study serves as a foundation for comparing the supply chain energy requirements and GHG emissions of today’s plastics manufacturing to tomorrow’s disruptive technologies, to inform the development of bio-based plastics and the circular economy for synthetic polymers.

Serine hydrolases cleave peptide and ester bonds and are ubiquitous in nature, with applications in biotechnology, in materials, and as drug targets. The serine hydrolase two-step mechanism employs a serine-histidine-aspartate/glutamate catalytic triad, where the histidine residue acts as a base to activate poor nucleophiles (a serine residue or a water molecule) and as an acid to allow the dissociation of poor leaving groups. This mechanism has been the subject of debate regarding how histidine shuttles the proton from the nucleophile to the leaving group. To elucidate the reaction mechanism of serine hydrolases, we employ quantum mechanics/molecular mechanics-based transition path sampling to obtain the reaction coordinate using the Aspergillus niger feruloyl esterase A (AnFaeA) as a model enzyme. The optimal reaction coordinates include terms involving nucleophilic attack on the carbonyl carbon and proton transfer to, and dissociation of, the leaving group. During the reaction, the histidine residue undergoes a reorientation on the time scale of hundreds of femtoseconds that supports the "moving histidine" mechanism, thus calling into question the "ring flip" mechanism. We find a concerted mechanism, where the transition state coincides with the tetrahedral intermediate with the histidine residue pointed between the nucleophile and the leaving group. Moreover, motions of the catalytic aspartate toward the histidine occur concertedly with proton abstraction by the catalytic histidine and help stabilize the transition state, thus partially explaining how serine hydrolases enable poor nucleophiles to attack the substrate carbonyl carbon. Rate calculations indicate that the second step (deacylation) is rate-determining, with a calculated rate constant of 66 s-1. Overall, these results reveal the pivotal role of active-site dynamics in the catalytic mechanism of AnFaeA, which is likely similar in other serine hydrolases.

Lignin recalcitrance presents a challenge for the development of a bioeconomy that employs lignocellulosic feedstocks. The efficiency of lignin deconstruction is improved by a reduction of molecula...

The accumulation of plastic waste in the environment has prompted the development of new chemical recycling technologies. A recently reported approach employed homogeneous organometallic catalysts ...

Reductive catalytic fractionation (RCF) is a promising approach to fractionate lignocellulose and convert lignin to a narrow product slate. To guide research towards commercialization, cost and sustainability must be considered. Here we report a techno-economic analysis (TEA), life cycle assessment (LCA), and air emission analysis of the RCF process, wherein biomass carbohydrates are converted to ethanol and the RCF oil is the lignin-derived product. The base-case process, using a feedstock supply of 2000 dry metric tons per day, methanol as a solvent, and H2 gas as a hydrogen source, predicts a minimum selling price (MSP) of crude RCF oil of $1.13 per kg when ethanol is sold at $2.50 per gallon of gasoline-equivalent ($0.66 per liter of gasoline-equivalent). We estimate that the RCF process accounts for 57% of biorefinery installed capital costs, 77% of positive life cycle global warming potential (GWP) (excluding carbon uptake), and 43% of positive cumulative energy demand (CED). Of $563.7 MM total installed capital costs, the RCF area accounts for $323.5 MM, driven by high-pressure reactors. Solvent recycle and water removal via distillation incur a process heat demand equivalent to 73% of the biomass energy content, and accounts for 35% of total operating costs. In contrast, H2 cost and catalyst recycle are relatively minor contributors to operating costs and environmental impacts. In the carbohydrate-rich pulps, polysaccharide retention is predicted not to substantially affect the RCF oil MSP. Analysis of cases using different solvents and hemicellulose as an in situ hydrogen donor reveals that reducing reactor pressure and the use of low vapor pressure solvents could reduce both capital costs and environmental impacts. Processes that reduce the energy demand for solvent separation also improve GWP, CED, and air emissions. Additionally, despite requiring natural gas imports, converting lignin as a biorefinery co-product could significantly reduce non-greenhouse gas air emissions compared to burning lignin. Overall, this study suggests that research should prioritize ways to lower RCF operating pressure to reduce capital expenses associated with high-pressure reactors, minimize solvent loading to reduce reactor size and energy required for solvent recovery, implement condensed-phase separations for solvent recovery, and utilize the entirety of RCF oil to maximize value-added product revenues. ispartof: Energy & Environmental Science vol:14 issue:8 pages:4147-4168 ispartof: location:England status: published

Recent works have established bio-based carboxylic acids as adaptable precursors to renewable biofuels and chemicals. However, the separation of carboxylic acids is a major energy and cost driver, accounting for 20-40% of the entire processing cost. Improved downstream separation technologies that reduce operating costs compared to conventional approaches are needed, particularly to enable bio-based commodity fuels and chemicals. Here, we combine techno-economic analysis (TEA) and an energy and environmental assessment with experimental results to compare weak-base adsorption (WBA) processes with the conventional strong ion exchange (IX) process for the recovery of the exemplary product, butyric acid. TEA indicates that WBA has the potential to reduce operating expenses from 34% to 6% relative to the selling price of butyric acid ($1.8 kg(-1)). Our energy analysis shows that the WBA process has 12.2-fold energy reduction and 9.2-fold GHG emission reduction compared to the conventional IX process.

The valorisation of the plant biopolymer lignin is now recognised as essential to enabling the economic viability of the lignocellulosic biorefining industry. In this context, the “lignin-first” biorefining approach, in which lignin valorisation is considered in the design phase, has demonstrated the fullest utilisation of lignocellulose. We define lignin-first methods as active stabilisation approaches that solubilise lignin from native lignocellulosic biomass while avoiding condensation reactions that lead to more recalcitrant lignin polymers. This active stabilisation can be accomplished by solvolysis and catalytic conversion of reactive intermediates to stable products or by protection-group chemistry of lignin oligomers or reactive monomers. Across the growing body of literature in this field, there are disparate approaches to report and analyse the results from lignin-first approaches, thus making quantitative comparisons between studies challenging. To that end, we present herein a set of guidelines for analysing critical data from lignin-first approaches, including feedstock analysis and process parameters, with the ambition of uniting the lignin-first research community around a common set of reportable metrics. These guidelines comprise standards and best practices or minimum requirements for feedstock analysis, stressing reporting of the fractionation efficiency, product yields, solvent mass balances, catalyst efficiency, and the requirements for additional reagents such as reducing, oxidising, or capping agents. Our goal is to establish best practices for the research community at large primarily to enable direct comparisons between studies from different laboratories. The use of these guidelines will be helpful for the newcomers to this field and pivotal for further progress in this exciting research area.

The inherent reactivity of lignin in conventional biomass processing commonly prevents isolation of native lignin and limits monomer yields from catalytic depolymerization strategies that target aryl-ether bonds. Here we show that flow-through solvolysis with methanol at 225 °C produces native-like lignin from poplar, enabling the study of intrinsic lignin properties and evaluation of steady-state lignin depolymerization processes.

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h−1 and 0.38 h−1, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.

Microbial terephthalic acid (TPA) catabolic pathways are conserved among the few bacteria known to turnover this xenobiotic aromatic compound. However, to date there are few reported cases in which this pathway has been successfully expressed in heterologous hosts to impart efficient utilization of TPA as a sole carbon source. In this work, we aimed to engineer TPA conversion in Acinetobacter baylyi ADP1 via the heterologous expression of catabolic and transporter genes from a native TPA-utilizing bacterium. Specifically, we obtained ADP1-derived strains capable of growing on TPA as the sole carbon source using chromosomal insertion and targeted amplification of the tph catabolic operon from Comamonas sp. E6. Adaptive laboratory evolution was then used to improve growth on this substrate. TPA consumption rates of the evolved strains, which retained multiple copies of the tph genes, were ~0.2 g/L/h (or ~1 g TPA/g cells/h), similar to that of Comamonas sp. E6 and almost 2-fold higher than that of Rhodococcus jostii RHA1, another native TPA-utilizing strain. To evaluate TPA transport in the evolved ADP1 strains, we engineered a TPA biosensor consisting of the transcription factor TphR and a fluorescent reporter. In combination with whole-genome sequencing, the TPA biosensor revealed that transport of TPA was not mediated by the heterologous proteins from Comamonas sp. E6. Instead, the endogenous ADP1 muconate transporter MucK, a member of the major facilitator superfamily, was responsible for TPA transport in several evolved strains in which MucK variants were found to enhance TPA uptake. Furthermore, the IclR-type transcriptional regulator DcaS was identified as a repressor of mucK expression. Overall, this work presents an unexpected function of a native protein identified through gene amplification, adaptive laboratory evolution, and a combination of screening methods. This study also provides a TPA biosensor for application in ADP1 and identifies transporter variants for use in metabolic engineering applications focused on plastic upcycling of polyesters.

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Esterase enzymes catalyze diverse hydrolysis reactions with important biological, commercial, and biotechnological applications. For the improvement of these biocatalysts, there is a need for widely accessible, inexpensive, and adaptable activity screening assays that identify enzymes with particular substrate specificities. Natural systems for biopolymer bioconversion, and likely those designed to mimic them, depend on cocktails of enzymes, each of which specifically targets the intact material as well as water-soluble subunits of varying size. In this work, we have adapted a UV/visible assay using pH-sensitive sulfonphthalein dyes for the real-time quantification of ester hydrolysis of bis-(2-hydroxyethyl) terephthalate (BHET), a subunit of polyethylene terephthalate (PET) plastic. We applied this method to a diverse set of known PET hydrolases and commercial esterases in a microplate format. The approach identified four PET hydrolases and one commercial esterase with high levels of specificity for BHET hydrolysis. Five additional PET hydrolases and three commercial esterases, including a thermophilic enzyme, effectively hydrolyzed both BHET and its monoester product MHET (mono-(2-hydroxyethyl) terephthalate). Specific activities were discernible within one hour and reactions reached an unequivocal endpoint well within 24 hours. The results from the UV/visible method correlated well with conventional HPLC analysis of the reaction products. We examined the suitability of the method toward variable pH, temperature, enzyme preparation method, mono- and multi-ester substrate type, and level of sensitivity

Self-driving labs (SDLs) combine fully automated experiments with artificial intelligence (AI) that decides the next set of experiments. Taken to their ultimate expression, SDLs could usher a new paradigm of scientific research, where the world is probed, interpreted, and explained by machines for human benefit. While there are functioning SDLs in the fields of chemistry and materials science, we contend that synthetic biology provides a unique opportunity since the genome provides a single target for affecting the incredibly wide repertoire of biological cell behavior. However, the level of investment required for the creation of biological SDLs is only warranted if directed towards solving difficult and enabling biological questions. Here, we discuss challenges and opportunities in creating SDLs for synthetic biology., Comment: 17 pages, 3 figures. Submitted for publication in Current Opinion in Biotechnology. Updated figure 3 in this version

Bacterial gene expression is orchestrated by numerous transcription factors (TFs). Elucidating how gene expression is regulated is fundamental to understanding bacterial physiology and engineering it for practical use. In this study, a machine-learning approach was applied to uncover the genome-scale transcriptional regulatory network (TRN) in Pseudomonas putida KT2440, an important organism for bioproduction. We performed independent component analysis of a compendium of 321 high-quality gene expression profiles, which were previously published or newly generated in this study. We identified 84 groups of independently modulated genes (iModulons) that explain 75.7% of the total variance in the compendium. With these iModulons, we (i) expand our understanding of the regulatory functions of 39 iModulon associated TFs (e.g., HexR, Zur) by systematic comparison with 1993 previously reported TF-gene interactions; (ii) outline transcriptional changes after the transition from the exponential growth to stationary phases; (iii) capture group of genes required for utilizing diverse carbon sources and increased stationary response with slower growth rates; (iv) unveil multiple evolutionary strategies of transcriptome reallocation to achieve fast growth rates; and (v) define an osmotic stimulon, which includes the Type VI secretion system, as coordination of multiple iModulon activity changes. Taken together, this study provides the first quantitative genome-scale TRN for P. putida KT2440 and a basis for a comprehensive understanding of its complex transcriptome changes in a variety of physiological states.

Here, we show that lignin-first biorefining of poplar can enable the production of dissolving cellulose pulp that can produce regenerated cellulose, which could substitute cotton. These results in turn indicate that agricultural land dedicated to cotton could be reclaimed for food production by extending poplar plantations to produce textile fibers. Based on climate-adapted poplar clones capable of growth on marginal lands in the Nordic region, we estimate an environmentally sustainable annual biomass production of similar to 11 tonnes/ha. At scale, lignin-first biorefining of this poplar could annually generate 2.4 tonnes/ha of dissolving pulp for textiles and 1.1 m(3) biofuels. Life cycle assessment indicates that, relative to cotton production, this approach could substantially reduce water consumption and identifies certain areas for further improvement. Overall, this work highlights a new value chain to reduce the environmental footprint of textiles, chemicals, and biofuels while enabling land reclamation and water savings from cotton back to food production.

The sequence of the promoter used to drive expression of glcDEF operon in the engineered strain MFL185 in the original article was incorrect. As described here, we performed additional experiments that indicate expression of this operon was increased in MFL185, as intended. Ultimately, this error is immaterial with respect to the findings and conclusions reported in the original article.

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (k(cat)/K(M)): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA’s binding affinities for the different guaiacols and was the inverse of GcoA(EP4)’s specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic–catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H(2)O(2) as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H(2)O(2)-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H(2)O(2) into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper–oxyl intermediate. The initial cleavage of H(2)O(2) and subsequent hydrogen atom abstraction from chitin by the copper–oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO–Cu(II) to LPMO–Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO–Cu(I) is 2,000-fold faster with H(2)O(2) than with O(2), the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H(2)O(2), whereas reoxidation by O(2) became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.

Technoeconomic and life-cycle analyses are presented for catalytic conversion of ethanol to fungible hydrocarbon fuel blendstocks, informed by advances in catalyst and process development. Whereas prior work toward this end focused on 3-step processes featuring dehydration, oligomerization, and hydrogenation, the consolidated alcohol dehydration and oligomerization (CADO) approach described here results in 1-step conversion of wet ethanol vapor (40 wt% in water) to hydrocarbons and water over a metal-modified zeolite catalyst. A development project increased liquid hydrocarbon yields from 36% of theoretical to >80%, reduced catalyst cost by an order of magnitude, scaled up the process by 300-fold, and reduced projected costs of ethanol conversion 12-fold. Current CADO products conform most closely to gasoline blendstocks, but can be blended with jet fuel at low levels today, and could potentially be blended at higher levels in the future. Operating plus annualized capital costs for conversion of wet ethanol to fungible blendstocks are estimated at $2.00/GJ for CADO today and $1.44/GJ in the future, similar to the unit energy cost of producing anhydrous ethanol from wet ethanol ($1.46/GJ). Including the cost of ethanol from either corn or future cellulosic biomass but not production incentives, projected minimum selling prices for fungible blendstocks produced via CADO are competitive with conventional jet fuel when oil is $100 per barrel but not at $60 per barrel. However, with existing production incentives, the projected minimum blendstock selling price is competitive with oil at $60 per barrel. Life-cycle greenhouse gas emission reductions for CADO-derived hydrocarbon blendstocks closely follow those for the ethanol feedstock.

Lignin and cellulose are two of the most abundant polymers on Earth and are found in close proximity within plant cell walls. Despite their ubiquity, relatively little is known quantitatively about...

The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70 wt% Tween® 20 and 30 wt% Span® 80. Pseudomonas putida KT2440 grew to an optical density (OD600) of 1.0 ± 0.2 when supplied with 1.6 wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002 g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism for most compounds, including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coefficients are consistent with concentration enrichment near lipid carbonyl groups, and permeability coefficients are sufficient to keep pace with cellular metabolism. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glycosylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compounds can passively traverse plant and microbial membranes on timescales commensurate with required biological activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compound functionalization.

Inverse bimetallic catalysts (IBCs), synthesized by sequential deposition of noble and oxophilic metals, offer potential reactivity enhancements to various reactions, including the reduction of car...

There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, the Ideonella sakaiensis PETase wild-type enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45 A resolution structure of the mutant was described, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies showed that reaction temperature had the strongest influence on performance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-based polyester recycling.

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP

Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts.

Correction for ‘Flow-through solvolysis enables production of native-like lignin from biomass’ by David G. Brandner et al., Green Chem., 2021, 23, 5437–5441, DOI: 10.1039/D1GC01591E.

Summary 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.

While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. In this work, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.