This study demonstrates an organic solvent-free processing strategy to valorize consolidated bioprocessing (CBP) residues, from switchgrass and poplar biomass, into functional poly(butylene succinate) (PBS)-based biocomposites using high-shear homogenization (HSH). HSH transformed the switchgrass and poplar CBP residues (CBP-R) into fine, uniformly distributed particles and microfibers. The composites of PBS with homogenized switchgrass residues (H-CBP-R-SG) or homogenized poplar residues (H-CBP-R-P) at a 70/30 weight ratio exhibited improved processability and mechanical integrity, with the Young's modulus for the PBS/H-CBP-R-SG and PBS/H-CBP-R-P nearly doubling to 0.66 +/- 0.07 GPa and 0.65 +/- 0.04 GPa, respectively, compared to neat PBS (0.36 +/- 0.02 GPa). Dynamic Mechanical Analysis (DMA) reveals a significant suppression of the tan delta peak magnitude, indicating that HSH-mediated physical activation facilitates stress transfer in composites typical of covalent chemical grafting systems. While the transition to a stiffness-dominated profile reduces ductility, the resulting composites exhibit the dimensional stability and resistance to thermal warping required for high-fidelity FDM 3D printing and injection molding. Beyond material performance, comprehensive techno-economic analysis (TEA) and life cycle assessment (LCA) confirmed that diverting CBP residues into composite products can improve the economic viability of the biorefinery without substantially increasing biorefinery global warming potential (GWP). At a 30 wt% blend ratio, incorporating residuals into PBS yielded a minimum selling price for the composite of $4.07 per kg compared to the conventional bioplastic price of $5.00 per kg. This approach aligns with circular bioeconomy principles by converting waste streams into value-added products. Furthermore, this innovative strategy addresses key challenges in bioplastic development, including cost, compatibility, and performance, while simultaneously advancing waste minimization strategies for sustainable manufacturing systems.

Glycosyltransferases (GTs) are the primary enzymes responsible for the biosynthesis of the complex polysaccharides in plant cell walls. Given the important role of GTs in plants, it is necessary to undertake their functional characterization to better understand plant cell wall synthesis pathways to develop improved feedstocks for efficient conversion into fuels and products to support the emerging bioeconomy. The GT47 family in plants represents a unique target for characterization due to the substantial diversity of donor and acceptor substrates observed within a single family. Here, we have carried out the biochemical characterization of MUR3 and XLT2 orthologs from the aquatic monocot Spirodela polyrhiza. Our findings support existing genetic and phylogenetic data classifying these enzymes as regio-specific galactosyltransferases involved in xyloglucan (XyG) sidechain biosynthesis. In addition, we have identified novel characteristics for both enzymes, such as in vitro arabinopyranosyltransferase activity and distinctiveness in xyloglucan reducing end specificity.

Clostridium thermocellum is one of the most efficient microorganisms for the deconstruction of cellulosic biomass. To achieve this high level of cellulolytic activity, C. thermocellum uses large multienzyme complexes known as cellulosomes to break down complex polysaccharides, notably cellulose, found in plant cell walls. The attachment of bacterial cells to the nearby substrate via the cellulosome has been hypothesized to be the reason for this high efficiency. The region lying between the cell and the substrate has shown great variation and dynamics that are affected by the growth stage of cells and the substrate used for growth. Here, we used both super-resolution imaging and machine-learning approaches to study the distribution of C. thermocellum cellulosomes at different stages of growth. We show that C. thermocellum initially retains its cellulosomes primarily on the cell surface but then relocates large cellulosome clusters to the interface with biomass, therefore depleting its cell surface of cellulosomes. These results indicate dynamic redistribution of cellulosomes during growth, with a functional shift toward substrate-associated degradation later during growth on biomass.

Liquid fuels from lignocellulosic feedstocks are required for transition to a sustainable bioeconomy. However, the recalcitrance of carbon-containing feedstock cell walls to deconstruction poses a barrier to cost effective biological conversion of plant biomass to biofuels. One-step consolidated bioprocessing (CBP) in which anaerobic thermophilic bacteria convert lignocellulosic biomass into liquid fuels is a platform for overcoming the recalcitrance of plant biomass. The amounts of hemicellulosic and pectic polysaccharides, two complex cell wall glycans that contribute to plant biomass recalcitrance and that are partially solubilized during CBP of switchgrass aerial biomass by Clostridium thermocellum were evaluated in the liquor, solid residues and residue washate recovered during a 120-h CBP process. After 120 h, 24

Cell-free biocatalysis is a rapidly evolving field with great potential for sustainably producing valuable chemicals. Some challenges in cell-free biocatalysis include reaction longevity, enzyme stability, and the cost of the biocatalysts. Here, the challenge of enzyme instability was addressed by employing thermophilic enzymes to improve the productivity of the lower mevalonate pathway, using limonene as an example isoprenoid product. The Classical mesophilic mevalonate pathway was compared to a newly assembled set of thermophilic enzymes comprising the Archaea I mevalonate pathway. The thermophilic pathway enzymes were thermostable to at least 60 °C and exhibited a 6× longer operating lifetime at 22 °C. Thus, despite lower initial activity rates at ambient temperature, the thermophilic pathway was longer-lived and resulted in a more productive cell-free reaction overall, achieving 1.7× higher yield of limonene compared to using enzymes from mesophiles. Moreover, the thermostable pathway retained activity for longer with the challenge of solvent exposure, namely, ethanol and isoprenol, which broadens the scope of accessible substrates and/or products in cell-free reactions employing this pathway. Altogether, we showed significant improvement in the stability and productivity of the lower mevalonate pathway, which will enable more efficient cell-free biosynthesis of isoprenoid products. This represents a valuable strategy to increase the robustness of cell-free systems by carefully sourcing biocatalysts from thermophilic organisms, which have proven to be resilient to challenges unique to cell-free reaction systems.

ABSTRACT Thermoanaerobacterium saccharolyticum is a thermophilic anaerobic bacterium that natively ferments a variety of hemicellulose substrates to organic acids and alcohols. It has recently been engineered to produce ethanol at high yield and titer; however, it uses a unique metabolic pathway for ethanol production that is poorly characterized. One of the distinctive aspects of this pathway is the presence of acetyl-CoA as an intermediate metabolite. In this organism, acetyl-CoA is converted to ethanol by a bifunctional AdhE enzyme. This enzyme has been a frequent target for mutations, and in many cases, the function of these mutations was unknown. Using a combination of genetic modifications, enzyme assays, and computational analysis, we have developed a better understanding of how mutations in AdhE affect ethanol production in the engineered homoethanologen strain. We identify a set of approximately interchangeable AdhE mutations (G544D, T597K, T597I, and T605I), whose function is to disrupt the activity of the alcohol dehydrogenase (ADH) domain of AdhE. This reduces NADH-linked ADH activity, which dramatically increases ethanol tolerance and changes the overall stoichiometry of acetaldehyde to ethanol conversion. Furthermore, our improved understanding of the function of these AdhE mutations calls into question a proposed feature of AdhE enzymes known as substrate channeling—direct transfer of acetaldehyde between the two domains of the AdhE enzyme. This improved the understanding of the role of AdhE mutations in T. saccharolyticum and provides deeper insights into the function of the unique ethanol production pathway in this organism. IMPORTANCE Many anaerobic bacteria maintain redox equilibrium by producing reduced organic compounds such as ethanol. The final two steps of ethanol production are mediated by a bifunctional enzyme, AdhE, and this enzyme is a frequent target of mutations in strains engineered for increased ethanol production. Paradoxically, these mutations increase ethanol production by eliminating the activity of one domain of the AdhE enzyme (the ADH domain). This provides additional support for a redox-imbalance theory of alcohol tolerance, which challenges the prevailing hypothesis that alcohol tolerance is associated with cell membrane effects.

Cell-free biocatalysis (CFB) is an efficient and environmentally friendly method to synthesize molecules such as pharmaceuticals, biochemicals, and biofuels through the in vitro use of enzyme cascades. These enzymes often require redox cofactors to drive chemical reactions. Natural redox cofactors (NAD(P)H) are expensive to isolate, motivating synthetic nicotinamide cofactor biomimetics (NCBs) as a cost-effective solution. A select handful of NCBs have been identified as potential NAD(P)H alternatives with comparable or improved redox capabilities, however, they display a tendency to degrade in common buffers. In this study, a library of 132 NCB candidates is systematically generated, over 85% of which have not been characterized in the literature, to expand the diversity of currently explored NCBs. The decomposition mechanism of NCBs in phosphate is evaluated using density functional theory (DFT), revealing protonation at the nicotinamide C5 position as a reporter of cofactor stability. Based on this result, we trained a linear regression model on DFT calculated descriptors to predict NCB stability in phosphate buffer, achieving mean absolute error (MAE) and root mean squared error (RMSE) values within computational accuracy. Analysis of key atomic descriptors and qualitative trends in our dataset informed the design of novel NCB candidates we propose with optimized stability. This work enables researchers to predict the relative stability of NCBs before synthesis, thereby streamlining the process to make CFB more affordable and viable at industry scales.

Clostridium thermocellum , a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe, however ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 Å structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

Clostridium thermocellum is a promising host for consolidated bioprocessing due to its ability to directly ferment cellulose into fuels and chemicals. However, natural product formation in this organism is limited. Here, we report engineering C. thermocellum for the production of 2,3-butanediol (23BD), a valuable industrial chemical. We functionally expressed a thermophilic 23BD pathway in this organism resulting in a 23BD titer of 19.7 mM from cellulose, representing a metabolic yield of 24%. We used a cell-free systems biology approach to identify limiting steps in the 23BD pathway, revealing that exogenous 23BD dehydrogenase (BDH) activity was essential for production, while native acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) activities were present but limiting in the parent strain. This approach also revealed redox balance limitations. We demonstrated that this improved understanding of redox balance limitations could be used to increase 23BD titer in vivo, showing that adding acetate could be used to increase 23BD yield. This work establishes a foundation for developing C. thermocellum into a robust platform for 23BD production directly from cellulose and highlights the utility of cell-free systems for guiding metabolic engineering in non-model organisms.

In biotechnology, the term 'cell-free' describes a wide range of established and emerging processes and technologies. However, broad application of the term 'cell-free' leads to confusion and ambiguity in the field, especially when applied to biomanufacturing. When engaging with investors, government agencies, and industry partners, we have found that the association of 'cell-free' with numerous technologies spanning diverse applications creates unnecessary confusion. As a result, we believe the lack of clarity complicates communication, impacts technology adoption, and creates challenges in securing funding and regulatory recognition. Specifically, in this opinion, we focus on terminology used to describe enzyme-driven biocatalytic approaches for manufacturing small molecules, often referred to as 'cell-free biomanufacturing.' This 'cell-free' approach harnesses biocatalytic pathways, in the absence of living cells, to synthesize small molecules used as pharmaceuticals, nutraceuticals, flavors, and fragrances, but also specific commodity chemicals and fuels. To clarify terminology, we introduce the new terms 'exozymes' and 'exozyme systems' as unambiguous descriptors for 'cell-free biomanufacturing.'

The field of synthetic biology is essential to the continued development of a bio-based economy, creating mechanisms to supply carbon needed in the economy by both converting existing end-of-life wastes as well as by creating novel, purpose-grown and sustainable feedstocks. Here, we first discuss the near- and long-term resources available for use as feedstocks for bioconversion as well as the output molecules needed for building the foundation of an expanded bio-based economy. We then outline the organisms and phenotypic traits that are needed for the performance-advantaged chassis organisms of the future. Furthermore, we detail the advances, challenges, and opportunities in both microbial and plant synthetic biology relevant to expanding the bio-based economy. Finally, we explore technologies that have and will further enable advances in synthetic biology and the greater bio-based economy.

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization. This work also highlights the critical role of intracellular acidification in shaping enzyme performance and microbial adaptation. The PET-FBA framework is implemented as a Python package at https://github.com/Chaowu88/etfba, with detailed documentation provided at https://etfba.readthedocs.io.

Substrate specificity is an essential characteristic of any enzyme's function and an understanding of the factors that determine this specificity is crucial for enzyme engineering. Unlike the structure of an enzyme which is directly impacted by its sequence, substrate specificity as an enzyme attribute involves a rather indirect relationship with sequence as it also depends on structural aspects that dictate substrate accessibility and active site dynamics. In this study, we explore the performance of classifier-based machine learning models trained on curated sequence and structural data for a class of glycosyltransferases (GTs), namely GT-Bs, to understand their substrate specificity determining factors. GTs enable the transfer of sugar moieties to other biomolecules such as oligosaccharides or proteins and are found in all kingdoms of life. In plants, GTs participate in the biosynthesis of plant cell wall biopolymers (e.g.: hemicelluloses and pectins) and are an integral part of the enzymatic machinery that enables the storage of carbon and energy as plant biomass. To elucidate the substrate specificity of uncharacterized GT-Bs, we constructed multi-label machine learning models (Support Vector Classifier, K-Nearest Neighbors, Gaussian Na & iuml;ve-Bayes, Random Forest) that incorporate both sequence and structural features. These models achieve good predictive accuracies on test datasets. However, despite our use of structural information, we highlight that there is further scope for improvement in training these models to draw interpretable relationships between sequence, structure and substrate specificity determining motifs in GT-Bs.

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call "cell-free systems biology", where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a three-enzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO2 from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

The use of nicotinamide cofactors in cell-free biocatalytic systems is necessitated by the high specificity that these enzymes show for their natural redox mediators. Unfortunately, isolation and use of natural cofactors is costly, which suggests that enhancing their stability is key to enabling their use in industrial processes. This study details NAD+ and NADH stability in three buffer systems (sodium phosphate, HEPES, and Tris) at 19 °C and 25 °C and for up to 43 d. In Tris, both NADH and NAD+ were found to be highly stable. NADH degradation rates of 4 μM/d (19 °C) and 11 μM/d (25 °C) were observed in Tris buffer, corresponding to >90% and 75% remaining after 43 d, respectively. Higher degradation rates (up to 34 μM/d) were observed when sodium phosphate or HEPES buffers were used. The effect of a mild increase in temperature was determined to be significant for long-term stability, and it was shown that degradation under these conditions can be easily monitored via UV–Vis, because the degradation proceeds via the oxidation/de-aromatization of the dihydropyridine ring. Overall, this work emphasizes that the choice of buffer system is consequential for bioreactor systems employing natural nicotinamide cofactors for extended periods of time.

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase enzyme, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADHlinked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

Abstract Background Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. Results In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. Conclusions Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

Plant secondary cell walls (SCWs) are composed of a heterogeneous interplay of three major biopolymers: cellulose, hemicelluloses, and lignin. Details regarding specific intermolecular interactions and higher-order architecture of the SCW superstructure remain ambiguous. Here, we use solid-state nuclear magnetic resonance (ssNMR) measurements to infer refined details about the structural configuration, intermolecular interactions, and relative proximity of all three major biopolymers within air-dried Populus wood. To enhance the utility of these findings and enable evaluation of hypotheses in a physics-based environment in silico, the NMR observables are articulated into an atomistic, macromolecular model for biopolymer assemblies within the plant SCW. Through molecular dynamics simulation, we quantitatively evaluate several variations of atomistic models to determine structural details that are corroborated by ssNMR measurements.

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk(on)) and desorption (k(off)) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.