Biotechnology and Bioengineering

PurposeLarge quantities of human pluripotent stem cells (hPSCs) are required for clinical applications. 3D suspension cultures are suitable for large scale manufacturing of hPSCs but yield, viability and quality are affected by the hydrodynamic environment. This paper characterizes the hydrodynamic environment inside vertical wheel bioreactors (VWBRs) as a function of size and agitation rates, measures its effect on cell aggregation and proliferation, and proposes the use of Lagrangian-based shear stress and energy dissipation rate (EDR) exposures to support scale-up. MethodsIn silico: Transient, 3D, turbulent flow simulations are conducted for two VWBR sizes (100, 500 mL) at five agitation rates between 20 and 80 rpm. Trajectories of cell aggregates of sizes from 200 to 1,000 microns are calculated, and shear stress and EDR exposures are collected along these trajectories. In vitro: ESI-017 hPSCs were cultured in VWBRs for 6 days. Aggregation efficiency and daily fold ratios were calculated based on cell counts and initial inoculation density. ResultsAggregate size, agitation rate and bioreactor size modulate cell aggregate exposures to EDR and shear stress, which significantly depart from maximum or volume average metrics used for scale-up. Combined in vitro/in silico results show EDR affects aggregation efficiency, cell counts and aggregate size, and has a small effect on daily fold ratios but a significant effect on total fold ratio. ConclusionHistory of trajectory-based cell aggregate exposures to EDRs provide a better scale-up basis for VWBRs than volume-averaged EDR. Shear stress does not significantly affect hPSC aggregation, proliferation and expansion in VWBRs under the tested conditions.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY(R) VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

The efficient production of food and biochemicals using microorganisms that utilize single-carbon feedstocks presents a promising approach for advancing a circular bioeconomy. Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast already widely used in industry, making it an attractive host for such applications. Recently, K. phaffii was converted into an autotrophic strain capable of assimilating CO2 into both biomass and secreted organic acids, using energy derived from dissimilation of methanol to CO2. In these strains, methanol oxidation is catalysed by an alcohol oxidase (Aox2), which transfers electrons to oxygen without conserving reducing equivalents. To address this limitation, in this study we explored redirecting methanol dissimilation through the native alcohol dehydrogenase (Adh2), coupling methanol oxidation with NADH generation to improve carbon efficiency. By deleting AOX2 and overexpressing ADH2, we generated Adh2-based autotrophic strains that exhibited growth rates comparable to the parental strain (0.007 h-{superscript 1}), while reducing specific CO2 production by 53% and increasing biomass yield (YX/MeOH) by 59%. We further applied this strategy to convert previously developed autotrophic strains producing itaconic acid and lactic acid into Adh2-dependent strains. Optimizing ADH2 expression through multicopy integration resulted in strains with approximately two-fold higher molar carbon efficiency (Y(X+P)/CO2) while achieving elevated product titers--2.2-fold for itaconic acid and 3.8-fold for lactic acid--relative to the parental strains. Our findings demonstrate that alcohol dehydrogenase-mediated methanol dissimilation can significantly improve yield and productivity of autotrophic K. phaffii strains, with broad implications for sustainable bioproduction from one-carbon substrates.

Lignin-derived aromatics are abundant in depolymerized lignin but remain remain untilized as carbon sources for commercial production of bulk chemicals. Among these aromatics, p-coumaric acid can be funnelled through the {beta}-ketoadipate pathway toward cis,cis-muconic acid (ccMA), a precursor of bio-based adipic and terephthalic acids. However, efficient ccMA production by Acinetobacter baylyi ADP1 is constrained by toxicity of catechol (the immediate precursor of ccMA), inefficient channelling of protocatechuate (PCA) metabolism towards ccMA production, and absence of PCA decarboxylase for converting PCA to catechol. Therefore, in this study, we engineered a modular co-culture system, combining engineered strains of A. baylyi and E. coli, for ccMA production from synthetic p-coumaric acid. Deletion of catB and catC genes and overexpression of catA in A. baylyi GJS_catA strain enabled near-stoichiometric conversion of catechol to ccMA ([~]90% carbon yield) with titres up to 56.4 mM ([~] 8 g/L) under controlled fed-batch feeding. The strain was further engineered (A. baylyi GJS2_catA) to convert p-coumaric acid to PCA. Due to the inactivity of heterologous PCA decarboxylase (aroY gene) in A. baylyi, this gene was incorporated in E. coli where it exhibited activity through PCA to catechol conversion. Upon its production by E.coli_aroY in the co-culture, catechol is instantaneously converted to ccMA by A. baylyi GJS2_catA strain. In a two-step process, 22 mM p-coumaric acid was initially converted to 20.6 mM PCA (A. baylyi GJS2_catA), which was further converted to catechol (E.coli_aroY) and finally to 18.55 mM ccMA (2.63 g L-{superscript 1}) by A. baylyi GJS2_catA. This process was validated by the valorization of lignin-derived p-coumaric acid to ccMA. While the modular strategy developed in this study substantially improves ccMA titres, it also highlights the bottlenecks in A. baylyi metabolic pathway engineering for lignin valorization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/709578v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@a83daborg.highwire.dtl.DTLVardef@168c6b6org.highwire.dtl.DTLVardef@1ce0abdorg.highwire.dtl.DTLVardef@23200b_HPS_FORMAT_FIGEXP M_FIG C_FIG

In this study, we employ a two-stage dynamic metabolic control strategy to enhance the NADPH dependent biosynthesis of ethylene glycol from xylose in engineered E. coli. We evaluated the use of metabolic valves to dynamically reduce the enzymes involved in competitive pathways which compete for substrates with ethylene glycol biosynthesis, as well as regulatory pathways aimed at increasing NADPH fluxes. The performance of our initial strains with limits in pathway expression levels was improved by the addition of competitive valves, but not by increases in NADPH flux. In contrast, improving pathway expression levels, led to strains improved significantly by our regulatory valves which improved NADPH flux, but not by the competitive valves. This is consistent with a central hypothesis that faster pathways in and of themselves can compete with other metabolic fluxes by being faster and are better aided by regulatory changes capable of change rates elsewhere in metabolism. In this case in NADPH flux. Lastly, upon scale up to fed-batch bioreactors, our optimized strain, featuring dynamic control of two regulatory valves produced 140 g/L of EG in 70 hours at 92% of the theoretical yield.

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

Selective encapsulation of target enzymes is an increasingly well studied field with a host of potential applications for biotechnology. Natively, many bacteria utilize bacterial microcompartments (BMCs) for enzyme encapsulation to enhance catalysis. BMCs are protein shells that enable selective localization of targeted metabolic enzymes and may improve catalytic rates by co-localizing pathway enzymes and/or serve to sequester toxic or volatile intermediates. The microcompartment shell of Haliangium ochraceum (HO) is a notable BMC chassis because of its modularity and versatility; it is easily expressed and assembled outside its native host and can accept a wide array of cargo. Recently, it was demonstrated that assembly of HO BMC shells can be easily achieved in vitro. Following up on our previous work on in vivo assembly of HO-BMCs with triose phosphate isomerase (TPI) as model enzyme cargo, here we have demonstrated the advantages of in vitro assembly (IVA) for targeted enzyme encapsulation. We achieved variable loading of BMC shells with targeted amounts of TPI and demonstrated enhanced thermal stability of encapsulated TPI versus free TPI up to 62{degrees}C.

Cyanobacteria have garnered interest as promising biological platforms for producing renewable biofuel, chemical feedstock, and bioactive molecules. For biotechnology applications, robust well-characterized genetic tools are required for genetically modifying cyanobacteria, but these tools are often developed for specific model strains. Here, we used broad host-range RSF1010-based plasmids to characterize a set of orthogonal constitutive promoters in diverse cyanobacterial strains. The promoters are random variants of the synthetic Escherichia coli PconII promoter. A library of PconII promoters driving a fluorescent reporter gene was first evaluated in Synechococcus elongatus and found to have a wide range of gene expression levels. A set of 25 promoter variants with graded strengths was selected after characterization in S. elongatus and three additional model cyanobacterial strains. To demonstrate the utility of these promoters, we isolated new genetically tractable cyanobacterial strains with high salt and alkalinity tolerance and transferred the subset of promoters into one of these newly isolated strains. Similar to the results with model strains, the subset of promoters had a wide range of expression levels in the non-model strain. These characterized promoters expand the genetic tools available for genetic engineering of model and non-model cyanobacterial strains. ImportanceThe use of cyanobacteria to produce renewable products will require engineered expression of many genes that affect cell growth, metabolism, and agronomic properties, leading to efficient production of biomass and desired products. Engineering the strength of gene transcription is an important element of overall gene expression levels. The set of constitutive promoters described here, with a wide range of expression strengths characterized in several diverse cyanobacterial strains, provides an important resource for genetic engineering required for biotechnology applications. Research AreasMicrobial genetics, plasmids and other genetic constructs, biotechnology Journal SecctionBiotechnology

The enzymatic depolymerization of polyethylene terephthalate (PET) presents a sustainable route for plastic circularity, but its industrial viability is disadvantaged by the need for thermostable enzymes that remain active under mild, energy-efficient conditions. While the Polyester Hydrolase Leipzig 7 (PHL7) rapidly degrades amorphous PET near its melting point, its poor protein expression, inactivation issues at temperatures above 60{degrees}C and slow depolymerization activity below 60{degrees}C limit its practical application. Here, we employ inverse folding models ProteinMPNN and LigandMPNN, informed by structural and evolutionary information, to redesign the sequence of PHL7, aiming to improve protein expression, thermal stability and activity. From 36 designed variants, we identified two (termed D5 and D11) with significantly enhanced PET depolymerization rates at lower temperatures, where enzymatic performance is typically limited. Remarkably, design D5 at 50{degrees}C achieved the same product yield as PHL7 at 70{degrees}C in 24 h PET microparticle degradation assays, with a shifted product profile favoring mono-(2-hydroxyethyl) terephthalate (MHET) over terephthalic acid (TPA). Molecular dynamics simulations revealed that the active redesigns exhibit enhanced local flexibility in key active site regions at 50{degrees}C, providing a mechanistic understanding of their low-temperature catalysis. This work demonstrates that computational sequence redesign can optimize biocatalysts for lower production costs and milder operational conditions. Furthermore, the D5 variant enables a potential route to resynthesize virgin PET via MHET polycondensation, offering an efficient circular economy pathway.

Mammalian cell lines are the preferred hosts for producing commercially relevant therapeutic proteins such as antibodies, multispecifics, and cytokine fusion proteins. Even though significant investment is made to optimize upstream and downstream processes, the optimal gene design parameters for heterologous recombinant protein expression remain poorly understood. We describe here a generic approach to gene optimization in which design features are systematically sampled and modulated iteratively using machine learning (ML). Synthetic genes encoding the Dasher fluorescent protein, differing only in synonymous codons, were used to interrogate the gene-sequence preferences of transient antibody-expressing HEK293 cells. Synonymous codon variations influenced expression by more than two orders of magnitude. This variation in protein yield was used to build ML models relating gene design features, which were then employed to design further-improved genes. The ML models were shown to be expression system-specific. Messenger RNA levels and ribosome occupancy were highly correlated with protein levels, suggesting that mRNA lifetime has a causal relationship with coding bias. Our results illustrate a novel, generally applicable method to improve gene expression via synonymous re-coding for any protein target or host cell.

Zymomonas mobilis is an ethanologenic Alphaproteobacterium with many interesting characteristics for fundamental research and applied microbial engineering. Although genetic engineering has been established for Z. mobilis since the 1980s, a rich set of inducible transcriptional regulators is still unavailable. In this work, seven different chemically inducible promoters have been systematically tested for their functionality in Z. mobilis. In particular, for the first time, NahR-PsalTTC, VanRAM-PvanCC, CinRAM-Pcin and LuxR-PluxB have been characterized in Z. mobilis, alongside the commonly used regulator-promoter pairs TetR-Ptet and LacI-PlacT7A1_O3O4, and the less commonly used XylS-Pm. All promoters investigated in this work are compatible with the Golden Gate modular cloning framework Zymo-Parts. Characterization was carried out with a shuttle vector backbone based on pZMO7, which has so far been rarely used for applications in Z. mobilis but seems to be completely stable without selection and generates high and uniform levels of expression. From the experimental results presented, it can be concluded that VanRAM-PvanCC and CinRAM-Pcin are particularly promising for broad use in the Z. mobilis community. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/712268v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16579e6org.highwire.dtl.DTLVardef@1262533org.highwire.dtl.DTLVardef@15456a2org.highwire.dtl.DTLVardef@3af98_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cultivated meat has undeniable potential to address some of the current detrimental impacts of animal farming, while addressing food security worldwide. However, one of the main challenges in cultivated meat production is manufacturing cost. The main contributor to cost is the culture media which comprises expensive components such as growth factors and animal-derived proteins. This study investigated alternative, food grade, high protein extracts as serum replacements in serum-free media formulations. The extracts were chosen to represent various sustainable sources of proteins: marine (spirulina e.g. cyanobacterium), plant (faba bean) and insect (mealworm flour). Different processing methods and different solvents were investigated for production of cell culture-compatible extracts which were then tested with mouse myoblasts (C2C12) and primary porcine myosatellites (pMyoSCs). A serum-free medium formulation containing 2.6% v/v spirulina extract was found to support long term growth of C2C12 cells for [~]10 population doublings compared to only [~]2 in the control. The processing steps were optimized, showing that a glycerine solution was best for free amino acid and protein yield (4950 {micro}M total free amino acids, 11.45 mg/mL protein concentration). This solution had a positive effect on C2C12 cells, increasing their growth by up to 20% when added to the B8 medium. However, this benefit did not translate to pMyoSCs, which showed no significant growth increases in short-term screening. This work demonstrates a method for converting food grade protein powders into effective culture media supplements and highlights the potential of spirulina-based extracts for the use in cultivated meat. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/702276v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@206658org.highwire.dtl.DTLVardef@11f28ceorg.highwire.dtl.DTLVardef@b00fd6org.highwire.dtl.DTLVardef@dfaaf4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Created in BioRender. Gordon-Petrovskii, W. (2025) https://BioRender.com/by7khs1 C_FIG

Pseudomonas putida KT2440, renowned for its diverse metabolic capabilities, is a promising platform for downstream processing and revalorization of recalcitrant molecules. In this study, we examined and optimized P. putida KT2440s ability to utilize long-chain alcohols. These molecules are byproducts of the degradation of polyethylene (PE), the most widely used plastic. Using them as feedstock for microbial growth would close the plastic-derived carbon cycle, reducing environmental pollution. First, we discovered that P. putida KT2440 can use long-chain alcohols as the sole carbon and energy source. Using adaptive laboratory evolution (ALE), we generated variants with improved growth rates on long-chain alcohols, specifically 1-hexadecanol and 1-eicosanol. Mutations that became fixed during ALE provided insights into the mechanism, highlighting the importance of cell-substrate interaction. By heterologously expressing a hydrocarbon transporter-encoding gene, we successfully reproduced the ALE-derived phenotype, demonstrating that the bottleneck in long-chain alcohol utilization is not substrate transformation but uptake. These findings lay the groundwork for the potential application of P. putida KT2440 for the degradation of PE.

Biological carbon fixation is currently limited to seven naturally occurring pathways, each with its own limitations and constraints. In recent years, computational analyses of known biochemical reaction networks have identified dozens of theoretical carbon fixation pathways, some of which may have the potential to outperform their natural counterparts. This mix-and-match approach, however, cannot account for those reactions that have not been reported to occur in nature, which heavily limits the possible solution space. Here, we use a bioretrosynthetic approach coupled with expert biochemical knowledge to identify several novel pathways that leverage enzyme promiscuity and the latent biochemical reaction space. We analyze the thermodynamic, stoichiometric, and kinetic parameters of these pathways and compare them to the ubiquitous Calvin-Benson-Bassham cycle and previously proposed synthetic CO2 fixation cycles, highlighting advantages and disadvantages. We identify several promising pathways that could potentially outcompete the Calvin cycle and other previously proposed synthetic CO2 fixation pathways in predicted biomass yield and/or overall pathway activity. In addition, unlike most of the previously proposed efficient mix-and-match pathways, the pathways proposed in this work do not require vitamin B12, which is an advantage for future implementation in plants or microalgae that typically lack B12 biosynthesis. This work highlights the need for enzyme engineering and design in the quest for efficient biological carbon fixation.

Cell-free expression systems (CFES) are increasingly used alongside conventional biotechnological approaches to accelerate early-stage prototyping and are particularly valuable in point-of-use settings. However, their broader adoption remains limited by time- and cost-intensive preparation, as well as stringent cryogenic storage requirements. To address this, several studies have explored lyophilization with protective additives to generate stable, solid-state CFES. These approaches had to balance the protection gained with a loss of activity due to the additives. In this study, we present a CFES that contains a tardigrade-derived Cytosolic-Abundant Heat-Soluble (CAHS) protein to protect the biosynthetic machinery in lysates from damages during drying. We show that the CAHS protein, without any other additives, preserves protein synthesis activity during low-cost room temperature desiccation, while unprotected lysates are affected in mRNA synthesis kinetics and translation yields. The diversity of tardigrade-derived protective proteins is a treasure trove for cell-free synthetic biology, in particular for making CFES more accessible and portable. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/715078v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@8ecc2eorg.highwire.dtl.DTLVardef@ff0432org.highwire.dtl.DTLVardef@6c940eorg.highwire.dtl.DTLVardef@6c5390_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cells have the potential to utilize biological pathways to synthesize semiconductor nanomaterials, such as CdS quantum dots. As in chemical reaction schemes, biogenic synthesis requires control of the concentration and redox state of starting materials during the nucleation and growth of nanoparticles. Biological pathways regulate these key processes of particle synthesis, and manipulation of such pathways enables biological control of multiple aspects of nanoparticle synthesis. Here, strains of Escherichia coli were engineered to biosynthesize cadmium sulfide (CdS) quantum dots through the coordinated action of three pathways controlling sulfide generation, cadmium uptake, and nanoparticle nucleation. When exposed to low, micromolar concentrations of external cadmium, strains combining all three pathways produced CdS quantum dots. The synthesis of nanoparticles, nanoparticle yield, and nanoparticle size depended on the combination of pathways found in each strain. Cells lacking all three pathways produced no detectable nanomaterials, cells with specific combinations of one or two pathways produced small particles in the range of 1.95 to 7.9 nm, and cells with all three pathways produced the largest particles with average diameters of 11.78 nm. These results demonstrate that cells can be engineered to control multiple aspects of biogenic nanoparticle synthesis and that these pathways act together to tune the biosynthesis of semiconductor nanomaterials within cells. ImportanceMicrobes synthesize materials, including metallic and semiconductor nanomaterials. This capability stems from the natural ability of microbes to interact with and precisely manipulate metal atoms. Here, multiple biological pathways were combined within a single strain of Escherichia coli, creating a cell capable of producing CdS nanoparticles. This engineered cell controls multiple steps of particle synthesis, including metal uptake, reduction of starting materials, and binding cadmium and sulfide ions to initiate particle formation. Metal uptake by the cells was improved through the modification of a metal ion transport protein, improving cadmium uptake across the outer membrane and creating higher concentrations of cadmium within the cell. Cells with all three pathways were able to produce CdS nanoparticles, called quantum dots, even when exposed to low concentrations of external cadmium. This biotechnology enables nanomaterial synthesis under environmentally friendly conditions and may improve technologies using bacteria to clean up toxic metals.

Our increasing global population combined with the UN Sustainable Development Goals of zero hunger and good health require greater protein intake per capita and higher protein production. Consequently, sustainable food alternatives such as cultivated meat (CM) are urgently required. However, large-scale CM cell-systems face key challenges, particularly high media costs driven by amino acids and the need for ethically-sourced growth factors. Microalgae offer promising solutions, producing high protein yields with all essential amino acids simply from light, CO2, water and nutrients or spent CM media. Here we present Chlorella BDH-1 grown in spent CM media waste as a substitute-source for reduced amino acids and fetal bovine serum in cell culture media, enabling a circular strategy through beneficial mammalian cell-algae co-cultivation. We identified optimal algal growth conditions for maximum protein yield and demonstrated that two recycling rounds using industry-derived spent CM media maximize microalgal biomass yield per unit volume of waste media. We obtained algal lysate, determined thermal processing as the most cost-effective and mammalian cell-beneficial approach, and identified consumed lysate components. Compared to standard media, our lysate increased mammalian cell proliferation over 2-fold in reduced serum and amino acid conditions, replacing costly cell media components. We finally closed the loop by demonstrating a synergistic effect of the algal lysate with our co-cultivation - which co-produces algal biomass. The combination boosted mammalian cell proliferation 1.45-fold, conservatively estimating a media cost reduction by [~]66%. These findings establish parameters to advance the field towards cost-effective sustainable circular cell culture systems with applications in CM production and other biotechnology fields requiring large-scale tissue culture. Technology Readiness:

Sucrose synthase (SuSy) has been suggested as a key enabling enzyme for uridine diphosphate glucose (UDP-Glc) regeneration in glycosyltransferase-catalyzed biotransformations. However, its stability and efficiency in industrially relevant conditions have not been characterized or engineered, limiting its industrial readiness. Here, we combined enzyme discovery and characterization with comprehensive semi-rational enzyme engineering strategies, to optimize SuSys catalytic activity, thermostability, solvent tolerance, and soluble expression. The engineered variants were significantly more stable than wild-type, with up to 13.6 {degrees}C increase in melting temperature, over two orders of magnitude improvement in half-lives at elevated temperatures, and approximately three orders of magnitude increase in total turnover number. Additionally, the optimized variants retained up to 75% relative activity at 60 {degrees}C in the presence of 25% (v/v) DMSO, which the wild-type shows near complete loss of activity. Structural and molecular dynamics analyses reveal how mutations modulate conformational dynamics and hydrophobic packing, favoring catalytically competent conformations. Using methyl anthranilate glycosylation as a representative biotransformation, we demonstrate that the engineered SuSy variants consistently outperform both wild-type SuSy and stoichiometric UDP-Glc systems, enabling efficient UDP-Glc regeneration at reduced enzyme and sugar donor loadings. Finally, techno-economic and environmental assessments further indicate that implementation of engineered SuSy reduces reaction cost by approximately 6- and 2-fold relative to UDP-Glc and wild-type systems, respectively, while achieving average reductions of 3- and 2-fold in environmental end-point impacts. These results established SuSy engineering as a critical enabler for sustainable glycosylation reactions.

Poly({varepsilon}-caprolactone) (PCL) is a well-known biodegradable polyester and is among the few polyesters susceptible to degradation in marine environments; however, marine-derived PCL-degrading enzymes remain poorly characterized. Here, we searched for PCL-degrading enzymes from the marine bacterium Alloacanivorax gelatiniphagus JCM 18425 using a genome-based approach. Five candidate genes were predicted, and one encoded protein, designated Ag0826, was identified as a PCL depolymerase. Recombinant Ag0826 was expressed, purified, and biochemically characterized. The enzyme exhibited optimal activity at 35-40{degrees}C and pH 8.0, although it showed limited thermal stability. Substrate specificity was compared with that of leaf-branch compost cutinase (LCC), a well-characterized poly(ethylene terephthalate) (PET) hydrolase, using various polyesters. Both enzymes exhibited largely overlapping substrate ranges with respect to the presence or absence of monomer conversion activity across the tested substrates. Ag0826 slightly degraded PET to terephthalic acid, indicating potential PET-hydrolyzing activity; its conversion rate, however, was substantially lower than that of LCC, suggesting that Ag0826 exhibits a substrate preference differing from LCC. Phylogenetic analysis based on amino acid sequences revealed that Ag0826 formed a separate clade from LCC and IsPETase (from Ideonella sakaiensis). At a broader level, Ag0826 was positioned near HaloPETase1 (from Halopseudomonas pachastrellae), which has been proposed as a Type III PET hydrolase; in contrast, residues corresponding to the substrate-binding subsites were similar but not identical between the two enzymes. These results suggest that Ag0826 broadly belongs to the group of known PET hydrolases, yet it exhibits a partially distinct sequence profile even within this enzyme family.

Coacervate droplets and lipid vesicles are two classes of self-assembled compartments that have been proposed as protocell models. Hybrid protocells, in which a coacervate core is surrounded by a lipid membrane, can integrate the advantages of both protocell systems while overcoming their limitations. Although hybrid protocell membranes have been produced with a variety of diacyl phospholipids related to modern biology and some single-chain amphiphiles inspired by prebiotic scenarios, little is known about how mixtures of single-chain amphiphiles impact hybrid protocell membrane formation and properties. Given the plausible diversity of amphiphiles in the prebiotic milieu, the resulting membranes would have inherently incorporated multiple lipids of different types, potentially altering the properties and viability of hybrid protocells in their environment. Here, we systematically increased the compositional heterogeneity of hybrid protocell membranes by using different prebiotically relevant single-chain amphiphiles of varying head groups and alkyl chain lengths. These membranes were assembled around model coacervate droplets generated from polyallylamine hydrochloride and adenosine diphosphate, and the effect of heterogeneity on membrane properties and stability was evaluated. Compared to protocells with homogeneous membranes, those with heterogeneous amphiphile membranes exhibited higher yields, smaller sizes, and greater sub-compartment formation. Also, they showed increased membrane order, retained similar lateral lipid diffusion, and showed population-level variability in permeability to small anionic molecules. Notably, heterogeneous membranes showed enhanced structural stability under acidic conditions, retaining key properties like size and sub-compartment heterogeneity, thereby broadening the pH range over which hybrid protocells remain intact. These findings suggest that amphiphile diversity not only would have influenced the structural properties of hybrid protocells but also created diversity within the protocell population and enhanced their robustness, thereby playing a crucial role in protocell evolution on early Earth.