Carrageenans are essential texturizers in various dairy products, where they form elastic gel networks through specific interactions with casein micelles, enabling effective structure formation even at low concentrations. Plant-based milk alternatives on the other side, such as almond-or oat-based products, recently experience a rising consumer demand, but are devoid of these protein-polysaccharide interactions, which poses major challenges for their efficient texturization. Consequently, new mild, adaptable, and label-friendly gelation strategies are required to achieve desirable texture and stability in such systems. Here, we present an enzymatic approach to trigger gel formation in non-dairy matrices using the carrageenan sulfatase CaCgS1 from Cellulophaga algicola DSM 14237. The enzyme catalyzes the desulfation of x-carrageenan to a x/(3-hybrid carrageenan under mild conditions, which triggers the gelation of the reaction product in-situ and yields a homogenous hydrogel with enhanced gel strength and thermal stability by an increased helical aggregation activity of the modified polymer chains. The increased gel strength relies exclusively on the cleavage of sulfate groups without the need to alter the ionic composition or increase the polymer concentration. Applied to plant-based milk formulations, this process enables the generation of homogeneous, stable textures at low polymer concentrations. The concept provides a novel method for structuring plant-based foods and expands the application scope of enzyme-induced gelation in modern food design, and beyond.

Carbocyclic nicotinamide cofactors are attractive NAD(P)+ analogues that retain native redox activity while offering substantially enhanced chemical and thermal stability. Their broader use, however, has been limited by demanding multistep chemical syntheses involving complex protection strategies and limited exploration of enzymatic alternatives. In this study, we studied both chemical and enzymatic approaches for the synthesis of carba-analogues. For the enzymatic route, we identify and characterize the key enzymes involved in cofactor assembly, evaluating their tolerance toward non-native, carbocyclic substrates and extending the assembly to further generate the phosphorylated analogue, cNADP+. In addition, extending the analysis to cofactor thermostability, carba-NAD+ displayed a remarkable halftime (t 1/2 > 1386 h at 50 °C), far exceeding that of NAD+ (t 1/2 = 76 h) and is accepted by a broad panel of oxidoreductases. Collectively, this work outlines a modular workflow and details the synthesis landscape for accessing thermostable, synthetic nicotinamide cofactor analogues, cNAD+ and cNADP+, unveiling new opportunities for their application in cofactor engineering and synthetic biocatalysis.

Poly(3-hydroxybutyrate) (PHB) is the most extensively studied polyhydroxyalkanoate and has long been promoted as a sustainable alternative to petrochemical polymers due to its excellent biocompatibility and biodegradability. Despite decades of research, PHB has not achieved broad commercial adoption, in part because its ( R )-isotactic microstructure leads to undesirable material properties such as high crystallinity and poor processability. This stereoregularity is widely assumed to be a consequence of the strict stereoselectivity of PHA synthases. Here, we provide the first evidence that challenges this long-standing assumption. In a proof-of-concept study, we demonstrate that stereoirregular PHB containing 6.84% ± 0.04% ( S )-3 hydroxybutyrate with a m dyad fraction of 11.8% ± 0.7% can be produced in fed-batch fermentation at a productivity of (58 ± 9) mg L −1 h −1 . Importantly, the material properties of the resulting stereoirregular PHB align with improvements predicted from chemically synthesized PHB. Notably, the melting temperature reduced from 179.3°C ± 0.7°C to 154.3°C ± 1.1°C, which was accompanied by decreased molar mass decomposition during processing, suggesting improved recyclability of the stereoirregular polymer. These findings overturn key assumptions about PHA synthase stereoselectivity and open the door to microbial production of PHB with tunable material properties.

Multienzyme cascades are emerging as alternatives to fermentation for converting biogenic feedstocks into value-added chemicals and fuels. A major bottleneck in redox transformations is the loss of costly and unstable cofactors in continuous-flow systems, typically necessitating co-immobilization and complex regeneration schemes. Self-sufficient biocatalytic nanomachines, created by fusing enzymes with tethered cofactors, offer a streamlined solution by enabling intramolecular cofactor retention and recycling. Here, we demonstrate that such nanomachines can be integrated into a complete redox-dependent cascade, exemplified by a 10-step glucose-to-isobutanol pathway. Oxidoreductases were fused into redox pairs using peptide linkers containing a single cysteine for covalent attachment of polyethylene glycol (PEG)-modified nicotinamide cofactors, with rational selection of monomeric enzymes ensuring structural compatibility and soluble expression. The resulting nanomachines remained catalytically competent and produced isobutanol in both batch and continuous-flow operation without the addition of free NAD+. A total turnover number of ∼1.4 × 104 was achieved for tethered PEG-NAD+, among the highest reported for immobilized nicotinamide cofactors. Use of the ultrastable analogue PEG-cNAD+ further demonstrates the modularity of the platform. Together, these results establish a proof-of-concept for cofactor-autonomous multienzyme redox cascades in continuous flow and define design principles for future cell-free synthesis systems.

5-Keto-4-deoxy glucarate dehydratase and 2-keto-3-deoxy-L-arabinonate dehydratase are members of the dihydrodipicolinate synthase superfamily of aldolase class I enzymes. The enzymes are part of the oxidative nonphosphorylated metabolic pathways for the production of 2-ketoglutarate from glucuronic acid and arabinose, respectively. They carry out a C4 dehydration on their substrates, 5-keto-4-deoxy glucarate and 2-keto-3-deoxy-L-arabinonate, and produce the same product, ketoglutarate semialdehyde. While the enzymes are similar in structure and reaction mechanism, they exhibit a high substrate specificity and have only been reported to be active on their native substrate. In order to investigate the high substrate specificity, we aimed to generate promiscuous variants of both enzymes that can accept multiple substrates and investigate the evolutionary development of the substrate specificity in the superfamily. Ancestral sequence reconstruction and GFN2-xTB calculations were used to develop candidates for further analysis of substrate scope. Both approaches yielded multiple promiscuous dehydratases with activity on both substrates.

The expanding application of deep eutectic solvents (DESs) in both upstream and downstream processes has brought increasing attention to the role of pH in these systems. Despite its importance, accurate pH determination in DESs remains a significant challenge, mainly due to the lack of standardized measurement protocols. This has resulted in limited and sometimes contradictory data in literature. In this study, we investigate the acid-base properties of representative DESs using two complementary techniques: pH electrode measurements and UV-vis spectrophotometry with pH-sensitive indicators. The DESs examined include hydrophilic systems based on choline chloride, betaine, and tetrabutylammonium chloride combined with glycerol or ethylene glycol, as well as a hydrophobic lidocaine-oleic acid system. Indicator solutions were titrated with hydrochloric acid, while absorbance and electrode pH values were recorded across each dye's transition range. The resulting absorbance-pH curves were fitted using an inverse Boltzmann model, showing excellent correlation (R-2 > 0.99) and enabling reliable interpolation. Additionally, Gaussian process (GP)-based machine learning (ML) confirmed strong agreement between methods within a 95% confidence interval. This integrated approach provides a robust framework for accurate pH assessment in DESs.

Xanthan is a structurally complex exopolysaccharide produced by Xanthomonas campestris and one of the most extensively studied microbial biopolymers. As a sustainable alternative to petroleum-based polymers, its broader application requires precise control of polysaccharide decoration, yet the enzymatic basis of these modifications remains incompletely understood. Here, we characterise the activity and substrate scope of GumG, an AT-3 domain-containing membrane-bound acetyltransferase responsible for xanthan O-acetylation. Using mass spectrometry in combination with in vitro and in vivo assays, we show that GumG mediates non-specific acetylation of the outer mannose residue and displays pronounced substrate promiscuity. GumG also exhibits limited propionyltransferase activity, enabling the biosynthesis of hybrid acetylated-propionylated xanthan at an 8.27:1 ratio. Molecular docking and analysis of 31 xanthan variants identify a cytoplasmic substrate-binding pocket defined by Val67 and Phe71 that governs donor specificity, and an engineered GumG variant (F71L) shows enhanced propionyltransferase activity. In addition, a periplasmic His40-Trp143-Asp246-His297 motif is proposed to constitute the catalytic center. Together, these findings provide mechanistic insight into GumG multifunctionality and establish a framework for engineering xanthan derivatives with tailored physicochemical properties.

Poly(3-hydroxybutyrate) (PHB) is a biocompatible and biodegradable polyhydroxyalkanoate, but its highly regular stereomicrostructure causes high crystallinity and poor processability. Here, we demonstrate that stereoirregular PHB can be produced microbially, challenging the view that strict PHA synthase stereospecificity makes such structures inaccessible. In a proof-of-concept study, we produced stereoirregular PHB containing 6.84% ± 0.04% (S)-3-hydroxybutyrate, with an m-dyad fraction of 11.8% ± 0.7%. Compared with conventional PHB, the stereoirregular material exhibited a reduced melting temperature, from 179.3 °C ± 0.7 to 154.3 °C ± 1.1 °C, and decreased molar-mass decomposition during processing. These results demonstrate that microbial synthesis can access stereochemically diverse PHB. Importantly, they show that biologically produced polymers can exhibit the improved processability and recyclability previously associated with chemically synthesized analogues. These findings overturn a long-standing assumption in PHB biosynthesis and provide a basis for biologically tuning polymer microstructure and performance through microbial engineering.

Amino acids are the building blocks of proteins and thus among the macronutrients to feed humankind. They have extensive industrial applications as animal feed and food additives like dietary supplements, and flavor enhancers and moreover as chemical precursors. We present the design of a synthetic enzyme cascade system for synthesizing a series of amino acids from methanol. This acts as a case study on how to contribute to the sustainable, renewable energy-based supply of food and feed. The modular nature of the cascade allows for plug-and-play module swapping and fine tuning of enzyme composition to customize the target compound. The one-pot enzymatic systems, capable of utilizing methanol, ammonia, and, partially, carbon dioxide, were employed to synthesize glycine, serine, L-aspartic acid, L-valine, L-glutamic acid, and L-proline. Considering the increasing availability of methanol produced from carbon dioxide through thermocatalytic and even electrocatalytic and photocatalytic processes, methanol represents a key intermediate in future CO2-based value chains. In this context, our study provides a pathway for amino acid synthesis and food and feed production based on methanol and CO2 as carbon building blocks with reduced environmental impact.

The genetic and physiological diversity of bacteria are critical resources for discovering new exopolysaccharides (EPS) as raw materials with biotechnological applications. However, uncovering new EPS is limited by their lack of production in laboratory cultures, as EPS are often cryptic, and their biosynthesis only proceeds upon unknown environmental cues. The dinucleotide cyclic diguanosine monophosphate (c-di-GMP) has emerged as a universal second messenger in bacteria and a common activator of many EPS. Here, a pleD* transformation to elevate intracellular c-di-GMP levels and a carbohydrate fingerprinting analysis were combined for high-throughput screening of 330 bacterial strains (~70% exhibiting robust growth under the conditions tested) in search of c-di-GMP-activated EPS. Nearly 10% of strains were revealed as promising candidates to overproduce novel EPS composites in a c-di-GMP-dependent manner. Under these conditions, Sphingomonas sp. SphC10 massively produced an EPS with an unusual monosaccharide composition compared with known biotechnologically relevant sphingans.

5‐Keto‐4‐deoxy glucarate dehydratase and 2‐keto‐3‐deoxy‐L‐arabinonate dehydratase are members of the dihydrodipicolinate synthase superfamily of aldolase class I enzymes. The enzymes are part of the oxidative nonphosphorylated metabolic pathways for the production of 2‐ketoglutarate from glucuronic acid and arabinose, respectively. They carry out a C4 dehydration on their substrates, 5‐keto‐4‐deoxy glucarate and 2‐keto‐3‐deoxy‐L‐arabinonate, and produce the same product, ketoglutarate semialdehyde. While the enzymes are similar in structure and reaction mechanism, they exhibit a high substrate specificity and have only been reported to be active on their native substrate. In order to investigate the high substrate specificity, we aimed to generate promiscuous variants of both enzymes that can accept multiple substrates and investigate the evolutionary development of the substrate specificity in the superfamily. Ancestral sequence reconstruction and GFN2‐xTB calculations were used to develop candidates for further analysis of substrate scope. Both approaches yielded multiple promiscuous dehydratases with activity on both substrates.

Navigating trade-offs between efficiency, specificity, and stability is a central challenge in biocatalyst design. Ancestral sequence reconstruction (ASR) is an effective approach for such design as it leverages the natural trajectories of evolution to infer sequences with different properties under a variety of selective pressures. Here, ASR was applied to systematically map the catalytic landscape of the NAD(P)H-dependent ketol-acid reductoisomerase (KARI) superfamily, which catalyze the rate-limiting step ((S)-2-acetolactate to (R)-2,3-dihydroxy-3-isovalerate (DHIV)), in the conversion of glucose, a renewable feedstock, to the platform chemical, isobutanol. Reconstructed ancestral variants exhibit a variety of properties not seen in extant KARIs, including enhanced catalytic efficiencies (up to 25-fold), longevity (total turnover numbers >10(6)), and melting temperatures approaching 100 degrees C (40 degrees C above benchmarks). Notably, we expose latent high-performance states within this fitness landscape; one ancestral variant (N79) displays solvent-mediated catalytic activation, exemplifying an enzyme that combines resource-efficient catalysis, process-level robustness, and compatibility with high-yield production of isobutanol.

The integration of pretreatment and saccharification into a single operational step offers a streamlined strategy for converting lignocellulosic residues into fermentable second-generation hydrolysates. In this study, dilute acid pretreatment and subsequent enzymatic hydrolysis were conducted in one pot to simplify processing while maximizing sugar recovery. Direct pH adjustment of the pretreated cereal straw after dilute acid pretreatment enabled full retention of intermediately released monomeric and oligomeric sugars, eliminating losses typically associated with intermediate solid-liquid separation and washing steps. At an initial dry matter load of 20 wt.%, the one-pot process yielded combined sugar concentrations of up to 130 g L-1 and achieved combined polymer conversions of up to 89%, demonstrating its effectiveness for high-gravity biomass processing. Detailed insight into the distribution of saccharides, organic acids, and lignocellulose-derived inhibitory compounds was achieved by applying complementary analytical methods to comprehensively characterize the produced hydrolysates. Using non-detoxified one-pot hydrolysate as the main carbon source in fermentation medium resulted in robust growth of Saccharomyces cerevisiae Ethanol Red®, highlighting its compatibility with industrial yeast strains. These results underscore the potential of integrated one-pot processing as a cost-effective route for producing fermentable hydrolysates from cereal straw at a high initial dry matter load.

The marine-derived fungus Peniophora sp. CBMAI 1063 is a hyper-producer of laccase. Laccases are multicopper oxidases able to oxidize different aromatic compounds while reducing molecular oxygen to water. Several laccases from terrestrial environments have been purified and characterized. However, little is known about marine-derived laccases. In this study, Pnh_Lac1 (Lac1) from the fungus Peniophora sp. CBMAI 1063 was heterologously expressed in Pichia pastoris, purified, characterized, and used for the degradation/detoxification of synthetic dyes. Lac1 (similar to 72 kDa) exhibited optimal activity at 60 degrees C and pH 3, with good thermostability (T-50(1h) = 56 degrees C) and high tolerance to metal ions and organic solvents. Lac 1 decolorized/degraded different classes of dyes, under low enzyme concentrations (0.2-0.02 U mL(-1)), with an excellent performance regarding the decolorization of Indigo Carmine (93% after 2 h) in the presence of syringaldehyde. Additionally, 65% of the azo dye Reactive Black 5 was degraded by the Lac1-mediator system into lower molecular weight metabolites, with a significant reduction in phytotoxicity. These results demonstrate that the marine-derived Lac1 is a fungal laccase highly active under low concentration, with the potential to mitigate environmental pollutants on biodegradation strategies based on biocatalysis.

Biocatalytic oxidation provides a potentially efficient platform for chemical production, but the air-liquid interface formed in such reactions often affects the biocatalyst performance. Here, we report the kinetic stability of alcohol dehydrogenase (ADH), NADH oxidase (NOX), and associated cofactors upon exposure to various gas-liquid interfaces in a defined interface apparatus. Air and oxygen were bubbled individually to distinguish their effects. The observed loss of enzyme from the solution and the activity were mostly attributed to the enzyme removal via the gas-liquid interface, foaming, and aggregation. An engineered ADH with enhanced interactions between subunits showed a longer half-life and improved kinetic stability at interfaces. Meanwhile, four different NOX enzymes were also tested, resulting in different stability profiles. This study shows the importance of selecting the appropriate enzymes for oxygen-dependent biocatalytic processes and further highlights the importance of the relevant process equipment and conditions to test biocatalysts for subsequent scale-up.

5-(Aminomethyl)-2-furancarboxylic acid (AMFCA) represents a biogenic replacement to fossil-based monomers for the production of semi-aromatic polyamides, thermoplastics widely used in the automotive, electrical and packaging industries. AMFCA has been synthesized via chemical, chemo-enzymatic and enzymatic one-pot two-step transformations. Herein, the conversion of 5-(hydroxymethyl)-furfural (HMF) to AMFCA is explored through a one-pot, one-step, cell-free, enzymatic process, reaching titers in the range of 3.35-4.62 g L-1. The four-enzyme cascade includes an engineered HMF oxidase from Methylovorus sp. MP688, an aldehyde dehydrogenase from Sphingobium sp. SYK-6 and an omega-transaminase from Chromobacterium violaceum as main biocatalysts. l-Alanine is used as an amine donor, sustainably regenerated by an alanine dehydrogenase, which also guarantees an intrinsic nicotinamide cofactor balance. As environmental metrics, the E-factor and the "Global Warming Potential" (GWP, kg CO2 per kg AMFCA) are introduced for both the upstream and downstream processes of AMFCA production for the first time. The sustainability assessment provides valuable insights into the hotspots to be improved for minimized environmental burden: operating at high substrate loadings, generation of wastewater effluents that can be mildly treated, optimized volume-to-product ratios in chromatographic steps, and introduction of renewable energy in the chemical plant.

Integration of electrochemical CO2 reduction with microbial fermentation enables conversion of CO2 into valuable chemicals but poses challenges at the electrolysis-fermentation interface. The electrolyte must ensure efficient CO2 reduction while remaining compatible with microbial growth. We investigated various electrolytes for coupling CO2 electroreduction to formate with formate fermentation by Methylorubrum extorquens TK 0001. Electrolyte performance was evaluated by formate production and microbial growth. A phosphate-based buffer demonstrated the best overall compatibility. Optimal microbial growth occurred at 0.1 mol L-1 KPi, with tolerance of up to 111 mmol L-1 formate. Continuous CO2 electrolysis in 1.0 mol L-1 KPi produced 2.0 mol L-1 formate in 48 h. Formate fermentation with M. extorquens showed biomass yield of 107 mg CDW gformate -1 and a growth rate of 0.10 h-1. These results highlight the crucial role of buffer composition and concentration in balancing efficient CO2 electroreduction with stable fermentation. Optimizing this electrochemical-biological interface enables direct utilization of CO2-derived formate as a substrate for sustainable microbial production, offering a promising scalable route for industrial biotechnology.

This study presents the development of coatings as biohybrid UV sensors containing E. coli biomass modified with photoconvertible fluorescent proteins as the responsive elements. These sensors indicate UV light exposure by a color shift from green to red, occurring within 15 min of irradiation with UV‐A light at 400 nm. The green coatings demonstrate the ability to undergo photoconversion after extended storage (>1 year), resulting in a stable and irreversible change in color. This research highlights the potential for more sustainable formulations incorporating biomass as a substitute filler. These fillers can be seen as a platform to equip coatings with special functionalities like providing color or UV sensing capability.