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We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6-2.8 angstrom) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma. Development of vaccines remains challenging because viral antigens can be unstable or aggregate. Here, authors present ancestral sequence reconstruction as a method to generate stable and soluble antigens using exclusively available sequence information., QC 20240206

Recycling plastics is the key to reaching a sustainable materials economy. Biocatalytic degradation of plastics shows great promise by allowing selective depolymerization of man-made materials into constituent building blocks under mild aqueous conditions. However, insoluble plastics have polymer chains that can reside in different conformations and show compact secondary structures that offer low accessibility for initiating the depolymerization reaction by enzymes. In this work, we overcome these shortcomings by microwave irradiation as a pre-treatment process to deliver powders of polyethylene terephthalate (PET) particles suitable for subsequent biotechnology-assisted plastic degradation by previously generated engineered enzymes. An optimized microwave step resulted in 1400 times higher integral of released terephthalic acid (TPA) from high-performance liquid chromatography (HPLC), compared to original untreated PET bottle. Biocatalytic plastic hydrolysis of substrates originating from PET bottles responded to 78 % yield conversion from 2 h microwave pretreatment and 1 h enzymatic reaction at 30 °C. The increase in activity stems from enhanced substrate accessibility from the microwave step, followed by the administration of designer enzymes capable of accommodating oligomers and shorter chains released in a productive conformation., QC 20231114

2,5-furandicarboxylic acid (FDCA) has gained great industrial interest as a renewable alternative to terephthalic acid (TPA) in the generation of bioplastics. However, chemical production of FDCA involves harsh reaction conditions not aligned with sustainable manufacturing. Herein, we demonstrate the use of whole-cell mediated synthesis of FDCA from 2-furoic acid (FA) as substrate. Our approach moves away from the use of isolated enzymes by supplementing the UbiD−UbiX system in E. coli with the gene of P. thermopropionicum HmfF (PtHmfF) known to generate FDCA. The resulting whole-cell system allows for production of FDCA under mild conditions by carboxylation of FA. We show how the enzymatically produced FDCA can be used to generate FDCA-based biopolymers along with a terpene-based diol monomer by enzymatic polycondensation catalyzed by Candida antarctica lipase B (CALB). This work high-lights how underutilized hemicellulose-derived C5 building blocks can be converted into renewable platform chemicals and materials by a simple cell factory in a CO2 sequestration process., QC 20230629

Bio-based plastics potentially have several positive impacts on the environment; however, in order to make a real difference, they need to have managed and sustainable end of life. This means they should from the start be designed for chemical, mechanical, and/or organic (biological) recycling. Development of energy-efficient and selective chemical recycling processes is a necessary part in reaching truly circular plastic flows. Polyesters are generally well suited for chemical recycling due to the presence of reversible ester bonds. Utilization of dynamic covalent chemistry to include a second, even more easily reversed bond, such as Schiff base (SB, imine bond), could further facilitate chemical recycling, enabling depolymerization back to monomeric products under mild conditions. Here, we present the synthesis of three vanillin-derived SB monomers SBM1, SBM2, and SBM3 and the corresponding polymers SBP3a-b, SBP4a-b, and SBP5a-b. Three different diamines and two potentially bio-sourced diesters were utilized to yield altogether six different polyester-imines with different aliphatic/aromatic contents. All the obtained SB-based polyesters were thermally stable at ∼290-330 °C and had a high char yield during the pyrolysis, which may indicate inherent flame resistance. All the polyesters were amorphous with glass transition temperatures from 36 to 76 °C. The chemical recyclability and hydrolytic degradation of the synthesized polyesters was evaluated by using real-time 1H NMR spectroscopy. Finally, the susceptibility of the synthesized polyester-imines to enzymatic degradation by PETase was demonstrated. The experimental results were further supported by induced-fit docking experiments to theoretically evaluate the potential productive binding of the produced polyester-imines and intermediates thereof to the active site of PETase., QC 20230629

On the occasion of Professor Bernhard Hauer′s (partial) retirement, we reflect on and highlight his distinguished career in biocatalysis. Bernhard, a biologist by training, has greatly influenced biocatalysis with his vision and ideas throughout his four-decade career. The development of his career went hand in hand with the evolution of biocatalysis and the application and development of enzymes for chemical processes. In this Account, we present selected examples of his early work on the development of enzymes and their application in an industrial setting, with a focus on his specific contributions to harnessing the catalytic power of enzymes for novel reactions and the understanding and engineering of flexible loops and channels on catalysis., QC 20230817

Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation., Not duplicate with DiVA 1608437QC 20230307

QC 20220111

Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent-copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the its timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 degrees C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme., QC 20220627

Generation of renewable polymers is a long-standing goal toward reaching a more sustainable society, but building blocks in biomass can be incompatible with desired polymerization type, hampering the full implementation potential of biomaterials. Herein, we show how conceptually simple oxidative transformations can be used to unlock the inherent reactivity of terpene synthons in generating polyesters by two different mechanisms starting from the same alpha-pinene substrate. In the first pathway, alpha-pinene was oxidized into the bicyclic verbanone-based lactone and subsequently polymerized into star-shaped polymers via ring-opening polymerization, resulting in a biobased semicrystalline polyester with tunable glass transition and melting temperatures. In a second pathway, polyesters were synthesized via polycondensation, utilizing the diol 1-(1'-chydroxyethyl)-3-(2'-hydroxyethyl)-2,2-dimethylcyclobutane (HHDC) synthesized by oxidative cleavage of the double bond of alpha-pinene, together with unsaturated biobased diesters such as dimethyl maleate (DMM) and dimethyl itaconate (DMI). The resulting families of terpenebased polyesters were thereafter successfully cross-Iinked by either transetherification, utilizing the terminal hydroxyl groups of the synthesized verbanone-based materials, or by UV irradiation, utilizing the unsaturation provided by the DMM or DMI moieties within the HHDC-based copolymers. This work highlights the potential to apply an oxidative toolbox to valorize inert terpene metabolites enabling generation of biosourced polyesters and coatings thereof by complementary mechanisms., QC 20220112

Although recovery of fibers from used textiles with retained material quality is desired, separation of individual components from polymer blends used in today's complex textile materials is currently not available at viable scale. Biotechnology could provide a solution to this pressing problem by enabling selective depolymerization of recyclable fibers of natural and synthetic origin, to isolate constituents or even recover monomers. We compiled experimental data for biocatalytic polymer degradation with a focus on synthetic polymers with hydrolysable links and calculated conversion rates to explore this path The analysis emphasizes that we urgently need major research efforts: beyond cellulose-based fibers, biotechnological-assisted depolymerization of plastics so far only works for polyethylene terephthalate, with degradation of a few other relevant synthetic polymer chains being reported. In contrast, by analyzing market data and emerging trends for synthetic fibers in the textile industry, in combination with numbers from used garment collection and sorting plants, it was shown that the use of difficult-to-recycle blended materials is rapidly growing. If the lack of recycling technology and production trend for fiber blends remains, a volume of more than 3400 Mt of waste will have been accumulated by 2030. This work highlights the urgent need to transform the textile industry from a biocatalytic perspective., QC 20211130

We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells., QC 20210419

Polar polythiophenes with oligoethylene glycol side chains are exceedingly soft materials. A low glass transition temperature and low degree of crystallinity prevents their use as a bulk material. The synthesis of a copolymer comprising 1) soft polythiophene blocks with tetraethylene glycol side chains, and 2) hard urethane segments is reported. The molecular design is contrary to that of other semiconductor-insulator copolymers, which typically combine a soft nonconjugated spacer with hard conjugated segments. Copolymerization of polar polythiophenes and urethane segments results in a ductile material that can be used as a free-standing solid. The copolymer displays a storage modulus of 25 MPa at room temperature, elongation at break of 95%, and a reduced degree of swelling due to hydrogen bonding. Both chemical doping and electrochemical oxidation reveal that the introduction of urethane segments does not unduly reduce the hole charge-carrier mobility and ability to take up charge. Further, stable operation is observed when the copolymer is used as the active layer of organic electrochemical transistors., QC 20210409

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology., QC 20210412

The polymerization of a bio-based terpene-derived monomer, sobrerol methacrylate (SobMA), was evaluated in the design of polymeric nanoparticles (nanolatexes). Their synthesis was accomplished by using emulsion polymerization, either by free-radical polymerization in the presence of a cationic surfactant or a cationic macroRAFT agent by employing RAFT-mediated polymerization-induced self-assembly (PISA). By tuning the length of the hydrophobic polymer, it was possible to control the nanoparticle size between 70 and 110 nm. The average size of the latexes in both wet and dry state were investigated by microscopy imaging and dynamic light scattering (DLS). Additionally, SobMA was successfully copolymerized with butyl methacrylate (BMA) targeting soft-core nanolatexes. The comparison of the kinetic profile of the cationically stabilized nanolatexes highlighted the differences of both processes. The SobMA-based nanolatexes yielded high T-g similar to 120 degrees C, while the copolymer sample exhibited a lower T-g similar to 50 degrees C, as assessed by Differential Scanning Calorimetry (DSC). Thereafter, the nanolatexes were adsorbed onto cellulose (filter paper), where they were annealed at elevated temperatures to result in polymeric coatings. Their morphologies were analysed by Field Emission Scanning Electron Microscopy (FE-SEM) and compared to a commercial sulfate polystyrene latex (PS latex). By microscopic investigation the film formation mechanism could be unravelled. Water contact angle (CA) measurements verified the transition from a hydrophilic to a hydrophobic surface after film formation had occured. The obtained results are promising for the toolbox of bio-based building blocks, focused on sobrerol-based monomers, to be used in emulsion polymerizations either for tailored PISA-latexes or facile conventional latex formation, in order to replace methyl methacrylate or other high T-g-monomers., QC 20210409

Enzyme-catalyzed transformations have a great potential in both the pharmaceutical and chemical industry to achieve complex and (stereo)selective synthesis under mild reaction conditions. Still, the implementation of biocatalysis in the prerequisite upgrading of inert synthons into activated monomers for polymer applications has not yet been fully realized. In this contribution, we show that scaled-up synthesis of bicyclic norcamphor lactone using an engineered Baeyer–Villiger monooxygenase (BVMO) is feasible to reach complete conversion of the corresponding ketone in 24 h in shake-flask. The lactone monomer obtained by enzyme catalysis was copolymerized with ε-caprolactone via ring-opening polymerization to study the impact of the additional ring on material properties. Moreover, four-arm star-like, homo and block copolymers were designed from ε-caprolactone, ε-decalactone, and norcamphor lactone and characterized for their structural and thermal properties. These newly explored macromolecules were functionalized with furan rings using the enzyme Candida antarctica lipase B which allowed the formation of thermolabile networks via the pericyclic reaction with bismaleimide by means of Diels–Alder chemistry. The bonding/debonding state of these star-like based materials can be tuned by a suitable selection of thermal treatment. The temperature-dependent reversibility was assessed by thermal analysis and solubility test. Our results presented here shed light on the high potential of the use of chemoenzymatic approaches in the synthesis of new functional materials with tuned physiochemical properties., QC 20200421