Amide bond formation is a basal transformation in synthetic chemistry and the pharmaceutical industry that is traditionally performed under harsh conditions, using excess amounts of amine and relying on coupling agents. Biocatalysis shows great potential in contributing to milder and more sustainable amide bond formation in water, in particular using the emerging family of amide bond synthetase (ABS) enzymes. Here, we use molecular dynamics, biocatalysis, and enzyme engineering to study amide bond formation in extant and ancestral ABS from Marinactinospora thermotolerans (McbA). Our results show that while being more thermostable, the C-terminal domain that delivers the amine substrate to the adenylated acid intermediate is more flexible in ancestral McbA, presumably leading to an extended amine scope as observed experimentally from a small panel of aliphatic and aromatic substrates. An engineered ancestor of McbA harboring a single mutation that presumptively represent a catalytic shift residue when going from ancestral to modern biocatalyst, show two to ten-fold improved conversions over its ancestral template while maintaining high thermostability, highlighting ancestral sequence reconstruction as a potent method in protein engineering. Kinetic experiments showed that the engineered ancestral enzyme had 2-fold higher apparent kcat values in amide formation compared to extant enzyme, concomitant with relaxed substrate inhibition and loss-of-dependency on magnesium. Finally, we optimize ATP recycling utilizing a single polyphosphate kinase to showcase how engineered ancestral McbA together with reaction optimization is amenable for pharmacophore synthesis at a preparative scale.

Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. The relatively low specific activity and tendency to accept O2 as a substrate have made Rubisco an attractive but challenging target for enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO2 and O2 levels. We address the challenge of designing a zero-shot input library of the Gallionella Rubisco, without prior experimental knowledge, by coupling the phylogenetically guided model EV mutation with "in silico evolution". This multisite mutagenesis library of Synechocystis (n = 16) was subjected to competitive growth in different gas feeds coupled to deep sequencing, in order to compare Rubisco variants. We identified an amino acid exchange that increased the thermostability of Gallionella Rubisco and conveyed resilience to otherwise detrimental amino acid exchanges. The platform is a first step toward high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts.

Accelerated production of recyclable and biodegradable polymers is crucial in combating the socioeconomic and environmental issues connected to traditional plastics. While renewable diacids have been in the spotlight for the generation of biobased polyesters with tailored properties by varying the alkyl chain length, capitalizing on diols from biomass for this purpose is underexplored and has mainly focused on linear and branched shorter chain alcohols. Here, we explored the potential of two (-)-alpha-pinene-derived diols (PDOs) as building blocks to generate biobased polyesters harboring bicyclic ring structures in their backbones that can mimic aromatic fossil-based plastics' properties. We demonstrate a concise synthesis of two novel unsymmetrical chiral PDOs on the 20-40 g scale, together with eight structurally differing heat-resistant polyesters, as reflected by high glass transition (T g ) temperatures (90 and 121 degrees C) for two of the polymers. The stereochemistry of PDO-derived polyesters is guided by intramolecular hydrogen bonding made possible by the protruding rings and the polyester backbone. Most of the synthesized polyesters (five) in this study showed potential as adhesives based on the analysis of tensile strength and adhesive properties on paper boards. The steric hindrance of the intact bicyclic alpha-pinene ring structure protruding from the backbone of the polymers can also aid in the degradation process, manifested by facile chemical recycling of these polyesters under mild conditions to recover both monomers. Finally, our results show how the generated rigid polymers are susceptible to enzymatic degradation by PETase and cutinase without any chemical pretreatment. Our results illuminate the potential of expanding the current scope of biobased monomers to bicyclic diols to generate biomaterials with tailor-made properties.

Plastic waste management is challenged by the inefficiencies and environmental impact of traditional chemical recycling methods. Here, the authors explore the chemoenzymatic cascade depolymerization approach, which offers a promising and sustainable solution for transforming plastic waste into valuable products.

Enzymatic degradation of polymers holds promise for advancing towards a bio-based economy. However, the bulky nature of polymers presents challenges in accessibility for biocatalysts, hindering depolymerization reactions. Beyond the impact of crystallinity, polymer chains can reside in different conformations affecting binding efficiency to the enzyme active site. We previously showed that the gauche and trans chain conformers associated with crystalline and amorphous regions of the synthetic polyethylene terephthalate (PET) display different affinity to PETase, thus affecting the depolymerization rate. However, structural-function relationships for biopolymers remain poorly understood in biocatalysis. In this study, we explored the biodegradation of previously synthesized bio-polyesters made from a rigid bicyclic chiral terpene-based diol and copolymerized with various renewable diesters. Herein, four of those polyesters spanning from semi-aromatic to aliphatic were subjected to enzymatic degradations in concert with induced-fit docking (IFD) analyses. The monomer yield following enzymatic depolymerization by IsPETase S238 A, Dura and LCC ranged from 2 % to 17 % without any further pre-treatment step. The degradation efficiency was found to correlate with the extent of matched substrate and enzyme conformations revealed by IFD, regardless of the actual reaction temperature employed. Our findings demonstrate the importance of conformational selection in enzymatic depolymerization of biopolymers. A straight or twisted conformation of the polymer chain is crucial in biocatalytic degradation by showing different affinities to enzyme ground-state conformers. This work highlights the importance of considering the conformational match between the polymer and the enzyme to optimize the biocatalytic degradation efficiency of biopolymers, providing valuable insights for the development of sustainable bioprocesses.

The continued growth in phthalate esters (PAEs) production (∼8 million tons per year) has led to the increased emissions of PAEs into atmospheric, soil, and aquatic environments, posing a serious threat to human and animal health. Microbial enzyme-mediated degradation is an effective remediation strategy for removing PAE contaminants in the environment. Several hydrolases from both culturable and non-culturable microorganisms have been identified with outstanding degradation capacities against PAEs. A hydrolase identified from Glutamicibacter sp. strain 0426 could completely degrade 300 mg/L of dibutyl phthalate (DBP) within 12 h at 32℃ and pH 6.9, and an esterase which was screened from a metagenomic library exhibited high hydrolytic activity (128 U/mg) toward DBP at 40℃ and pH 7.5. However, there are still only a limited number of PAE-degrading enzymes that have been fully characterized so far. Herein, we show the significant influence of PAEs on plastic recycling and environmental pollution. We review recent advances in the identification and isolation of PAE-degrading enzymes from diverse environments. We highlight the potential of metagenomic analyses for exploring novel and powerful PAE hydrolases. Moreover, we discuss a possible enzyme-catalyzed reaction mechanism for PAE hydrolysis given the scarce experimental evidence. The substrate specificity among different monoalkyl/dialkyl PAE hydrolases is attributed to the steric hindrance and electrostatic repulsion affecting the PAE binding to an enzyme. Furthermore, we discuss different directed evolution strategies for improving the performance of PAE-degrading enzymes. Several challenges and future directions in research on PAE-degrading enzymes are also identified.

The demands on chemical industry to transform towards safety and sustainability will require multi-disciplinary research and development where experts on chemistry and chemical engineering, toxicology, ecotoxicology, and life cycle assessment collaborate to develop novel production methods, chemicals and materials. Here we summarise the results of the Mistra SafeChem programme which has yielded considerable output in the areas of catalysis/bio-catalysis, hazard screening for humans and ecosystems and life cycle assessment with chemical footprints, both as individual scientific achievements and as part of an integrated approach to assess safety and sustainability of novel chemicals and chemical synthesis processes, in chemical value chains and across collaborations between industry- academia/ industry-industry. The outcomes from the programme are summarised and discussed and experiences from dialogues on the future of safe and sustainable chemistry are presented.

More than 8 billion tons of plastic waste has been generated, posing severe environmental consequences and health risks. Due to prolonged exposure, microplastic particles are found in human blood and other bodily fluids. Despite a lack of toxicity studies regarding microplastics, harmful effects for humans seem plausible and cannot be excluded. As small plastic particles readily translocate from the gut to body fluids, enzyme-based treatment of serum could constitute a promising future avenue to clear synthetic polymers and their corresponding oligomers via their degradation into monomers of lower toxicity than the material they originate from. Still, whereas it is known that the enzymatic depolymerization rate of synthetic polymers varies by orders of magnitude depending on the buffer and media composition, the activity of plastic-degrading enzymes in serum was unknown. Here, we report how an engineered PETase, which we show to be generally trans-selective via induced fit docking, can depolymerize two different microplastic-like substrates of the commodity polymer polyethylene terephthalate (PET) into its non-toxic monomer terephthalic acid (TPA) alongside mono(2-hydroxyethyl)terephthalate (MHET) in human serum at 37 °C. We show that the application of PETase does not influence cell viability in vitro. Our work highlights the potential of applying biocatalysis in biomedicine and represents a first step towards finding a future solution to the problem that microplastics in the bloodstream may pose.

Dynamic polyimine-amide networks with exceptional properties, including high flexibility, excellent thermal stability and dual closed-loop circularity were designed by combining dynamic covalent imine-functionalities with amide-chemistry. The solvent free up-scalable synthesis started from carboxyl-functionalization of lignin-derivable aldehydes followed by melt polycondensation with a triamine to form two dynamic networks (PIAX1 and PIAX2, respectively). While previously reported vanillin-derived polyimine thermosets were typically non-flexible and brittle, our polyimine-amides are flexible with elongation at break 380 % for PIAX1 and 65 % for PIAX2, where the higher flexibility of PIAX1 is deduced to the lower glass transition temperature and crosslinking density. Both materials illustrate fast stress relaxation even at low temperature, down to 50 °C in the case of PIAX1. Retained and even improved mechanical properties were observed after several cycles of thermal reprocessing, e.g., after three reprocessing cycles by hot pressing, PIAX1 recovered 116 % of original tensile stress and 126 % of original elongation at break, while chemical recycling under acidic conditions at room temperature yielded repolymerizable trialdehydes and triamines. Furthermore, rapid self-healing and shape memory behaviour at low temperature were demonstrated for PIAX1. A promising molecular design, further tuneable by choice of aldehyde and triamine, is demonstrated enabling high performance and dual closed-loop circularity.

Amide bond synthesis is ranked as the second most important challenge in key green chemistry research areas identified by the ACS Green Chemistry Institute. While developing more sustainable amide bond forming reactions has been in focus, significantly less attention has been given to human toxicity and environmental aspects of the underlying amine and acid substrates and their corresponding coupled products, a potentially important contribution to the overall sustainability of the amide-bond-forming reactions. Here, we explore biocatalytic amide bond formation from a safer-and-more-sustainable-by-design perspective in which commercially available amines and acids as well as their corresponding amide products were evaluated in silico based on potential human toxicity and environmental fate and exposure. This in silico filtering resulted in a panel of 188 amine and 54 acid building blocks that could be classified as safe, referred to herein as “safechems”. To enable couplings of safechems, we generated a panel of robust and promiscuous ancestral ATP-dependent amide bond synthetases (ABS) using McbA from Marinactinospora thermotolerans SCSIO 00652 as a template. Ancestral ABS enzymes exhibited complementary specificities in the coupling of a representative safechem subset of 17 amines and 16 acids while showing an increased thermostability of up to 20 °C compared to the extant biocatalyst. Finally, the pool of safechems and their corresponding amides were evaluated by USEtox (the UNEP-SETAC toxicity model), analysing not only the intrinsic properties of the compounds but evaluating their complete impact pathway including fate, exposure and effects. The amides were in general predicted as more toxic compared to the starting acids and amines through non-additive effects, emphasising that focusing on the toxicity of the building blocks alone is not sufficient to strive towards low human and ecotoxicity impact. Pursuing a safer and more sustainable by design perspective in the implementation of safechems did not prevent us from generating an array of novel products with potentially potent applications as exemplified here by enzymatic synthesis of substructures that are part of drug candidates for e.g. cancer treatment.