Improving plastic degrading enzymes via directed evolution.

Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.

Stereoselective amino acid synthesis by photobiocatalytic oxidative coupling.

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1. So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9. Here we report photobiocatalytic asymmetric sp3-sp3 oxidative cross-coupling between organoboron reagents and amino acids. This reaction requires the cooperative use of engineered pyridoxal biocatalysts, photoredox catalysts and an oxidizing agent. We repurpose a family of pyridoxal-5'-phosphate-dependent enzymes, threonine aldolases10-12, for the α-C-H functionalization of glycine and α-branched amino acid substrates by a radical mechanism, giving rise to a range of α-tri- and tetrasubstituted non-canonical amino acids 13-15 possessing up to two contiguous stereocentres. Directed evolution of pyridoxal radical enzymes allowed primary and secondary radical precursors, including benzyl, allyl and alkylboron reagents, to be coupled in an enantio- and diastereocontrolled fashion. Cooperative photoredox-pyridoxal biocatalysis provides a platform for sp3-sp3 oxidative coupling16, permitting the stereoselective, intermolecular free-radical transformations that are unknown to chemistry or biology.

Proteome partitioning constraints in long-term laboratory evolution.

Adaptive laboratory evolution experiments provide a controlled context in which the dynamics of selection and adaptation can be followed in real-time at the single-nucleotide level. And yet this precision introduces hundreds of degrees-of-freedom as genetic changes accrue in parallel lineages over generations. On short timescales, physiological constraints have been leveraged to provide a coarse-grained view of bacterial gene expression characterized by a small set of phenomenological parameters. Here, we ask whether this same framework, operating at a level between genotype and fitness, informs physiological changes that occur on evolutionary timescales. Using a strain adapted to growth in glucose minimal medium, we find that the proteome is substantially remodeled over 40 000 generations. The most striking change is an apparent increase in enzyme efficiency, particularly in the enzymes of lower-glycolysis. We propose that deletion of metabolic flux-sensing regulation early in the adaptation results in increased enzyme saturation and can account for the observed proteome remodeling.

Boron catalysis in a designer enzyme.

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis1-3. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts4,5. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.

Experimental evolution for the recovery of growth loss due to genome reduction.

As the genome encodes the information crucial for cell growth, a sizeable genomic deficiency often causes a significant decrease in growth fitness. Whether and how the decreased growth fitness caused by genome reduction could be compensated by evolution was investigated here. Experimental evolution with an Escherichia coli strain carrying a reduced genome was conducted in multiple lineages for approximately 1000 generations. The growth rate, which largely declined due to genome reduction, was considerably recovered, associated with the improved carrying capacity. Genome mutations accumulated during evolution were significantly varied across the evolutionary lineages and were randomly localized on the reduced genome. Transcriptome reorganization showed a common evolutionary direction and conserved the chromosomal periodicity, regardless of highly diversified gene categories, regulons, and pathways enriched in the differentially expressed genes. Genome mutations and transcriptome reorganization caused by evolution, which were found to be dissimilar to those caused by genome reduction, must have followed divergent mechanisms in individual evolutionary lineages. Gene network reconstruction successfully identified three gene modules functionally differentiated, which were responsible for the evolutionary changes of the reduced genome in growth fitness, genome mutation, and gene expression, respectively. The diversity in evolutionary approaches improved the growth fitness associated with the homeostatic transcriptome architecture as if the evolutionary compensation for genome reduction was like all roads leading to Rome.

Automated in vivo enzyme engineering accelerates biocatalyst optimization.

Achieving cost-competitive bio-based processes requires development of stable and selective biocatalysts. Their realization through in vitro enzyme characterization and engineering is mostly low throughput and labor-intensive. Therefore, strategies for increasing throughput while diminishing manual labor are gaining momentum, such as in vivo screening and evolution campaigns. Computational tools like machine learning further support enzyme engineering efforts by widening the explorable design space. Here, we propose an integrated solution to enzyme engineering challenges whereby ML-guided, automated workflows (including library generation, implementation of hypermutation systems, adapted laboratory evolution, and in vivo growth-coupled selection) could be realized to accelerate pipelines towards superior biocatalysts.

Serial adaptive laboratory evolution enhances mixed carbon metabolic capacity of Escherichia coli.

Microbes have inherent capacities for utilizing various carbon sources, however they often exhibit sub-par fitness due to low metabolic efficiency. To test whether a bacterial strain can optimally utilize multiple carbon sources, Escherichia coli was serially evolved in L-lactate and glycerol. This yielded two end-point strains that evolved first in L-lactate then in glycerol, and vice versa. The end-point strains displayed a universal growth advantage on single and a mixture of adaptive carbon sources, enabled by a concerted action of carbon source-specialists and generalist mutants. The combination of just four variants of glpK, ppsA, ydcI, and rph-pyrE, accounted for more than 80% of end-point strain fitness. In addition, machine learning analysis revealed a coordinated activity of transcriptional regulators imparting condition-specific regulation of gene expression. The effectiveness of the serial adaptive laboratory evolution (ALE) scheme in bioproduction applications was assessed under single and mixed-carbon culture conditions, in which serial ALE strain exhibited superior productivity of acetoin compared to ancestral strains. Together, systems-level analysis elucidated the molecular basis of serial evolution, which hold potential utility in bioproduction applications.

Improvement of Chalcone Synthase Activity and High-Efficiency Fermentative Production of (2S)-Naringenin via In Vivo Biosensor-Guided Directed Evolution.

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2S)-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2S)-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2S)-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2S)-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, SjCHS1S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2S)-naringenin by SjCHS1S208N was constructed in Saccharomyces cerevisiae, combined with CHS precursor pathway optimization, increasing the (2S)-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2S)-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.

Directed-evolution approach to empower EGFR targeting for immunotherapy.

Advances in directed-evolution technologies are enabling new strategies to isolate binding proteins that recognize disease-associated states of a target protein. In this issue of Cell Reports Methods, Dobersberger et al. devised a yeast display-based selection scheme to discover proteins that engage the cancer-associated activated state of a receptor to enable design of safe and effective immunotherapies.

Engineering is evolution: a perspective on design processes to engineer biology.

Careful consideration of how we approach design is crucial to all areas of biotechnology. However, choosing or developing an effective design methodology is not always easy as biology, unlike most areas of engineering, is able to adapt and evolve. Here, we put forward that design and evolution follow a similar cyclic process and therefore all design methods, including traditional design, directed evolution, and even random trial and error, exist within an evolutionary design spectrum. This contrasts with conventional views that often place these methods at odds and provides a valuable framework for unifying engineering approaches for challenging biological design problems.

Evolution-guided engineering of trans-acyltransferase polyketide synthases.

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis-acyltransferase (cis-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans-AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans-AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

Continuous evolution of compact protein degradation tags regulated by selective molecular glues.

Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.

Biocatalytic strategy for the construction of sp3-rich polycyclic compounds from directed evolution and computational modelling.

Catalysis with engineered enzymes has provided more efficient routes for the production of active pharmaceutical agents. However, the potential of biocatalysis to assist in early-stage drug discovery campaigns remains largely untapped. In this study, we have developed a biocatalytic strategy for the construction of sp3-rich polycyclic compounds via the intramolecular cyclopropanation of benzothiophenes and related heterocycles. Two carbene transferases with complementary regioisomer selectivity were evolved to catalyse the stereoselective cyclization of benzothiophene substrates bearing diazo ester groups at the C2 or C3 position of the heterocycle. The detailed mechanisms of these reactions were elucidated by a combination of crystallographic and computational analyses. Leveraging these insights, the substrate scope of one of the biocatalysts could be expanded to include previously unreactive substrates, highlighting the value of integrating evolutionary and rational strategies to develop enzymes for new-to-nature transformations. The molecular scaffolds accessed here feature a combination of three-dimensional and stereochemical complexity with 'rule-of-three' properties, which should make them highly valuable for fragment-based drug discovery campaigns.

Empowering Protein Engineering through Recombination of Beneficial Substitutions.

Directed evolution stands as a seminal technology for generating novel protein functionalities, a cornerstone in biocatalysis, metabolic engineering, and synthetic biology. Today, with the development of various mutagenesis methods and advanced analytical machines, the challenge of diversity generation and high-throughput screening platforms is largely solved, and one of the remaining challenges is: how to empower the potential of single beneficial substitutions with recombination to achieve the epistatic effect. This review overviews experimental and computer-assisted recombination methods in protein engineering campaigns. In addition, integrated and machine learning-guided strategies were highlighted to discuss how these recombination approaches contribute to generating the screening library with better diversity, coverage, and size. A decision tree was finally summarized to guide the further selection of proper recombination strategies in practice, which was beneficial for accelerating protein engineering.

Accelerated evolution of chosen genes.

Orthogonal replication enables rapid continuous biomolecular evolution in Escherichia coli.

Establishing a synthetic orthogonal replication system enables accelerated evolution in E. coli.

The evolution of new function in living organisms is slow and fundamentally limited by their critical mutation rate. Here, we established a stable orthogonal replication system in Escherichia coli. The orthogonal replicon can carry diverse cargos of at least 16.5 kilobases and is not copied by host polymerases but is selectively copied by an orthogonal DNA polymerase (O-DNAP), which does not copy the genome. We designed mutant O-DNAPs that selectively increase the mutation rate of the orthogonal replicon by two to four orders of magnitude. We demonstrate the utility of our system for accelerated continuous evolution by evolving a 150-fold increase in resistance to tigecycline in 12 days. And, starting from a GFP variant, we evolved a 1000-fold increase in cellular fluorescence in 5 days.

In Vitro Enzyme Self-Selection Using Molecular Programs.

Directed evolution provides a powerful route for in vitro enzyme engineering. State-of-the-art techniques functionally screen up to millions of enzyme variants using high throughput microfluidic sorters, whose operation remains technically challenging. Alternatively, in vitro self-selection methods, analogous to in vivo complementation strategies, open the way to even higher throughputs, but have been demonstrated only for a few specific activities. Here, we leverage synthetic molecular networks to generalize in vitro compartmentalized self-selection processes. We introduce a programmable circuit architecture that can link an arbitrary target enzymatic activity to the replication of its encoding gene. Microencapsulation of a bacterial expression library with this autonomous selection circuit results in the single-step and screening-free enrichment of genetic sequences coding for programmed enzymatic phenotypes. We demonstrate the potential of this approach for the nicking enzyme Nt.BstNBI (NBI). We applied autonomous selection conditions to enrich for thermostability or catalytic efficiency, manipulating up to 107 microcompartments and 5 × 105 variants at once. Full gene reads of the libraries using nanopore sequencing revealed detailed mutational activity landscapes, suggesting a key role of electrostatic interactions with DNA in the enzyme's turnover. The most beneficial mutations, identified after a single round of self-selection, provided variants with, respectively, 20 times and 3 °C increased activity and thermostability. Based on a modular molecular programming architecture, this approach does not require complex instrumentation and can be repurposed for other enzymes, including those that are not related to DNA chemistry.

Ultrahigh-Throughput Directed Evolution of Polymer-Degrading Enzymes Using Yeast Display.

Enzymes that degrade synthetic polymers have attracted intense interest for eco-friendly plastic recycling. However, because enzymes did not evolve for the cleavage of abiotic polymers, directed evolution strategies are needed to enhance activity for plastic degradation. Previous directed evolution efforts relied on polymer degradation assays that were limited to screening ∼104 mutants. Here, we report a high-throughput yeast surface display platform to rapidly evaluate >107 enzyme mutants for increased activity in cleaving synthetic polymers. In this platform, individual yeast cells display distinct mutants, and enzyme activity is detected by a change in fluorescence upon the cleavage of a synthetic probe resembling a polymer of interest. Highly active mutants are isolated by fluorescence activated cell sorting and identified through DNA sequencing. To demonstrate this platform, we performed directed evolution of a polyethylene terephthalate (PET)-depolymerizing enzyme, leaf and branch compost cutinase (LCC). We identified activity-boosting mutations that substantially increased the kinetics of degradation of solid PET films. Biochemical assays and molecular dynamics (MD) simulations of the most active variants suggest that the H218Y mutation improves the binding of the enzyme to PET. Overall, this evolution platform increases the screening throughput of polymer-degrading enzymes by 3 orders of magnitude and identifies mutations that enhance kinetics for depolymerizing solid substrates.

Quantification of evolved DNA-editing enzymes at scale with DEQSeq.

We introduce DEQSeq, a nanopore sequencing approach that rationalizes the selection of favorable genome editing enzymes from directed molecular evolution experiments. With the ability to capture full-length sequences, editing efficiencies, and specificities from thousands of evolved enzymes simultaneously, DEQSeq streamlines the process of identifying the most valuable variants for further study and application. We apply DEQSeq to evolved libraries of Cas12f-ABEs and designer-recombinases, identifying variants with improved properties for future applications. Our results demonstrate that DEQSeq is a powerful tool for accelerating enzyme discovery and advancing genome editing research.

Illuminating protein space with a programmable generative model.

Three billion years of evolution has produced a tremendous diversity of protein molecules1, but the full potential of proteins is likely to be much greater. Accessing this potential has been challenging for both computation and experiments because the space of possible protein molecules is much larger than the space of those likely to have functions. Here we introduce Chroma, a generative model for proteins and protein complexes that can directly sample novel protein structures and sequences, and that can be conditioned to steer the generative process towards desired properties and functions. To enable this, we introduce a diffusion process that respects the conformational statistics of polymer ensembles, an efficient neural architecture for molecular systems that enables long-range reasoning with sub-quadratic scaling, layers for efficiently synthesizing three-dimensional structures of proteins from predicted inter-residue geometries and a general low-temperature sampling algorithm for diffusion models. Chroma achieves protein design as Bayesian inference under external constraints, which can involve symmetries, substructure, shape, semantics and even natural-language prompts. The experimental characterization of 310 proteins shows that sampling from Chroma results in proteins that are highly expressed, fold and have favourable biophysical properties. The crystal structures of two designed proteins exhibit atomistic agreement with Chroma samples (a backbone root-mean-square deviation of around 1.0 Å). With this unified approach to protein design, we hope to accelerate the programming of protein matter to benefit human health, materials science and synthetic biology.