Enzyme-directed evolution and the screening of novel natural enzymes have long been significantly limited by the tools and methodologies used for screening. Fluorescence-activated droplet sorting is a high-throughput enzyme engineering technique based on microfluidic technology. It employs droplet encapsulation of cells, addressing challenges in detecting cell-secreted proteins or extracellular metabolites that are difficult for traditional fluorescence cell surface display (FACS) to handle. The sorting throughput can reach up to 30 kHz. FADS technology allows the individual separation of enzyme variants in droplets, with each droplet acting as a microreactor for enzyme reactions. When combined with fluorescence activity measurements within the droplets, precise classification of these variants based on desired characteristics becomes possible. This method of distinguishing and isolating high-performance enzymes significantly enhances the accuracy and efficiency of enzyme identification and selection. Following phenotype detection and sorting, individual cells remain encapsulated in separate droplets, preserving their independence and seamlessly connecting with downstream single-cell cultivation, sequencing, and other omics studies. The FADS platform, which integrates a two-channel detection system, was reported to enable the screening of esterases with high selectivity for preferentially producing enantiomers of ibuprofen from a mutant library of directed evolution (approximately 10⁷ mutants).

Fig. 1 General concepts and implementation methods of fluorescence-activated droplet sorting. (Julie V L, et al.,2021)

In recent years, FADS technology has found increasingly widespread applications in the screening of enzymes, metabolic products, and antibodies. By combining principles such as droplet generation, fluorescence activation, and advanced sorting mechanisms, FADS serves as a powerful tool for the evolution and screening of enzymes and fundamentally transforms the field of enzyme screening.

Phage-assisted continuous evolution technology is a protein directed evolution technique based on bacteriophages. It relies on establishing an adaptive link between the desired activity of the target protein and the infectivity of the phage carrying the corresponding gene for that protein. This simulation of natural evolution involves the association of the desired protein activity with the adaptive fitness of the phage, where random mutations in DNA occur at rates significantly higher than those found in nature in each generation. The expressed proteins are then selected based on their in situ adaptability. This stands in sharp contrast to traditional directed evolution methods, where mutagenesis and selection occur as discrete steps, necessitating the introduction of mutations by scientists in each iteration of evolution. PACE has been successfully applied to protein evolution and is theoretically applicable to the evolution of any genetically encoded molecule, including nucleic acid aptamers.

Fig. 1 Improvement of enzymes using phage-based continuous evolution and selection technologies to engineer enzymes. (Jones K A, et al.,2021)

PACE utilizes bacteriophages as a carrier for the continuous evolution of enzymes. This technique involves constructing diverse bacteriophage libraries carrying variants of the target enzyme gene, followed by iterative cycles of phage display, amplification, and mutation. The application of selective pressure during the phage display process leads to the emergence of enzyme variants with improved catalytic properties. The iterative nature of PACE simulates natural evolution, and each step of the evolution cycle can be executed continuously without manual intervention. This enables enzymes to rapidly adapt and optimize, ultimately resulting in enhanced catalytic efficiency, substrate specificity, and stability for various biocatalytic applications.

Microtiter plate screening technology is a high-throughput screening technique used to analyze biological and chemical reactions in small volumes. This technique is often done in a 96-well or 384-well plate format, hence the name. It's heavily used in areas such as drug discovery, clinical diagnostics, genomics, and enzyme-directed evolution. The rise of automation and machine learning propels MTPS technology to different heights, allowing unprecedented scalability, precision, and speed while minimizing human intervention and resource usage.

MTPS enables rapid and high-throughput screening of large-scale mutant libraries, facilitating the selection of enzymes with the desired properties. By analyzing thousands of variants simultaneously, it rapidly accelerates the process of enzyme evolution. MTPS technology can effectively aid in enzyme rational design by allowing scientists to measure and compare the enzyme function of numerous mutant derivatives. By providing a way to conduct numerous assays concurrently, MTPS helps to validate the predicted performance of rationally designed enzymes. This accelerates the enzyme engineering process, allowing the creation of tailor-made enzymes for bioscience research, drug discovery, diagnostics, and different industrial applications.

Fig. 1 Microtiter plate reaction screening and optimization of surfactant chemistry. (Brocklehurst C E, et al., 2018)

Compartmentalized self replication (CSR) technology is a high-throughput screening method used for the evolution of proteins and whole organisms. It follows the principle of Darwinian evolution, promoting random mutation and selective enrichment. Researchers isolate genetically distinct entities in microcompartments to undergo independent replication and selection. The use of massive parallelism in CSR accelerates the evolution process. CSR has already had applications in enzyme engineering, therapeutics, biofuels and the creation of novel biosynthetic pathways. Researchers are currently developing other methods such as emulsification and microfluidics, to facilitate the evolution of CSR technology.

Fig. 1 General CSR concept and procedure schematic. (Abil Z, et al., 2018)

The application of CSR technology has revolutionized enzyme development, introducing improved precision and capacity for customization. This system allows for the directed evolution of enzymes in a compartmentalized manner, providing techno-biological control over the replication, expression, and clonal selection of mutagenic DNA. Through CSR, an array of enzymatic traits - including substrate selectivity, catalytic efficiency, and thermal stability - can be optimized for individual or combined purposes. By leveraging this technology, scientists can engineer enzymes with accelerated evolutionary pace in a laboratory setting, helping to overcome the limitations of natural selection.

Computer-aided rational design (CARD) technology seamlessly integrates computational modeling and experimental techniques, orchestrating the meticulous design and optimization of materials endowed with distinct properties. Rooted in the twin pillars of the structure-function relationship and the prediction-verification iteration, it strategically harnesses computational models to foresee outcomes, validates these prognostications through empirical experiments, and then cyclically hones the models in response to feedback. The evolutionary trajectory of CARD is intricately intertwined with progress in computer science, molecular biology, and engineering. Its application burgeons in realms like novel drug molecule discovery and advanced materials design. Researchers continually weave the threads of machine learning and artificial intelligence into the fabric of CARD, perpetually enhancing its predictive prowess and operational efficiency.

In the realm of enzyme development, CARD stands as a revolutionary force, reshaping the landscape by amplifying comprehension, manipulation, and creation of enzymes. Employing mathematical modeling and simulations, it navigates the intricate nuances of biochemical reactions and workflows, ushering in a more targeted and efficient era in enzyme design. This technology not only facilitates the rational tweaking of existing enzymes but also envisions the potential ramifications of molecular modifications, aiding in the birth of novel biocatalysts. Through meticulous data analysis and forecasting, CARD truncates the arduous and expensive path of experimental testing. It acts as a compass in enzyme engineering, discerning the most promising routes for catalytic enhancement, stability augmentation, and substrate specificity with surgical precision.

Fig. 1 Experimental protein engineering strategies and idealized schemes for design-test-build-learn cycles using computational optimization of enzymes. (Scherer M, et al., 2021)