The ability to design efficient enzymes from scratch would have a profound effect on chemistry, biotechnology and medicine. Rapid progress in protein engineering over the past decade makes us ...optimistic that this ambition is within reach. The development of artificial enzymes containing metal cofactors and noncanonical organocatalytic groups shows how protein structure can be optimized to harness the reactivity of nonproteinogenic elements. In parallel, computational methods have been used to design protein catalysts for diverse reactions on the basis of fundamental principles of transition state stabilization. Although the activities of designed catalysts have been quite low, extensive laboratory evolution has been used to generate efficient enzymes. Structural analysis of these systems has revealed the high degree of precision that will be needed to design catalysts with greater activity. To this end, emerging protein design methods, including deep learning, hold particular promise for improving model accuracy. Here we take stock of key developments in the field and highlight new opportunities for innovation that should allow us to transition beyond the current state of the art and enable the robust design of biocatalysts to address societal needs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in ...chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.
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IJS, KISLJ, NUK, UL, UM, UPUK
High selectivity and exquisite control over the outcome of reactions entice chemists to use biocatalysts in organic synthesis. However, many useful reactions are not accessible because they are not ...in nature’s known repertoire. In this Review, we outline an evolutionary approach to engineering enzymes to catalyze reactions not found in nature. We begin with examples of how nature has discovered new catalytic functions and how such evolutionary progression has been recapitulated in the laboratory starting from extant enzymes. We then examine non‐native enzyme activities that have been exploited for chemical synthesis, with an emphasis on reactions that do not have natural counterparts. Non‐natural activities can be improved by directed evolution, thus mimicking the process used by nature to create new catalysts. Finally, we describe the discovery of non‐native catalytic functions that may provide future opportunities for the expansion of the enzyme universe.
Exploiting hidden talents: The engineering of enzymes to catalyze reactions not known in nature will expand the range of transformations that can be promoted by biocatalysis. This Review presents a common pathway by which new enzyme activities evolve in nature and examples of the use of a similar approach to create enzymes for non‐natural reactions (see picture).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Enzymes generated by natural recruitment and protein engineering have greatly contribute in various sets of applications. However, their insufficient stability is a bottleneck that limit the rapid ...development of biocatalysis. Novel approaches based on precise and global structural dissection, advanced gene manipulation, and combination with the multidisciplinary techniques open a new horizon to generate stable enzymes efficiently. Here, we comprehensively introduced emerging advances of protein engineering strategies for enzyme stabilization. Then, we highlighted practical cases to show importance of enzyme stabilization in pharmaceutical and industrial applications. Combining computational enzyme design with molecular evolution will hold considerable promise in this field.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Recent developments in computational chemistry and biology have come together in the “inside‐out” approach to enzyme engineering. Proteins have been designed to catalyze reactions not previously ...accelerated in nature. Some of these proteins fold and act as catalysts, but the success rate is still low. The achievements and limitations of the current technology are highlighted and contrasted to other protein engineering techniques. On its own, computational “inside‐out” design can lead to the production of catalytically active and selective proteins, but their kinetic performances fall short of natural enzymes. When combined with directed evolution, molecular dynamics simulations, and crowd‐sourced structure‐prediction approaches, however, computational designs can be significantly improved in terms of binding, turnover, and thermal stability.
The “inside‐out” approach to computer‐based enzyme design unites the newest developments in the areas of computational chemistry and biology. This has enabled the design of proteins that catalyze reactions not accelerated in nature. The achievements and limitations of the current technology are highlighted and compared to other methods.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The directed evolution of enzymes is now routinely used to develop new catalysts with various applications, such as in environmentally friendly production of chemicals and renewable fuels. In her ...Nobel lecture, F. Arnold describes how lessons from nature inspired the development of methods for directed evolution.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Despite enormous progress in protein engineering, complemented by bioprocess engineering, the revolution awaiting the application of biocatalysis in the fine chemical industry has still not been ...fully realized. In order to achieve that, further research is required on several topics, including (1) rapid methods for protein engineering using machine learning, (2) mathematical modelling of multi-enzyme cascade processes, (3) process standardization, (4) continuous process technology, (5) methods to identify improvements required to achieve industrial implementation, (6) downstream processing, (7) enzyme stability modelling and prediction, as well as (8) new reactor technology. In this brief mini-review, the status of each of these topics will be briefly discussed.
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Rational protein engineering requires a holistic understanding of protein function. Here, we apply deep learning to unlabeled amino-acid sequences to distill the fundamental features of a protein ...into a statistical representation that is semantically rich and structurally, evolutionarily and biophysically grounded. We show that the simplest models built on top of this unified representation (UniRep) are broadly applicable and generalize to unseen regions of sequence space. Our data-driven approach predicts the stability of natural and de novo designed proteins, and the quantitative function of molecularly diverse mutants, competitively with the state-of-the-art methods. UniRep further enables two orders of magnitude efficiency improvement in a protein engineering task. UniRep is a versatile summary of fundamental protein features that can be applied across protein engineering informatics.
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Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in ...regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.
Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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