Biocatalysis harnesses enzymes to make valuable products. This green technology is used in countless applications from bench scale to industrial production and allows practitioners to access complex organic molecules, often with fewer synthetic steps and reduced waste. The last decade has seen an explosion in the development of experimental and computational tools to tailor enzymatic properties, equipping enzyme engineers with the ability to create biocatalysts that perform reactions not present in nature. By using (chemo)-enzymatic synthesis routes or orchestrating intricate enzyme cascades, scientists can synthesize elaborate targets ranging from DNA and complex pharmaceuticals to starch made in vitro from CO2-derived methanol. In addition, new chemistries have emerged through the combination of biocatalysis with transition metal catalysis, photocatalysis, and electrocatalysis. This review highlights recent key developments, identifies current limitations, and provides a future prospect for this rapidly developing technology. Description Editor’s summary As nature’s catalysts, enzymes are adaptable, specific, and interoperable. There have been important advances in the identification of transformations catalyzed by enzymes, and new methods have been developed for identifying, engineering, or creating de novo suitable enzymes for a wide range of applications. In a review, Buller et al. collect this recent progress in biocatalysis research and look ahead toward increasing the use of computational tools, accelerating design test cycles, and expanding chemistry beyond what is familiar in nature. Engineered enzymes provide a green chemistry solution to the synthesis of complex organic molecules and can be used in the degradation of waste plastic and chemicals. —Michael A. Funk Engineered enzymes provide a green chemistry solution for synthesis of complex molecules. BACKGROUND Biocatalysis is an approach to synthetic chemistry in which enzymes carry out chemical reactions. Historically, enzymes from natural sources have been used to break down oils and proteins in laundry detergents, produce semisynthetic antibiotics, and create simple chiral precursors for the pharmaceutical industry. In the past 5 years, the number of available protein sequences has increased by a staggering 20-fold, accelerating the discovery of enzymes with useful activities and properties. Directed evolution, the cornerstone of our ability to tailor enzymatic properties, is allowing researchers to tailor enzymes for the synthesis of complex molecules, the modification of biological therapeutics, and the breakdown of plastic waste. Machine learning–driven protein-structure prediction, coupled with advances in automation and high-throughput screening, is further advancing our ability to create enzymes with desired function. The ability to add nonbiological catalytic elements to enzymes means that enzyme engineers no longer have to rely solely on natural catalytic machinery. Scientists have thus gained the capacity to redesign, reimagine, and repurpose enzymes. Illustrative applications include enzyme cascades to manufacture the antivirals islatravir and molnupiravir, biocatalysts that can generate and control radicals, and enzymes that exploit photocatalysis to affect stereocontrolled C–C couplings, hydroaminations, or Diels-Alder reactions. ADVANCES The past 5 years have witnessed a surge in the development of data-driven tools enabling the accelerated discovery, engineering, and deployment of enzymes for applications in chemistry, medicine, and food technology. By leveraging enzymes’ ability to control the environment of a chemical reaction, scientists have developed successful platforms for the construction of complex small molecules from suitable starting materials or even CO2. In addition to their use in (chemo)-enzymatic cascade reactions, precisely tailored biocatalysts can manufacture new therapeutic modalities, such as antisense oligonucleotide therapeutics, and bioconjugates. DNA synthesis, which still relies on phosphoramidite chemistry, is being reinvented with template-independent deoxynucleotidyl transferases to make the process faster and cheaper. Machine learning, already dominating protein-structure prediction and design, is finding applications in enzyme engineering, including improvement of functions such as enantioselectivity, activity, and stability. OUTLOOK In the decade ahead, biocatalysis research and applications will continue to profit from advances in data mining, machine learning, and DNA reading and writing. The combinatorial design of enzymes and the ease with which new enzyme variants can be experimentally generated lends itself to train data-intense machine-learning algorithms. Ideally, the sequence-function data of variants screened in a directed-evolution campaign could be used to predict which variants to evaluate next. Machine learning works best with clean and reproducible data, mandating the standardization and reporting of data and the further development of experimental techniques, including molecular biology methods, automation, and high-throughput screening assays. The toolbox of drug discovery is expanding beyond traditional small molecules (molecular weights <500 g/mol) to include RNA therapeutics, protein degraders, cyclopeptides, antibody drug conjugates, and gene therapy. Consequently, the synthetic complexity of lead molecules and clinical candidates is increasing with a growing percentage of chiral and beyond-rule-of-5 molecules. Enzymatic synthesis will play a key role beyond its present impact in small-molecule drug discovery and development. The discovery of new enzyme families and the rational design of new enzyme functions will expand the toolbox of available biocatalysts. Development and deployment of retrosynthetic tools containing enzymatic reactions will be an important step toward democratizing biocatalysis and making it available to the nonexpert. Fueled by these innovations, nature’s catalysts will be profitably used to address current challenges, including the fight against diseases, provision of affordable clean energy, and reduction of industrial and consumer waste. The accelerating development of biocatalysis. In silico and experimental tools developed in the last decade allow the fast creation of tailored enzymes. In addition to the concise synthesis of complex small molecules in elegant (chemo)-enzymatic cascades, newly accessible applications include enzymatic DNA synthesis, the generation of therapeutic oligonucleotides, and up- and recycling of plastic waste. [Figure created with Canva]

From nature to industry: Harnessing enzymes for biocatalysis

Since the discovery of Stimulator of Interferon Genes (STING) as an important pivot for cytosolic DNA sensation and interferon (IFN) induction, intensive efforts have been endeavored to clarify the molecular mechanism of its activation, its physiological function as a ubiquitously expressed protein, and to explore its potential as a therapeutic target in a wide range of immune-related diseases. With its orthodox ligand 2'3'-cyclic GMP-AMP (2'3'-cGAMP) and the upstream sensor 2'3'-cGAMP synthase (cGAS) to be found, STING acquires its central functionality in the best-studied signaling cascade, namely the cGAS-STING-IFN pathway. However, recently updated research through structural research, genetic screening, and biochemical assay greatly extends the current knowledge of STING biology. A second ligand pocket was recently discovered in the transmembrane domain for a synthetic agonist. On its downstream outputs, accumulating studies sketch primordial and multifaceted roles of STING beyond its cytokine-inducing function, such as autophagy, cell death, metabolic modulation, endoplasmic reticulum (ER) stress, and RNA virus restriction. Furthermore, with the expansion of the STING interactome, the details of STING trafficking also get clearer. After retrospecting the brief history of viral interference and the milestone events since the discovery of STING, we present a vivid panorama of STING biology taking into account the details of the biochemical assay and structural information, especially its versatile outputs and functions beyond IFN induction. We also summarize the roles of STING in the pathogenesis of various diseases and highlight the development of small-molecular compounds targeting STING for disease treatment in combination with the latest research. Finally, we discuss the open questions imperative to answer. 

/pdf/multifaceted-functions-of-sting-in-human-health-and-disease-2ktxogrb.pdf

Multifaceted functions of STING in human health and disease: from molecular mechanism to targeted strategy

Biocatalysis is a highly valued enabling technology for pharmaceutical research and development as it can unlock synthetic routes to complex chiral motifs with unparalleled selectivity and efficiency. This perspective aims to review recent advances in the pharmaceutical implementation of biocatalysis across early and late-stage development with a focus on the implementation of processes for preparative-scale syntheses.

The Evolving Nature of Biocatalysis in Pharmaceutical Research and Development

Stereochemically and structurally complex cyclic dinucleotide-based stimulator of interferon genes (STING) pathway, is were designed and synthesized to access a previously unexplored chemical space. The assessment of biochemical affinity and cellular potency, along with computational, structural, and biophysical characterization, was applied to influence the design and optimization of novel STING agonists, resulting in the discovery of MK-1454 as a molecule with appropriate properties for clinical development. When administered intratumorally to immune-competent mice-bearing syngeneic tumors, MK-1454 exhibited robust tumor cytokine upregulation and effective antitumor activity. Tumor shrinkage in mouse models that are intrinsically resistant to single-agent therapy was further enhanced when treating the animals with MK-1454 in combination with a fully murinized antimouse PD-1 antibody, mDX400. These data support the development of STING agonists in combination with pembrolizumab (humanized anti-PD-1 antibody) for patients with tumors that are partially responsive or nonresponsive to single-agent anti-PD-1 therapy.

Discovery of MK-1454: A Potent Cyclic Dinucleotide Stimulator of Interferon Genes Agonist for the Treatment of Cancer.

Selective functionalization of aliphatic C-H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C-H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on preoxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C-H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)-H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity toward a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity-determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

Enzymatic Nitrogen Insertion into Unactivated C-H Bonds.

A kinase-cGAS cascade to synthesize a therapeutic STING activator

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A kinase-cGAS cascade to synthesize a therapeutic STING activator