In vitro enzyme cascades possess great benefits, such as their synthetic capabilities for complex molecules, no need for intermediate isolation, and the shift of unfavorable equilibria towards the products. Their performance, however, can be impaired by, for example, destabilizing or inhibitory interactions between the cascade components or incongruous reaction conditions. The optimization of such systems is therefore often inevitable but not an easy task. Many parameters such as the design of the synthesis route, the choice of enzymes, reaction conditions, or process design can alter the performance of an in vitro enzymatic cascade. Many strategies to tackle this complex task exist, ranging from experimental to in silico approaches and combinations of both. This review collates examples of various optimization strategies and their success. The feasibility of optimization goals, the influence of certain parameters and the usage of algorithm-based optimizations are discussed.

The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski's horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.The chemical synthesis of 2 3 -cGAMP contains eight reaction steps and suffers from low yields [8,9], whereas the enzymatic reaction reaches nearly full conversion within a few hours [6]. In recent studies, human cGAS was investigated predominantly with regards to the identification of key residues [10,11] and the kinetic mechanism [12]. Although several homologous enzymes are known, only murine cGAS [6,7], porcine cGAS [10,13], and chicken cGAS [14] received more attention. For the murine and porcine homologue, crystal structures are also available and, in parts, crucial amino acids with relevance for the catalytic function are known. Alignments of amino acid sequences of identified cGAS homologues revealed a low-sequence homology in regions without determined function [10,15]. This high level of variation and the markedly reduced activity of human cGAS in comparison to murine cGAS [11] demonstrate the possibility that other cGAS homologues might be available with enhanced properties or different characteristics and functions. For characterization, cGAS enzymes were synthesized with eukaryotic cell lines or expressed recombinantly in Escherichia coli. Eukaryotic genes tend to be difficult to express in bacterial systems, though fusion-tags, such as maltose-binding protein (MBP, 42.5 kDa) [16], glutathione S-transferase (GST, 26 kDa) [2], and small ubiquitin-like modifier (SUMO, 12 kDa) [1], were used for better solubility and functional expression of cGAS. Challenges remain to find suitable expression systems for eukaryotic protein production with good yield and purity, and under conditions conducive to functional protein studies. To realize higher throughput in heterologous protein synthesis, other methods than traditional recombinant expression can be considered.

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that catalyzes the synthesis of the cyclic GMP-AMP dinucleotide 2'3'-cGAMP. 2'3'-cGAMP functions as inducer for the production of type I interferons. Derivatives of this important second messenger are highly valuable for pharmaceutical applications. However, the production of these analogues requires complex, multistep syntheses. Herein, human cGAS is shown to react with a series of unnatural nucleotides, thus leading to novel cyclic dinucleotides. Most substrate derivatives with modifications at the nucleobase, ribose, and the α-thio phosphate were accepted. These results demonstrate the catalytic promiscuity of human cGAS and its utility for the biocatalytic synthesis of cyclic dinucleotide derivatives.

Biocatalysis is constantly providing novel options for the synthesis of active pharmaceutical ingredients (APIs). In addition to drug development and manufacturing, biocatalysis also plays a role in drug discovery and can support many active ingredient syntheses at an early stage to build up entire scaffolds in a targeted and preparative manner. Recent progress in recruiting new enzymes by genome mining and screening or adapting their substrate, as well as product scope, by protein engineering has made biocatalysts a competitive tool applied in academic and industrial spheres. This is especially true for the advances in the field of nonribosomal peptide synthesis and enzyme cascades that are expanding the capabilities for the discovery and synthesis of new bioactive compounds via biotransformation. Here we highlight some of the most recent developments to add to the portfolio of biocatalysis with special relevance for the synthesis and late-stage functionalization of APIs, in order to bypass pure chemical processes.

Enzyme cascades are an emerging synthetic tool for the synthesis of various molecules, combining the advantages of biocatalysis and of one-pot multi-step reactions. However, the more complex the enzyme cascade is, the more difficult it is to achieve adequate productivities and product concentrations. Therefore, the whole process must be optimized to account for synergistic effects. One way to deal with this challenge involves data-driven models in combination with experimental validation. Here, Bayesian optimization was applied to an ATP-producing and -regenerating enzyme cascade consisting of polyphosphate kinases. The enzyme and co-substrate concentrations were adjusted for an ATP-dependent reaction, catalyzed by mevalonate kinase (MVK). With a total of 16 experiments, we were able to iteratively optimize the initial concentrations of the components used in the one-pot synthesis to improve the specific activity of MVK with 10.2 U mg−1. The specific activity even exceeded the results of the reference reaction with stoichiometrically added ATP amounts, with which a specific activity of 8.8 U mg−1 was reached. At the same time, the product concentrations were also improved so that complete yields were achieved.