# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Expansion of Product Spectrum
#### 2.1.1 Natural Metabolism as Source of Enzymes and Biochemical Pathways
This one-step biocatalytic process has been proven superior in terms of step- and atom-economy in a five-step synthesis, which only yielded a mediocre 25% and 55% _ee_. These examples impressively show that biocatalysis has emerged to the extent that it can support many active ingredient syntheses at an early stage or bypass pure chemical processes in order to build up entire natural and active ingredient scaffolds in a targeted and preparative manner. 1.2 Expansion of Product Spectrum by Engineered Biocatalysts, as Exemplarily Illustrated for Nonribosomal Peptide Syntheases  
Many therapeutically relevant peptides, such as the anticancer agent bleomycin, the antibiotic vancomycin or the immunosuppressiveant cyclosporin A, are biosynthesized by large enzyme complexes, which are composed of nonribosomal peptide synthetases (NRPS) [59]. In recent years, various methods have been developed that allow the rational (re-)programming of these fascinating biocatalysts and, hence, the generation of tailored, bioactive drugs. Before we describe these innovative approaches (Table 2), it is necessary to briefly introduce the thiotemplate-based assembly logic of NRPS. NRPS are modularly organized enzymes that are capable of fusing a variety of amino acid substrates for the generation of peptidic structures. Each module represents a function unit and is solely responsible for the incorporation of one amino acid monomer into the growing peptide chain [60]. For this, a module comprises multiple catalytic domains with different functions. The adenylation (A) domain catalyzes a two-step reaction. After ATP-dependent activation of its substrate, the resulting aminoacyl adenylate is covalently bound to a flexible 4\({}^{\prime}\)-phosphopantetheine arm of a neighboring peptidyl carrier protein (PCP) or thiolation (T) domain. A condensation (C) domain mediates the amide linkage between the PCP-tethered substrate and the PCP-bound aminoacyl intermediate of the preceding module [60; 61]. This elongation process is consecutively carried out until the terminal NRPS module is reached (Figure 9). Here, a thioesterase (TE) domain detaches the full-length polypeptide from the enzyme complex by hydrolysis or intramolecular lactamization. Some NRPS modules are known to harbor additional domains, which carry out specific modifications such as a methyltransferasease (MT) or an epimerase (E) domain [61]. The assembly of peptides by NRPS is more error-prone than ribosomal synthesis, which is due to the substrate flexibility of A domains. In nature, this promiscuity leads to structural diversity within certain families of natural products, when two related amino acids compete for incorporation [62]. Moreover, the substrate flexibility can also be exploited for the generation of natural product derivatives in a process called precursor-directed biosynthesis [63]. Figure 8: Cyclisation of the ketone to the terpenoid within a single biocatalytic step in contrast to a five-step synthesis [25]. Figure 9: Penicillin biosynthesis in _Penicillin chrysogenum_ as textbook example of NRPS assembly logic. Domain notation: A, adenylation; T, thiolation; C, condensation; E, epimerase; TE, thioesterase. However, the substrate tolerance inherent to NRPS is limited, which is why methods were sought to expand the substrate range of these enzymes. Initial attempts involved the swapping of entire A domains, yet these efforts were met with only limited success. The modest outcome could be attributed to the disruption of interdomain communication and the decreased activity of the engineered NRPS [61]. Furthermore, it has long been assumed that the C domains contribute to substrate proofreading [71]. This presumption stemmed from the observation that substitutions of cognate C-A or A-T-C domain pairs were more successful in NRPS engineering than substitutions of A domains alone. Recently, however, the model of substrate-specifying C domains was challenged both by protein evolution studies [72; 73] and by kinetic modeling [74].