# 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
It turned out that C domains influence the rate of product formation, but have no direct effect on substrate selection [74]. Clearly, this insight has important implications for NRPS engineering. Even under the assumption that A domains act as the sole gatekeepers in NRPS biosynthesis [74], the processivity of the corresponding assembly lines depends, to a large extent, on protein-protein interactions between C, A, and other domains. For this, appropriate recognition or linker sequences are essential. Communication-mediating (COM) domains were initially discovered in 2004, after the construction of NRPS mutants with truncated C-terminal regions [75]. Although a strategy for the biocombinatorial assembly of hybrid NRPS was already described at this time [75; 76], it was not until 2018 when the structural basis of the interactions between N- and C-terminal docking domains (DDs) was resolved [77]. In recent years, several studies have demonstrated the feasibility of reprogramming NRPS by swapping or mutating the COM domains and DDs, respectively [77; 78; 79]. However, it also became clear that this approach, albeit powerful, is limited in its applicability with regard to NRPS enzymes that consist of multiple modules. One solution to this problem was the splitting of multimodule NRPS by natural DD pairs [64]. Moreover, alignments of interdomain linker regions from NRPS found in _Photorhabus_ and _Xenorhabditis_ bacteria led to the identification of fusion positions between C and A domains that can be exploited for the assembly of customized NRPS [65]. The necessary building blocks are referred to as exchange units (XUs) and consist of three domains (A-T-C or A-T-C/E). By obeying few compatibility rules, the fusion of different XUs leads to functional NRPS (Figure 10A) that produce novel peptides with acceptable yields [65; 66]. The combinatorial concept underlying XUs (Figure 10A) could be developed even further after the discovery of a highly conserved fusion point inside the C domains. By merging appropriate NRPS parts at these sites, highly specific and efficient peptide synthetases can be generated [67]. Despite this progress, the construction of chimeric NRPS was still quite laborious. The cloning of the required DNA constructs had to deal with the giant size of NRPS biocatalysts and with repetitive sequence stretches alike. To reduce the complexity of these systems, their splitting into discrete, yet functional subunits was thus desirable. This goal was eventually achieved by furnishing the previously defined XUs with synthetic peptide sequences that can interact with each other via a coiled-coil structural motif [68]. With the help of such synthetic zippers (SZs; Figure 10B), a new type of NRPS architecture has been established that is both productive and easy to handle in the laboratory [68]. An interesting alternative to the use of SZs is to endow split NRPS modules with zinc finger tags [69]. In this way, DNA-templated NRPS can be engineered (Figure 10C), which enables a sequence-controlled biosynthesis of tailored peptides [69]. Except for the replacement of entire domains, modules or XUs, less invasive approaches were also tested, such as the mutagenesis of A domains [80]. An important milestone in this research area was the development of screens for the rapid testing of randomly mutated A domains. The respective assays are based on yeast surface display and enable the selective labeling of catalytically active A domains with fluorophores. In this way, desirable mutations can be readily identified via fluorescence activated cell sorting [81]. Another, more targeted strategy is the swapping of the flavodoxin-like subdomain (FSD). The FSD resides within A domains and is known to harbor the key residues for substrate binding [82; 83]. Unlike entire A domain swaps, FSD replacements are well tolerated and typically do not interfere with interdomain communication [83].