# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Expanding Biocatalytic Capabilities in Diversification and Late-Stage Functionalization  
Late-stage functionalization (LSF) of non-activated carbon centers in natural products and active pharmaceutical ingredients (APIs) is a fundamental tool for drug discovery and drug synthesis [84; 85]. However, the direct installation of typically hydroxy-, methyl-, or chloro-groups as well as the formation of C-O, C-N, C-S or C-C bonds in complex scaffolds, challenge synthetic organic chemistry in terms of selectivity and mild reaction conditions to ensure the integrity of sensitive functional groups in drug molecules [86; 87]. In addition to metal-catalyzed C-H activation, photocatalysis, or electrosynthesis, biocatalysis has proven to be an advantageous tool for LSFs. High chemo-, regio-, and stereoselectivity, and ambient conditions in aqueous media turned enzyme-catalyzed reactions into promising alternatives to conventional synthesis [86]. As the state-of-the-art native resources do not harbor enzyme-catalyzed equivalents to all synthetic LSFs, identification of novel naturally occurring biocatalysts, in combination with the design of new-to-nature enzymes with desired catalytic activity, are necessary to make biocatalysis more competitive [88]. A selection of current approaches is described in the Table 3.  
Members of the class of oxidoreductase, such as cytochrome P450 monooxygenenses (P450s), lacases, unspecific peroxygenases (UPOs) or lytic polysaccharide monooxygenases, hold great potential for industrial and pharmaceutical applications [89]. To date, the large and versatile group of P450s are the most common biocatalysts for C-H functionalizations [88; 90]. These heme enzymes are widely distributed across all domains of life and selectively insert oxygen into various molecules, as well as catalyze heteroatom dealkylations and C-C bond cleavage [91]. Due to their catalytic properties, they have been the subject of latest investigations ranging from genome mining and enzyme activity screening to computation and protein engineering [92]. To broaden the available enzyme portfolio, new P450s were recently identified by genome mining and the transcriptional analysis of wild-type bacteria and fungi (Figure 11) [93; 94; 95; 96; 97; 98; 99]. [PERSON] et al. demonstrated the potential of genome mining in combination with strain screening by testing 84 microorganisms  
Figure 10: Recently described strategies for the engineering of NRPS. (**A**) Construction of chimeric NRPS from exchange units (XUs). (**B**) Linkage of mono-modular NRPS with synthetic zippers (SZs). (**C**) Generation of DNA-templated NRPS with zinc fingers. (**D**) Swapping of flavodoxin-like subdomains inside A domains with the help of CRISPR-Cas9. for their conversion of seven pharmaceutical compounds (amodiaquine, cyclospor A, tamoxifen, haloperidol, ritonavir, rapamycin, and testosterone) [94]. They identified three promising strains, which accepted all seven substrates, as well as six additional strains converting six substrates [94]. Further transcriptional analysis and heterologous expression in _E. coli_ C42 (DE3) revealed a novel P450, belonging to the CYP105C family, that converts the anti-HIV agent ritonavir to hydroxy ritonavir as the main product (19% yield in the wild-type strain _Actinosymrena mirum_[94]; -1.6% yield in _E. coli_) [98]. In a two-step screening approach focusing on the discovery and characterization of new P450s, the [PERSON] group identified and described the product selectivity of two P450s from _Streptomyces platensis_ with a high preference for hydroxylation (CYP105D, 58% hydroxy ritonavir) or demethylation (CYP107Z, 53% demethylated and 7% didemethylated amitriptyline) [95]. However, since the crux of screening is that \"you only get what you screen for\", the actual catalytic capability of a strain or an enzyme cannot be displayed in activity screenings with a limited number of substrates.