# 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
Enzyme engineering was initiated based on the homology model structure and, after only three rounds of engineering, the required criterion of 100 G\(\cdot\)L\({}^{-1}\) substrate loading could be reached. The final enzyme variant _Sp_KDOs have recently been employed in a combined chemo-enzymatic total synthesis of the potential APIs tambromycin [44] and cepafungin I [45], which are studied for use in cancer treatment (Figure 4B). Hydroxy-L-lysines can also be used as intermediates for the generation of chiral amino alcohols which represent multifunctional chiral building blocks [46]. [PERSON] et al. demonstrated the identification of novel KDOs utilizing cell-free protein synthesis, which has the potential to drastically speed up the screening of enzymes [47; 48]. KDOs have been efficiently used in a whole-cell biocatalyst format for the preparative synthesis of hydroxy-L-lysines with product concentrations of multiple grams per liter [40; 48]. Moreover, [PERSON] et al. recently employed KDOs as immobilized enzymes [20]. This led to a remarkable increase in their stability. Immobilized enzymes could be reused in repetitive batches up to seven times, and in preparative biotransformation reactions, 32.4 g-L-1 of product were produced, with a specific space-time yield of 100 G-L-1-h-1 per gram of immobilized enzyme with a KDO from _Catenulispora acidophilia_[20]. [PERSON] et al. demonstrated the first successful engineering of a KDO from _Niastella koreensis_ by semi-rationally engineering the active site. The specific activity of the enzyme was improved more than four-fold [43]. The group of [PERSON] and coworkers made substantial progress in engineering \(\alpha\)KGDs and contributed to understanding the structure-function relationship of the enzymes [21]. They set out to engineer a l-proline _cis_-4-hydroxylase from _Sinorhizobium melilioti_ for hydroxylation of a novel substrate, the non-proteinogenic amino acid l-homophenylalanine (Figure 5A) [21]. The respective hydroxylated product represents an intermediate for the  
Figure 4: (**A**) Biocatalytic reaction of the KDO from _Catenulispora acidophilia_[39]. (**B**) Molecules of pharmacological interest with chiral hydroxy-L-lysine as building block. \(\alpha\)-KG, \(\alpha\)-ketoglutarate. synthesis of potential kynureinase or kynureine monooxygenase inhibitors, which might be of use in the treatment of certain neurodegenerative disorders and cancer. In the study, the W40M/I103L enzyme variant exhibited a \(>\)100-fold improved activity and a \(>\)300-fold improved catalytic efficiency. Moreover, the turnover number was optimized 10-fold. Interestingly, a single amino acid exchange (W40Y) led to a shift from the hydroxylated product to the desaturated product, showcasing the first \(\alpha\)KGD to be engineered from hydroxylase to desaturase activity. In another study, the same L-proline _cis_-4-hydroxylase was engineered for halogenation of the native substrate (Figure 5B) [22]. One amino acid exchange (D108G) led to halogenation activity, which was further elevated over several rounds of enzyme engineering. The final enzyme variant SmP4H-7 showed a 12-fold improvement in the activity and a 98-fold improvement in the catalytic efficiency compared to the starting biocatalyst [22]. The mentioned studies demonstrate the power of enzyme engineering of \(\alpha\)KGDs, allowing the conversion of new substrates and even re-programming of the native enzyme reaction. Biocatalytic Scaffold Formation  
Many published total syntheses of natural compounds are labeled biomimetic. Certainly, this does not only mean that a certain synthesis step also occurs in a similar way in biosynthesis; rather, the word is also used to denote elegance, effectiveness or even non-conventionality.