# Recent Advances in Biocatalysis for Drug Synthesis
## 2 Biocatalysis for Drug Synthesis
### Synthetic Enzyme Cascades
The glyceraldehyde 3-phosphate derivative was obtained from the cascades starting point 2-ethylglycerol through oxidation and phosphorylation by pantothenate kinase (PanK) and the correct stereocenter was introduced by galactose oxidase (GOase). The enzymes of this main pathway were engineered by directed evolution to meet certain criteria like increased activity, the acceptance of non-natural substrates or high substrate concentrations and stereoselectivity. Four further enzymes were included, namely horseradish peroxidase (HRP), catalase, acetate kinase (AcK) and sucrose phosphorylase (SP) for, e.g., cofactor regeneration or an equilibrium shift towards islatravir by removing the released phosphate during the last step. In addition, some biocatalysts of the nine-enzyme cascade were immobilized for simpler product isolation. Finally, islatravir was received with a 51% overall yield without any isolation steps out of simple achiral starting materials [141]. Along with these rather rational approaches for the design of synthetic routes, natural pathways can be used as template as well. The in vitro enzymatic syntheses of the cannabinoids cannabigerolic acid (CBGA) and cannabigerovaninic acid (CBGVA) were realized  
by following and modifying the glycolysis and mevalonate pathway towards geranyl pyrophosphate (GPP) [151]. GPP is used for the prenylation of fed aromatic substrates leading to the cannabinoids yielding titers over 1 g L\({}^{-1}\). Cannabinoidss have interesting pharmaceutical properties and tetrahydroccannabinol (THC) and cannabidiol (CBD) are already approved drugs, though, many more clinical trials investigate other cannabinoidss for the treatment of various diseases as well [156]. However, this large cascade consists of 25 enzymes, lowering the commercial viability. In 2020, the system was improved to synthesize CBGA and CBGVA by twelve enzymes, also allowing the production of the aromatic precursors in situ (Figure 14D) [152]. This one-pot cascade can be separated into four modules. The precursors GPP and olivetolic acid (OA) or divarinic acid (DA) have to be built up by two separate routes with four and five enzymes, respectively. The synthesis of GPP follows a simplified isoprenoid biosynthesis pathway starting with isoprenol. OA or DA are built up from malonate and butyrate or hexanoate. The need for ATP, an important cofactor for the cannabinoid synthesis, could be reduced by three equivalents, by clever pathway adjustment using a non-natural reaction of the malonate decarboxylase (MdcA) \(\alpha\) subunit and a further module regenerates ATP from acetyl-phosphate. The final step of the system prenylates OA or DA to the cannabinoid product by CBGA synthase. Remarkably, the system produced CBGA and CBGVA in the \(\sim\)0.5 g L\({}^{-1}\) range in 10 h, which is nearly two orders of magnitude higher than by using an engineered yeast, where the highest reported titer was 8 mg L\({}^{-1}\)[152; 157]. Another example for the ability of biocatalysis to shorten synthesis routes is the generation of cyclic dinucleotides (CDNs). CDNs are ubiquitously occurring second messenger molecules. In vertebrates, the cyclic GMP-AMP dinucleotide 2'3'-cGAMP induces the production of interferons and cytokines as part of the innate immune system by binding to the STING receptor [158]. This makes 2'3'-cGAMP a promising candidate for pharmaceutical applications as potent immune-oncology therapeutics. Some derivatives are currently investigated for their activity against cancer and as vaccine adjuvants [159]. While the chemical synthesis of cyclic dinucleotides is possible, it requires multiple reaction steps, complex protective group chemistry and it suffers from low yields [160]. [PERSON] et al. demonstrated the production of several 2'3'-cGAMP derivatives in one reaction step using a truncated version of human cyclic GMP-AMP synthase (cGAS), exploiting the enzymes natural substrate promiscuity [161]. Thereby, 2'3'-cGAMP, cyclic GMP-2'-F-AMP and cyclic GMP-8-NH\({}_{2}\)-AMP were produced in a preparative scale.