Chiral amines are important chemical building blocks found in a vast array of biologically-active pharmaceutical ingredients, and a sustainable route to their synthesis is of wide appeal. The use of biocatalysis as an alternative to traditional chemical methods boasts many advantageous qualities, such as the ability to catalyse a myriad of highly selective transformations in aqueous environments, and increased atom economies to reduce waste. As such, the primary aim of this study was to develop multi-enzymatic routes towards the synthesis of chiral secondary amine scaffolds. It is becoming increasingly possible to combine several sequential biocatalytic reactions, via de novo enzymatic pathways, for the generation of complex value-added compounds from simple precursors. These cascades take advantage of generally compatible reaction conditions, and can be implemented in an in vivo, in vitro or hybrid in vitro / in vivo cascade design. Through the adaptation of the design-build-test-analyse (DBTA) cycle, several multi-enzyme cascades were established for the preparation of chiral pyrrolidine and piperidine compounds, exploiting a range of carboxylic acid reductase (CAR), Ï-transaminase (Ï-TA), imine reductase (IRED), galactose oxidase (GOase), and alcohol dehydrogenase (ADH) enzyme homologs. In particular, a CAR-TA-IRED single whole cell cascade for the production of chiral secondary amines through the conversion of simple linear keto acids was achieved, with all cofactor requirements being met by the microbial host cell. Cascade optimisation was achieved through sequential parameter investigation, and control over the expression levels of recombinant cascade enzymes was accomplished through gene duplications. An alternate in vitro GOase-IRED cascade was developed for the successful synthesis of the valuable drug precursor 3-aminopiperidine. Amide bond formation remains a key target for biocatalytic processes due to current routes requiring harsh reaction conditions, and as such the ATP-independent N-acyltransferase CapW was identified as a potential new addition to the ever-expanding biocatalytic toolbox. Soluble recombinant expression of active CapW in E. coli cells was achieved here for the first time, and was verified via a screen for hydrolytic activity against Î²-lactamase substrate Nitrocefin, paving the way for the production of increased amounts of this protein for future applications such as crystallisation studies.