Previously, we have reported the combination of ammonia borane as a non-selective reductant alongside stereoselective amine oxidase enzymes to achieve the deracemisation of substituted N-heterocycles. Further biotransformations were run in which at the point of enamine formation, ammonia borane was added alongside an (R)-selective engineered variant of 6-hydroxy-D-nicotine oxidase (6-HDNO E350L/E352D), with a further addition of catalase from bovine liver (CBL) to alleviate the build-up of hydrogen peroxide resulting from sequential oxidations. For some substrates, this deracemisation allowed for asymmetric 2-phenylpiperidine derivatives to be formed in good ee (see Supporting Information). During the progress of this work, our library of IRED biocatalysts was also largely expanded through a metagenomics approach. (S)-Nicotine ( ) was identified as a potential natural product target for our methodology and the relevant keto-alcohol, 3-(pyridin-3oyl)propanol (19), was synthesised. After establishing both the wild-type and engineered choline oxidase enzymes were capable of oxidising this substrate, we screened various IRED biocatalysts from our library in biotransformations alongside AcCO6. We identified the putative IRED from Aspergillus lentulus (pIR-09) as capable of catalysing the annulation of methylamine with the keto-aldehyde to yield nicotine. While this enzyme produced the non-natural (R)-enantiomer, we once again observed the undesired enamine as the major product. As we were confident of the ability of ammonia borane to outcompete the stereoselective reduction of pIR-09, we attempted in situ deracemisation once more, allowing us to access the correct enantiomer 20 in a two-step chemoenzymatic synthesis from simple starting materials (Scheme 6). Although the overall yield in this synthesis was modest, it provides a clear blueprint in how this methodology may be applied to access asymmetric saturated amine heterocycles in good purity and ee, with enzyme engineering technologies available to improve reaction metrics. Scheme 6. Two-step chemoenzymatic synthesis of (S)-nicotine.