This project focuses on the improvement of the enzyme hyoscyamine 6β-hydroxylase (H6H) via directed evolution techniques for the development of an environment-friendly, economical and high-yielding biocatalytic process for scopolamine production. H6H is a critical enzyme for biosynthesis of scopolamine, a valuable tropane alkaloid used in medicine to treat conditions such as motion sickness, gastrointestinal spasms, mydriasis induction and premedication in anesthesia. Currently, scopolamine production depends on field cultivation, leaf extraction and purification from contaminants and other tropane alkaloids. This methods has key limitations and a completely in vitro process would be advantageous to the pharmaceutical industry. While chemical synthesis remains not viable due to difficulties in obtaining enantiopure scopolamine in high yield, biotechnological approaches have demonstrated that enzymatic synthesis of scopolamine is achievable. However, enzyme activity improvement is needed to establish a high-yielding biocatalytic process for its production. Among protein engineering, evolutionary methods are a well stablished set of techniques to improve the catalytic properties and other features for biocatalyst optimization. Indeed, many engineered enzymes tailored via directed evolution are currently used in the pharmaceutical industry. In this context, the overall goal of the project is the development of an alternative biocatalytic process for efficient scopolamine synthesis. Briefly, we propose to carry out random mutagenesis via error-prone polymerase chaine reaction (epPCR) and site-saturation mutagenesis (SSM) of putative active site positions in H6H. Additionally, beneficial mutations of improved variants will be combined by recombination methods. Firstly, catalytic activity of mutants will be screened utilizing high-throughput technology and then validated by HPLC-UV. Our goal is to achieve a > 10-fold improvement in activity for H6H-catalyzed conversion of hyoscyamine to scopolamine. In addition, evolutionary methods are powerful tools for elucidating protein structure-function relationships, an aspect of H6H that is not thoroughly elucidated. According to the vast bibliography on enzyme activity improvement through evolutionary methods and the expert opinion and extensive working experience in these strategies of Dr. Rudi Fasan, we foresee that evolutionary methods could provide a means to greatly improve the catalytic activity of H6H in the two-step conversion of hyoscyamine to scopolamine. In conclusion, the most significant contribution of this project will be the directed evolution of improved hyoscyamine 6β-hydroxylase based biocatalysts for establishing an efficient bioprocess for scopolamine production. In addition, these experiments will provide valuable information about structure-function relationships in the enzyme.