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\begin{document}

\title{Improvement of an Escherichia coli whole-cell biocatalyst for geranyl
glucoside production using directed evolution}



\author[1]{Julian Ruediger}%
\author[1]{Wilfried Schwab}%
\affil[1]{Technical University of Munich}%


\vspace{-1em}



  
  \date{June 17, 2024}


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\begin{abstract}
The biotechnological production of glycosides is an economically
competitive manufacturing alternative to classical chemical synthesis.
Through continuous production improvement, glycosides can now be used in
low-cost products by various industries. However, many production
systems still suffer from low yields. Directed evolution, coupled with a
suitable screening method, can tackle this challenge. We generated
glycosyltransferase mutants through error-prone-PCR and screened the
library using a small-scale whole-cell biotransformation system. The
screening of only 176 colonies yielded three putative candidates.
Detailed investigations revealed that the reason for the increase in
product titer was mainly due to different expression effects of the
mutagenized genes rather than improved enzyme kinetics. In total, a
60-fold increase in product formation was achieved. Therefore, in
addition to the quality of the mutant library, an efficient and stable
expression system is crucial to achieve high concentrations of active
enzyme and product, as formation of inclusion bodies and other inactive
forms of the biocatalyst reduces productivity.%
\end{abstract}%



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