Establishment of cell-free expression systems derived from non-standard bacterial organisms for natural product synthesis and screening with electroanalytical methods

Abstract

Cell-free expression systems are powerful and cost-efficient in vitro tools for the rapid production of high-value biomolecules. Historically, cell-free expression has relied solely on the cellular machinery of model bacterial organisms like Escherichia coli. However, there has been a recent surge in cell-free expression systems derived from non-standard bacterial organisms that can be used for highly specialized applications such as the synthesis of small molecule natural products. Given that over two-thirds of the world's antibiotics are produced by members of the Streptomyces genus, it would be exceedingly valuable to have the capacity to synthesize important, and potentially novel, natural products on-demand without the need to maintain live bacterial cultures or employing costly synthetic chemistry methods. Thus, the development of a robust cell-free expression system for natural product synthesis is warranted. Herein, the establishment and optimization of several candidate cell-free expression systems derived from the crude cell extracts of Streptomyces and Pseudomonas species for natural product synthesis is reported. In addition, the viability of mixtures of crude cell extracts as novel chassis for cell-free natural product synthesis is evaluated in order to establish a path towards the production of otherwise inaccessible biomolecules. In an effort to develop sensitive and high-throughput screening methods for cell-free natural product synthesis, the use of electroanalytical methods such as square-wave and cyclic voltammetry to track the cell-free expression of the redox-active natural product pyocyanin is reported using crude cell extracts from Pseudomonas aeruginosa. Similar methods are then applied towards building a natural product discovery pipeline with Streptomyces violaceoruber. Overall, this work establishes several cell-free expression systems not reported in the literature and provides a unique approach towards their optimization for natural product synthesis using highly sensitive electroanalytical methods over the standard fluorescence-based routes. Further development of this technology would be beneficial to a multitude of scientific disciplines.