Team:Wageningen UR

Introduction

The discovery of secondary metabolites has had a profound impact on the development of human medicines. However, the widespread deployment of pharmaceuticals derived from secondary metabolites is severely limited by our ability to implement economically viable large-scale production systems. While advances in metabolic engineering and process technology have allowed the biobased production of fuels and chemicals to occur at an unprecedented scale, the production of many useful secondary metabolites has lagged behind. This is in part due to the slow growth and low productivity of the organisms that naturally produce these compounds, but also due to the complexity of the biochemical pathways involved in their biosynthesis. Therefore, using synthetic biology tools to identify, optimize and transplant these biosynthetic pathways into more productive host organisms is a major opportunity to overcome current technological limitations and ultimately to disseminate crucial medical treatments to people without the means to afford pharmaceuticals produced via traditional organic synthesis.

In order to pave the way for next-generation metabolic engineering, it was necessary to go beyond established standards. Escherichia coli is undoubtedly the most well-characterized organism in existence and has been the primary workhorse of fundamental synthetic biology research. However, despite the spectacular achievements in understanding and manipulating all levels of its physiology, when it comes to industrial-scale production, E. coli is far from an ideal cell factory. In contrast, yeasts and fungi are naturally more robust, more productive, and are easier to separate in downstream processing. The filamentous fungus Aspergillus niger is widely used in the production of organic acids due to its unmatched ability to secrete metabolites. Due to its inherent biosynthetic capabilities, the recent development of effective genome engineering tools makes it an ideal platform for advanced metabolic engineering applications.

In order to establish A. niger as a viable platform organism, we took a threefold approach:
a) The modularization and refactoring of a heterologous biosynthetic enzyme complex.
b) The development of a versatile molecular toolkit to facilitate the analysis of its physiology including in-situ ATP and pH biosensors, chromoproteins to screen transformants, and fluorescent cytoskeleton proteins to visualize A. niger’s cellular structure.
c) An evolutionary engineering method to develop a host strain with reduced mycelial cohesiveness for improved productivity.