Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous Host.

Abstract

A suite of molecular techniques have been developed in recent decades, which allow gene clusters coding for the biosynthesis of fungal natural products to be investigated and characterized in great detail. Many of these involve the manipulation of the native producer, for example, to increase yields of natural products or investigate the biosynthetic pathway through gene disruptions. However, an alternative and powerful means of investigating biosynthetic pathways, which does not rely on a cooperative native host, is the refactoring and heterologous expression of pathways in a suitable host strain. This protocol aims to walk the reader through the various steps required for the heterologous expression of a fungal biosynthetic gene cluster, specifically using Aspergillus oryzae strain NSAR1 and the pTYGS series of expression vectors. Briefly, this process involves the design and construction of up to four multigene expression vectors using yeast recombination, PEG-mediation transformation of A. oryzae protoplasts, and chemical extraction of the resulting transformants to screen for the presence of metabolites.