School of Biological Sciences, University of Bristol, Bristol, UK.
Methods Mol Biol. 2022;2489:23-39. doi: 10.1007/978-1-0716-2273-5_2.
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.
近几十年来，已经开发出了一系列分子技术，这些技术可以对编码真菌天然产物生物合成的基因簇进行详细的研究和表征。其中许多技术涉及对天然产物产生菌的操作，例如，提高天然产物的产量或通过基因敲除来研究生物合成途径。然而，一种替代的、强大的研究生物合成途径的方法是在合适的宿主菌株中对途径进行重构和异源表达，而不需要合作的天然宿主。本方案旨在为读者提供一个用于真菌生物合成基因簇异源表达的各个步骤，具体使用米曲霉 NSAR1 菌株和 pTYGS 系列表达载体。简而言之，这个过程包括使用酵母重组设计和构建多达四个多基因表达载体，通过 PEG 介导的原生质体转化，以及通过化学提取来筛选转化子中代谢物的存在。
