Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous Host.
Investigating Fungal Biosynthetic Pathways Using Heterologous Gene Expression: Aspergillus oryzae as a Heterologous Host.
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DOI：
10.1007/978-1-0716-2273-5_2
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发表时间：
2022-01-01
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影响因子：
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通讯作者：
Williams, Katherine
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文献类型：
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作者：
de Mattos-Shipley, Kate M J;Lazarus, Colin M;Williams, Katherine
近几十年来发展了一套分子生物学技术，可以对真菌天然产物生物合成的基因簇进行详细的研究和表征。其中许多涉及对天然生产者的操纵，例如，增加天然产物的产量或通过基因破坏研究生物合成途径。然而，研究生物合成途径的另一种强大手段是在合适的宿主菌株中重构和异源表达途径，这种手段不依赖于合作的天然宿主。该方案旨在引导读者通过真菌生物合成基因簇的异源表达所需的各种步骤，特别是使用Aspergillus sp.菌株NSAR 1和pTYGS系列表达载体。简单地说，这个过程包括设计和构建多达四个多基因表达载体，使用酵母重组、PEG介导的A.原生质体，并对所得转化体进行化学提取以筛选代谢物的存在。
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.