Strategies for Engineering Natural Product Biosynthesis in Fungi Elizabeth Skellam  Trends in Biotechnology  Volume 37, Issue 4, Pages 416-427 (April 2019) DOI: 10.1016/j.tibtech.2018.09.003 Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Examples of Biologically Active Fungal Natural Products Grouped by Biosynthetic Origin. Trends in Biotechnology 2019 37, 416-427DOI: (10.1016/j.tibtech.2018.09.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 Key Figure: An Overview of Engineering Strategies at the DNA and Protein Level to Influence the Production of Natural Products (NPs) in Native Hosts, in Heterologous Hosts, or In Vitro Traditionally NPs were identified through screening fungal chemical extracts for biologically active compounds. In the post-genomic era, there are numerous strategies for accessing NPs that are silent under normal laboratory conditions or from fungi that are difficult to cultivate. Starting at the DNA level, fungi can be manipulated at the global level to try to exert an effect over numerous NP pathways or engineered more precisely by considering a specific biosynthetic gene cluster (BGC). At the protein level, artificial enzymes can be constructed to engineer novel NPs or novel precursors can be introduced to diversify the structures of NPs. The desired result is the discovery of novel biologically active NPs; information about their chemical structure and bioactivities is used to understand the corresponding BGC or biosynthetic enzyme. The protein image in the second panel was adapted under Creative Commons Attribution 3.0 Unported license from user Dcrjsr; the original image is available at https://commons.wikimedia.org/wiki/File:TriosePhosphateIsomerase_Ribbon_pastel_trans.png. Trends in Biotechnology 2019 37, 416-427DOI: (10.1016/j.tibtech.2018.09.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 Schematic Representation of Fungal Cell Biology and Secondary Metabolism. Typically, the genes encoding the enzymes that synthesize a specific natural product (NP) are found physically clustered in the genome and are expressed under the same physiological conditions. Every gene in the biosynthetic gene cluster (BGC) has its own promoter and the regulation of coexpression of all pathway-specific genes is orchestrated through the binding of a transcription factor, which may also be encoded in the BGC. Furthermore, global transcriptional regulators may exert an effect over multiple BGCs involved in NP biosynthesis. This regulation of BGCs is tightly controlled and may be the result of environmental signals such as nutrient availability, light, pH, oxidative stress, competing organisms, etc. In eukaryotes, biosynthetic pathways may be compartmentalized at inter- and intracellular levels and the NPs may accumulate in specific tissues. A key feature of several of the major classes of fungal biosynthetic enzymes is that they are large, multifunctional enzymes containing distinct catalytic sites [e.g., polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs)]. Hybrid enzymes containing two fused modules are also common (e.g., PKS-NRPS), as are collaborative pairs of megasynthases where essentially each enzyme can be considered as a module. Trends in Biotechnology 2019 37, 416-427DOI: (10.1016/j.tibtech.2018.09.003) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 4 In Vivo and In Vitro Approaches for Engineering Fungal Biosynthetic Pathways Using a Native or Heterologous Host. The megasynthase represents a polyketide synthase (PKS), a nonribosomal peptide synthetase (NRPS), a PKS-NRPS, a terpene synthase, etc.; T1–T4 represent genes encoding different tailoring enzymes; TF, transcription factor. (A) Transcription factor/global regulator activation/replacement. (B) Native promoter replacement. (C) Gene inactivation. (D) Cloning and expression of the native biosynthetic gene cluster (BGC) in a heterologous host. (E) Combinatorial biosynthesis of genes from different BGCs expressed in a heterologous host. (F) Module swapping (e.g., in a PKS-NRPS). (G) Domain swapping (e.g., in a NR-PKS). (H) Enzyme deconstruction and reconstruction in vitro (e.g., in a NR-PKS). Trends in Biotechnology 2019 37, 416-427DOI: (10.1016/j.tibtech.2018.09.003) Copyright © 2018 Elsevier Ltd Terms and Conditions