Strategies for Engineering Natural Product Biosynthesis in Fungi

Abstract

Fungi are a prolific source of bioactive compounds, some of which have been developed as essential medicines and life-enhancing drugs. Genome sequencing has revealed that fungi have the potential to produce considerably more natural products (NPs) than are typically observed in the laboratory. Recently, there have been significant advances in the identification, understanding, and engineering of fungal biosynthetic gene clusters (BGCs). This review briefly describes examples of the engineering of fungal NP biosynthesis at the global, pathway, and enzyme level using in vivo and in vitro approaches and refers to the range and scale of heterologous expression systems available, developments in combinatorial biosynthesis, progress in understanding how fungal BGCs are regulated, and the applications of these novel biosynthetic enzymes as biocatalysts. Fungal NPs and Human Health NPs (see Glossary) or NP derivatives represented 25% of all FDA-approved drugs in the years spanning 1981 to 2014 [1]. Fungi are prolific producers of some of these important drugs, such as antibiotics (e.g., penicillin, pleuromutilin), cholesterol-lowering drugs (e.g., lovastatin, compactin), and immunosuppressants (e.g., mycophenolic acid, cyclosporine). Fungal NPs have many biological activities, ranging from carcinogens (e.g., aflatoxins), deadly toxins (e.g., a-amanitin), and industrial fungicides (e.g., strobilurin) to food colorants (e.g., azaphilones), hormones (e.g., gibberellin), and psychedelics (e.g., psilocybin) [2,3]. In nature, these NPs serve to protect fungi from predators (e.g., insects), UV radiation, and competition from other microorganisms and have evolved to secure their survival in niche habitats; they just happen to have had a profound effect on human health. The broad spectrum of biological activities reflects their structural diversity, which arises from their biosynthetic classification (e.g., polyketides, peptides, terpenes, alkaloids) (Figure 1). The complexity of fungal secondary metabolism presents numerous challenges for traditional drug discovery. Fungi often produce NPs at very low levels or in response to specific environmental cues that are difficult to reproduce in the laboratory [4]. The prominence of NPs in the pharmaceutical industry and re-emerging interest in NPs as lead structures in drug discovery [5] require methods to engineer improved NP analogs and activate silent biosynthetic pathways. In recent years there have been substantial advances in strategies for engineering fungal NP biosynthesis (Figure 2, Key Figure), which are highlighted in this review. These strategies can be divided into three broad categories: (i) inducing global transcriptional perturbations (e.g., through epigenetic modifications or the overexpression of global transcriptional regulators), which effectively 'awakens' the biosynthetic machinery and often affects multiple pathways resulting in the production of a variety of NPs, but is unpredictable and difficult to control Highlights

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

“…Successful heterologous expression demands apposite choice of host and vector systems, frequently the addition of foreign promoters for better regulation, and the toolkits and technologies helping to streamline the process. 1 Recent advances in increasing expression levels and product yield oen come from gene manipulation in the heterologous host. However, product yield from most reported fungal genetic studies still has great potential to be optimized.…”

“…azaphilones). 1 Almost all valuable fungal products are secondary metabolites (SMs) and are dened as organic molecules synthesized for purposes other than growth, development, or reproduction. 2 A lot of genes that encode for enzymes involved in the synthesis of SMs are arranged in biosynthetic gene clusters (BGCs) in the fungal genome, such as the ergot alkaloid BGC.…”

“…Fungal BGCs can be tightly regulated or completely silent in their native species, which explains the disparity between the number of predicted BGCs and known SMs. 1 For example, the plant endophytic fungus Dothideomycete sp. CRI7 exhibited the ability to produce a novel tricyclic polyketide and its biosynthetic precursor azaphilone derivatives.…”

Nonproteinaceous effectors: the terra incognita of plant–fungal interactions

“…However, this approach could result in unexpected phenotypes as a result of SM overproduction (Griffiths et al ., ). One strategy to overcome both in planta detection limits and functional redundancy is to take advantage of BGC expression in heterologous hosts like Saccharomyces cerevisiae or Aspergillus oryzae (Skellam, ). This method allows the production of large quantities of fungal SMs in vitro that can be further isolated and characterized chemically.…”

Manganese(II) Oxidizing Bacteria as Whole-Cell Catalyst for β-Keto Ester Oxidation

“…Conventional chemical production provides organic compounds that fulfil fundamental demands of modern society in pharmaceutical, agricultural, material and other fields, however, often at the expense of environment pollution and energy consumption. As such, biocatalysis provides a more favorable alternative considering its merits such as high catalytic activity and selectivity, mild reaction conditions (physiological pH and temperature), and environmental credentials (enzymes, organic solvent-free medium) [4][5][6]. In particular, whole-cell biocatalysis possesses unique advantages and extraordinary attractiveness.…”