Fungi are well known for their ability to produce a multitude of natural products On the one hand their potential to provide beneficial antibiotics and immunosuppressants has been maximized by the pharmaceutical industry to service the market with cost-efficient drugs On the other hand identification of trace amounts of known mycotoxins in food and feed samples is of major importance to ensure consumer health and safety Although several fungal natural products, their biosynthesis and regulation are known today, recent genome sequences of hundreds of fungal species illustrate that the secondary metabolite potential of fungi has been substantially underestimated Since expression of genes and subsequent production of the encoded metabolites are frequently cryptic or silent under standard laboratory conditions, strategies for activating these hidden new compounds are essential This review will cover the latest advances in fungal genome mining undertaken to unlock novel products

Strategies for mining fungal natural products

Peptides and proteins are chiral molecules with their structure determined by the composition and configuration of the amino acids constituting them. Natural amino acids (except glycine) display two chiral types (l- and d-enantiomers). For example, the presence of octopine, a derivative of l-arginine and d-alanine in octopus, or peptidyl poly-d-glutamic acid in a bacterial cell wall was demonstrated in the 1920s and 1930s, respectively. Nevertheless, an old dogma in biology was that proteins (in a strict sense) are composed of amino acids in the l-configuration exclusively, until a d-alanyl residue was reported in a frog skin opioid peptide in the early 1980s, and since, numerous d-amino acid containing peptides (DAACPs) have been discovered in multicellular organisms. Several hypotheses may be formulated to explain the origin of a d-residue in the peptide/protein chain. It may result from different mechanisms such as incorporation of a d-amino acid, non-enzymatic racemisation associated with ageing or diseases and enzymatic posttranslational modification. In the last case, the DAACPs are synthesised via a ribosome-dependent manner, and a normal codon for l-amino acid is present in the mRNA at the position where the d-residue is processed in the mature peptide by peptidyl aminoacyl l-d isomerisation, a peculiar and subtle posttranslational modification. In this review, the different pathways of biogenesis of DAACPs not only in bacteria but also in multicellular organisms are discussed, along with the description of the cellular specificity, the enzyme specificity and the substrate specificity of peptidyl aminoacyl l-d isomerisation.

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Biogenesis of D-amino acid containing peptides/proteins: where, when and how?

Penicillium chrysogenum (renamed P. rubens) is the most studied member of a family of more than 350 Penicillium species that constitute the genus. Since the discovery of penicillin by Alexander Fleming, this filamentous fungus is used as a commercial β-lactam antibiotic producer. For several decades, P. chrysogenum was subjected to a classical strain improvement (CSI) program to increase penicillin titers. This resulted in a massive increase in the penicillin production capacity, paralleled by the silencing of several other biosynthetic gene clusters (BGCs), causing a reduction in the production of a broad range of BGC encoded natural products (NPs). Several approaches have been used to restore the ability of the penicillin production strains to synthetize the NPs lost during the CSI. Here, we summarize various re-activation mechanisms of BGCs, and how interference with regulation can be used as a strategy to activate or silence BGCs in filamentous fungi. To further emphasize the versatility of P. chrysogenum as a fungal production platform for NPs with potential commercial value, protein engineering of biosynthetic enzymes is discussed as a tool to develop de novo BGC pathways for new NPs.

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Engineering of the Filamentous Fungus Penicillium chrysogenum as Cell Factory for Natural Products.

In industry, filamentous fungi have a prominent position as producers of economically relevant primary or secondary metabolites. Particularly, the advent of genetic engineering of filamentous fungi has led to a growing number of molecular tools to adopt filamentous fungi for biotechnical applications. Here, we summarize recent developments in fungal biology, where fungal host systems were genetically manipulated for optimal industrial applications. Firstly, available inducible promoter systems depending on carbon sources are mentioned together with various adaptations of the Tet-Off and Tet-On systems for use in different industrial fungal host systems. Subsequently, we summarize representative examples, where diverse expression systems were used for the production of heterologous products, including proteins from mammalian systems. In addition, the progressing usage of genomics and functional genomics data for strain improvement strategies are addressed, for the identification of biosynthesis genes and their related metabolic pathways. Functional genomic data are further used to decipher genomic differences between wild-type and high-production strains, in order to optimize endogenous metabolic pathways that lead to the synthesis of pharmaceutically relevant end products. Lastly, we discuss how molecular data sets can be used to modify products for optimized applications.

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Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi.

Bacteria of the genus Rhodococcus are well known for their ability to degrade a large range of organic compounds. Some rhodococci are free-living, saprophytic bacteria; others are animal and plant pathogens. Recently, several studies have shown that their genomes encode putative pathways for the synthesis of a large number of specialized metabolites that are likely to be involved in microbe-microbe and host-microbe interactions. To systematically explore the specialized metabolic potential of this genus, we here performed a comprehensive analysis of the biosynthetic coding capacity across publicly available rhododoccal genomes, and compared these with those of several Mycobacterium strains as well as that of their mutual close relative Amycolicicoccus subflavus. Comparative genomic analysis shows that most predicted biosynthetic gene cluster families in these strains are clade-specific and lack any homology with gene clusters encoding the production of known natural products. Interestingly, many of these clusters appear to encode the biosynthesis of lipopeptides, which may play key roles in the diverse environments were rhodococci thrive, by acting as biosurfactants, pathogenicity factors or antimicrobials. We also identified several gene cluster families that are universally shared among all three genera, which therefore may have a more ‘primary’ role in their physiology. Inactivation of these clusters by mutagenesis might help to generate weaker strains that can be used as live vaccines. The genus Rhodococcus thus provides an interesting target for natural product discovery, in view of its large and mostly uncharacterized biosynthetic repertoire, its relatively fast growth and the availability of effective genetic tools for its genomic modification.

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Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus.

cIntense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: d-(l-a-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.

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Rï¿½mon Boer

Papers

Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase