1. Taxonomy of Aspergillus, Penicillium and Talaromyces and its Significance for Biotechnology

Jos Houbraken, Robert A. Samson and Neriman Yilmaz

Various fungi are used in biotechnology for their ability to produce a variety of small molecules and enzymes. The order Eurotiales contains the species-rich genera Aspergillus, Penicillium and Talaromyces and some species belonging to those genera are utilised for biotechnology. Application of the single name nomenclature has led to numerous name changes for many fungi. This chapter will provide an overview of important name changes for the genera Aspergillus, Penicillium, Talaromyces and other related genera. The number of newly described species has also increased significantly in the last decade. A sequence based approach is currently recommended to correctly identify species of these genera. This chapter will also provide an overview of molecular identification techniques for isolates belonging to these genera.

2. Comparative Genomics, Resequencing and Fast Forward Genetics in Aspergillus and Penicillium

Scott E. Baker and Erin L. Bredeweg

New methods have accelerated the pace of DNA sequencing for comparative genomics and genetics of fungi. High throughput genome sequencing enables comparative analysis of multiple strains of the same species and mutagenized strain lineages with interesting phenotypes. With their compact genomes, species from the genera Aspergillus and Penicillium are ideal for these multi-strain genome analyses that connect genotypes with phenotypes of interest to fungal biology and biotechnology.

3. Diversity and Mechanisms of Genomic Adaptation in Penicillium

Jeanne Ropars, Ricardo C. Rodríguez de la Vega, Manuela López-Villavicencio, Jérôme Gouzy, Joëlle Dupont, Dominique Swennen, Emilie Dumas, Tatiana Giraud and Antoine Branca

Penicillium is a diverse fungal genus with hundreds of species occurring worldwide in various substrates, from soil to food, and with various lifestyles, from necrotrophic pathogenicity to endophytic mutualism. Several species are important for human affairs, being widely used in industry, such as the penicillin-producer P. rubens, the two cheese starters P. camemberti and P. roqueforti, and the mold used for fermenting sausages, P. nalgiovense. Other species are food spoilers that produce harmful mycotoxins or cause damages in fruit crops. Currently, 30 genomes of Penicillium belonging to 18 species are available. In this chapter, we reconstruct a phylogenetic tree based on available Penicillium genomes and outline the main features of the genomes, such as gene and transposable element content. We then review the recent advances that the available genomic and transcriptomic resources in the Penicillium genus have allowed regarding our understanding of the genomic processes of adaptation, including changes in gene content, expression and strikingly frequent and recent horizontal gene transfers. In addition, we summarize recent studies using genetic markers on the level of genetic diversity, mode of reproduction and population structure within Penicillium species. Overall, the Penicillium genus appears highly suitable models for studying the mechanisms of adaptation.

4. Approaches for Comparative Genomics in Aspergillus and Penicillium

Jane L. Nybo, Sebastian Theobald, Julian Brandl, Tammi C. Vesth and Mikael R. Andersen

The number of available genomes in the closely related fungal genera Aspergillus and Penicillium is rapidly increasing. At the time of writing, the genomes of 62 species are available, and an even higher number is being prepared. Fungal comparative genomics is thus becoming steadily more powerful and applicable for many types of studies. In this chapter, we provide an overview of the state-of-the-art of comparative genomics in these fungi, along with recommended methods. The chapter describes databases for fungal comparative genomics. Based on experience, we suggest strategies for multiple types of comparative genomics, ranging from analysis of single genes, over gene clusters and CaZymes to genome-scale comparative genomics. Furthermore, we have examined published comparative genomics papers to summarize the preferred bioinformatic methods and parameters for a given type of analysis, highly useful for new fungal geneticists. Moreover, the chapter contains a detailed overview of comparative genomics studies of key fungal traits such as primary metabolism, secondary metabolism, and secretome analysis. Finally, we gaze into a possible future of the field by comparing the current state of fungal comparative genomics to the development in bacterial genomics, where the comparison of hundreds of genomes has been performed for a while.

5. Blue Mold to Genomics and Beyond: Insights into the Biology and Virulence of Phytopathogenic Penicillium Species

Wayne M. Jurick II, Jiujiang Yu and Joan W. Bennett

Apples and pears are economically important pome fruits grown and consumed worldwide. The United States is the second largest producer of apples and pears in the world behind China. Fruit decay caused by blue molds, Penicillium expansum and other Penicillium spp., results in significant economic losses to the apple and pear fruit packing and processing industries. Since blue mold fungi produce several mycotoxins, they also are a food safety concern. In recent years, various '-omics' technologies have been used to learn more about fungal virulence and toxigenic potential, and to find possible news ways to control Penicillium species that infect pome fruits. Several P. expansum genomes have been sequenced and compared with the genomes of other sequenced Penicillium species. In this chapter, the current status of blue mold biology, control, epidemiology, genomics and functional genomics are discussed.

6. Post-genomic Approaches to Dissect Carbon Starvation Responses in Aspergilli

Jolanda M. van Munster, Anne-Marie Burggraaf, Istvan Pocsi, Melinda Szilágyi, Tamas Emri and Arthur F.J. Ram

Most filamentous fungi have a saprophytic lifestyle and proliferate on organic materials from plants. They contain a large arsenal of enzymes that are expressed and secreted in response to available carbon sources. Regulatory networks in which carbon-specific transcription factors and wide-domain transcription factors play essential roles tightly control the expression of these enzymes. While a suitable carbon source is present, the entire metabolism of the fungus is directed to grow as fast as possible via hyphal tip extension. At a certain point however, the available exogenous carbon source will be limited, resulting in a carbon starvation response. Filamentous fungi react to carbon starvation with some very specific responses including the induction of glycosyl hydrolases involved in fungal cell wall degradation (autolysis) and the onset of asexual spore formation. Recent advances in genome-wide transcriptomic and proteomic approaches have revealed new insights into molecular mechanisms involved in these fungal-specific carbon starvation responses. Together with the genetic accessibility of filamentous fungi, the omics technologies have made a large contribution in broadening our understanding of the carbon starvation response in recent years. In this review, we have focussed on summarizing and integrating the most important cellular responses of filamentous fungi towards carbon starvation. As most studies to date have been carried out with Aspergillus nidulans and Aspergillus niger, the focus will be further on these two species and to compare their responses to carbon starvation.

7. Genetics and Physiology of Sulfur Metabolism in Aspergillus

Andrzej Paszewski, Jerzy Brzywczy, Marzena Sieńko and Sebastian Piłsyk

Aspergillus nidulans, as many other fungi, can utilize sulfate as a sulfur source through a highly energy consuming sulfate assimilation pathway producing sulfide which is then incorporated into cysteine. When cysteine is in excess this pathway is repressed by the hierarchical sulfur metabolite repression (SMR) system, in which the SCF ubiquitin ligase activated by cysteine brings about degradation of the MetR transcription factor specific for sulfate assimilation pathway genes. Mutational impairment of SMR causes a permanent derepression of the sulfate assimilation pathway and leads to a direct synthesis of homocysteine from sulfide as an alternative route of synthesis of sulfur amino acids. This derepression results in overproduction of sulfide and sulfur-containing organic compounds associated with a considerable dissipation of ATP and NADPH. Transcriptomic analysis of mutants dysregulated in SMR has revealed considerable remodeling of cellular metabolism manifested by changes in the levels of several hundred transcripts. Among the up‑regulated genes the most remarkable are those encoding proteins normally involved in responses to various environmental stresses. Expression of genes encoding enzymes involved in homocysteine metabolism is independent of SMR and is activated by this amino acid, which prevents its accumulation above a toxic level.

8. Production of Feruloyl Esterases by Aspergillus Species

Miia R. Mäkelä, Luis Alexis Jiménez Barboza, Ronald P. de Vries and Kristiina S. Hildén

Ferulic acid esterases are enzymes capable of releasing ester-linked hydroxycinnamic acids from certain polysaccharide moieties. These carbohydrate polymers, xylan and pectin, are ubiquitous in nature, and are mostly found as major constituents of plant cell walls. Plant cell walls are the most common renewable carbon source on earth. They encompass many different types of biopolymer complexes harnessed together such as cellulose, hemicelluloses, pectin, lignin and proteins. Together these compounds form lignocellulose, which is one of the most resilient materials found in nature. Ferulic acid is one of the possible linkages between plant cell wall polymers and therefore is a major factor in the recalcitrance of cell wall against microbial attack. Saprobic plant cell wall degrading fungi therefore produce feruloyl esterases that are capable of removing ferulic acid from the polysaccharides in order to weaken the integrity of the cell wall. In this study we evaluated the variation in feruloyl esterase production of a set of Aspergillus| species, a fungal genus with many industrial applications, during growth on plant biomass in the presence and absence of ferulic acid.

9. Secondary Metabolite Formation by the Filamentous Fungus Penicillium chrysogenum in the Post-genomic Era

Marta M. Samol, Oleksandr Salo, Peter Lankhorst, Roel A.L. Bovenberg and Arnold J.M. Driessen

The filamentous fungus Penicillium chrysogenum is a major industrial producer of the β-lactam antibiotic penicillin. Penicillin was discovered by Alexander Fleming in 1928, and was among the first medications to be effective against bacterial infections. Penicillins are still widely used today but are now produced by high β-lactam yielding strains that emerged from extensive classical strain improvement programs lasting for several decades. In 2008, the genome of P. chrysogenum was sequenced revealing an unexploited reservoir of nonribosomal peptide synthetases and polyketide synthases genes that specify potential bioactive compounds. In recent years, several pathways have been resolved that are responsible for the production of a wide variety of secondary metabolites.

10. The pH Modulation by Fungal Secreted pH Effecting Molecules: A Mechanism Affecting Pathogenicity and Mycotoxin Accumulation During Colonization by Penicillium expansum

Dov Prusky, Shiri Barad, Nofar Glam, Nancy Keller and Amir Sherman

A postharvest pathogen as Penicillium can start its attack process as soon as spores land on wounded tissue; other pathogens as Colletotrichum breach the unripe fruit cuticle, remain quiescent for months until the fruit ripens and then colonize the host. Both post-harvest fungal pathogens initiate their development by secreting non-proteinaceous pH modulating factors as organic acids or ammonia, which acidify or alkalinize the ambient host environment. These fungal secreted pH effecting molecules (SEM) modulate the host environment, activate secondary metabolism and regulate an arsenal of enzymes in order to increase fungal pathogenicity under any specific conditions. This arsenal includes genes and processes that compromise host defenses, enhance programmed cell death (PCD), contribute to intracellular signaling, produce cell-wall-degrading enzymes, regulate specific transporters, induce redox protectant systems and generate factors needed by the pathogen to cope effectively with the hostile environment found within the host. In Penicillium, genes that contribute to gluconic acid (GLA) and secondary metabolites accumulation found to be pH regulated by the specific transcription factor PacC, indicating the capability to respond under acidifying modes of attack. In this chapter we will analyze Penicillium capability to modulate the environment pH as a mechanism for colonization.

11. Evolutionary Adaptation as a Tool to Generate Targeted Mutant Strains as Evidence by Increased Inulinase Production in Aspergillus oryzae

Helena Culleton, Eline Majoor, Vincent A. McKie and Ronald P. de Vries

Inulin is found widely distributed in nature as a storage polysaccharide and consists of a linear polymer of β-1,2-linked D-fructose molecules which can be hydrolyzed by endo-/exo-inulinases and fructofuranosidase (invertase) to give D-fructose and fructooligosaccharides. In this study we aimed to improve the inulin degradation potential of Aspergillus oryzae through the upregulation of exo-inulinase production using an evolutionary adaptation method. This method has the advantage over UV or chemical mutagenesis in that it is likely to have a lower number of random mutations as only beneficial mutations will provide a competitive advantage and will therefore become dominant in the culture. As an organism with no predicted endo-inulinase function, improved inulin degradation in A. oryzae would be largely dependent on the overproduction of this enzyme. Subsequent generation growth of Aspergillus oryzae (Rib40) on inulin for 9 weeks successfully resulted in exo-inulinase overproducing mutants.