Molekulargenetische Analysen an dem Mykophenolsäure produzierenden Pilz brevicompactum

Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften an der Fakultät für Biologie und Biotechnologie der Ruhr-Universität Bochum

Internationale Graduiertenschule Biowissenschaften Ruhr-Universität Bochum Lehrstuhl für Allgemeine und Molekulare Botanik

vorgelegt von Yasaman Mahmoudjanlou

aus Teheran, Iran

Bochum Mai, 2020

Referent: Prof. Dr. Ulrich Kück Korreferent: Prof. Dr. Dominik Begerow Molecular genetics of the biotechnologically relevant Penicillium brevicompactum

Dissertation to obtain the degree Doctor Rerum Naturalium (Dr. rer. nat.) Submitted at the Faculty of Biology and Biotechnology Ruhr-University Bochum

International Graduate School of Biosciences Ruhr University Bochum Department of General and Molecular Botany

submitted by Yasaman Mahmoudjanlou

from Tehran, Iran

Bochum May, 2020

1st Supervisor: Prof. Dr. Ulrich Kück 2nd Supervisor: Prof. Dr. Dominik Begerow Danksagung Mein Dank gilt zu allererst meinem Doktorvater Herrn Prof. Dr. Ulrich Kück, für die außerordentlich interessante Themenstellung, die zahlreichen wissenschaftlichen Anregungen und Diskussionen sowie die verantwortungsvollen Aufgaben, welche ich in meiner Zeit als wissenschaftlicher Mitarbeiter am Lehrstuhl wahrnehmen durfte. Aus unserer gemeinsamen Arbeit konnte ich sehr viele Erkenntnisse schöpfen, welche mich auch über das wissenschaftliche Arbeiten hinaus geprägt haben. Zu jeder Zeit konnte ich mich auf Ihre Unterstützung und Ihr herausragendes Engagement verlassen.

Bei Herrn Prof. Dr. Dominik Begerow bedanke ich mich für die Übernahme des Korreferats und seine stets offene Tür.

Besonderer Dank gilt auch Herrn Prof. Dr. Christian Frisvad, dass er mich so herzlich in der Technical University of Denmark in Lyngby aufgenommen hat und es mir ermöglichte, mein methodisches Repertoire enorm zu erweitern.

Auch bedanke ich mich bei allen aktuellen und ehemaligen Mitarbeitern des Lehrstuhls für Allgemeine und Molekulare Botanik. Allen voran Ingeborg Godehardt und Susanne Schlewinski für die Unterstützung bei meinen Experimenten. Dr. Tim Dahlmann für seine guten Ratschläge, gehaltvolle Diskussionen und seine enorme Hilfsbereitschaft, nicht zuletzt bei der Korrektur der vorliegenden Arbeit. Weiterhin gilt mein Dank PD Dr. Ines Teichert für das äußerst gewissenhafte Korrekturlesen und ihre überaus wertvollen Anregungen. PD Dr. Minou Nowrousian für anregende Diskussionen und ihre Hilfsbereitschaft in allen Belangen.

Allen meinen Doktorschwestern und Doktorbrüdern möchte ich für die tolle gemeinsam verbrachte Zeit danken! Barbara Ramšak, für gute Ratschläge, welche mir halfen einige herausfordernde Probleme zu lösen und ein immer offenes Ohr – „These Penicillium!“. Dr. Sarah Schmidt, für ihr herzliches, freundliches Wesen und die gemeinsam verbrachte Zeit in Bibliothek und Cafeteria. Valentina Stein, für die regelmäßige Unterstützung bei der Durchführung meiner Experimente und ihr außergewöhnliches Engagement. Ramona Lütkenhaus, für die kontinuierliche Hilfsbereitschaft im Zusammenhang mit Experimenten und/oder diversen Laborgeräten. Nicht zuletzt gilt mein Dank Dr. Daria Radchenko, Ramona Märker, Xuemei Lin, Maria Shariatnasery und Hendrik Strotmeier für die stets inspirierende Zusammenarbeit.

Meiner Familie und insbesondere Fabian Jasper-Möller danke ich, für die großartige und fortwährende Unterstützung, welche die vorliegende Arbeit erst ermöglicht haben. Table of Contents I

Table of Contents

Abbreviations ...... II

I. Introduction ...... 1 1. Biology of biotechnologically relevant Penicillium species ...... 1 1.1 Asexual versus sexual reproduction systems in Penicillia ...... 1 1.1.1 Asexual reproduction ...... 1 1.1.2 Sexual reproduction ...... 3 1.1.3 Parasexuality ...... 5 1.2 ...... 6 1.2.1 Classification and phylogeny ...... 7 1.2.1.1 Classical classification ...... 7 1.2.1.2 Generic classification...... 7 1.2.1.3 Infrageneric classification ...... 8 1.2.2 Nomenclature ...... 9 1.2.3 Identification ...... 10 1.3 Penicillia as producers of industrially relevant secondary metabolites and elaborator of food products ...... 10 2. Genetic modification of Penicillium species as a tool for functional analysis and strain improvement .. 15 2.1 Conventional generation of recombinant strains ...... 15 2.2 Directed genetic modification ...... 17 3. Summary ...... 23

II. Scope of Thesis ...... 24

III. Mahmoudjanlou et al. 2019 ...... 28

IV. Mahmoudjanlou et al. 2020 ...... 29

V. Discussion ...... 30 1. Improvement of a transformation system for P. brevicompactum ...... 30 2. Evidence of a heterothallic sexual state and cryptic sexuality in P. brevicompactum ...... 36 3. Different attempts for induction of a sexual cycle in P. brevicompactum were unsuccessful ...... 39 4. Mating type genes are a suitable molecular marker for identification and phylogeny of Penicillium species ...... 42 5. MAT1-1-1 controls asexual development of P. brevicompactum ...... 45 6. P. brevicompactum MAT1-1-1 transcription factor is associated with germ tube formation and pellet morphology ...... 47

VI. Summary ...... 50

VII. Zusammenfassung ...... 51

VIII. References ...... 53

IX. Supplementary Data ...... 75

X. Eigenanteil an Publikationen ...... 78

XI. Curriculum Vitae ...... 79

XII. Erklärung ...... 81 Abbreviations II

Abbreviations Δ deletion AMH anti-Mullerian hormone BiFC bimolecular fluorescence complementation bp base pair CAI codon adaptation index cDNA complementary DNA ChIP-seq chromatin Immunpräzipitation DNA-Sequenzierung CRISPR clustered regularly interspaced palindromic repeats CYA Czapek yeast agar DNA deoxyribonucleic acid dsRNA double-stranded EMSA electrophoretic mobility shift essays EPS Exopolysaccharides flbA fluffy low brlA expression GC3 third position of codons GFP green fluorescence protein HMG high mobility group HSP70 Heat shock protein 70 InsP3 inositol-l,4,5-trisphosphate ITS Internal transcribed spacer JGI Joint Genome Institute MAT mating type mRNA messenger RNA miRNA microRNA MPA Mycophenolic acid MS Murashige-Skoog NHEJ Non-homologous end joining ORF open reading frame PAM protospacer adjacent motif Abbreviations III

PCR polymerase chain reaction pre-miRNA precursor miRNA pri-miRNA primary transcripts of miRNA psiBα precocious sexual inducer RIP repeat-induced point mutation RNA ribonucleic acid RdRPs RNA-dependent RNA polymerases RISC RNA-induced silencing complex RNA ribonucleic acid RNAi RNA interference RNA-seq RNA sequencing RT-PCR reverse transcription polymerase chain reaction sgRNA guide RNA siRNA small interfering RNA ssRNA Single strand RNA StrA striatin s. str. Sensu stricto syn. synonym TF transcription factor tracr-RNA trans-activating RNA UV ultraviolet V-8 vegetable juice medium Y1H yeast one-hybrid Y2H yeast two-hybrid I. Introduction 1

I. Introduction

1. Biology of biotechnologically relevant Penicillium species Over 200 years ago, Link (1809) introduced the genus Penicillium, meaning ‘brush’ according to the formation of asexual reproductive structures, with broom-like chains of conidia. This genus belongs to the division and contains a wide range of miscellaneous species and is amongst the most predominant fungi on the earth (Houbraken et al. 2014a). Penicillia are ubiquitous fungi that can reside in different habitats like soil, plants, food products, feed, air, and indoor environments. Many species of this genus are of great economic significance (Frisvad and Samson 2004; Visagie et al. 2014). Although much of their economic impact is deleterious like food spoilage, mycotoxin production, and biodeterioration, their applications in biotechnology, food, medicine, and pharmaceutical industry make them economically beneficial as well (Samson and Pitt 2000; Kück et al. 2014). Various Penicillium species with biotechnological, medical and, industrial importance are frequently isolated and used. Therefore, understanding their life cycle and establishment of a practical and stable taxonomy are of great value (Houbraken et al. 2014a). In this introduction, the developmental stages, life cycle, as well as taxonomic classification, and the beneficial usage of Penicillium species are explained in detail.

1.1 Asexual versus sexual reproduction systems in Penicillia Penicillium species can generate different developmental structures. They grow vegetatively as a branching filamentous structure, called hypha. Hyphae are segmented into cells by internal cross-walls called septa. Septa contain septal pores that mediate the protoplasm exchange between the cells. The septate hyphae grow in different directions on the substrate and form an interwoven network. This organization is designated as mycelium. Additional mycelial developmental events are involved in the sexual and asexual reproduction of Penicillium species (Raper and Thom 1949).

Penicillia are able to reproduce sexually or asexually. Most species, however, are supposed to propagate mainly asexually (Dyer and Kück 2017).

1.1.1 Asexual reproduction The asexual reproduction takes place by asexual sporulation. Sporulation is accomplished by the formation of mitosporic, unicellular, uninucleate spores, the conidia, on the conidiophores. They are produced by mitotic divisions of flask-shaped cells (phialides) and form conidial chains with a basipetal arrangement (younger conidia close to the phialide and older one away I. Introduction 2 from it) (Thom 1910; Houbraken et al. 2014a). The mature conidia remove from the phialides and can be scattered by the wind. They can germinate on an appropriate substratum by the formation of germ tubes. The nuclei, which have been created by mitosis of conidia migrate into the germ tubes. Parallel to elongation of germ tubes, septation takes place and the hyphae finally form a septate branched mycelium (Raper and Thom 1949).

For asexual sporulation, development of asexual reproductive structures called conidiophores is crucial. Conidiophores arise from hyphae of the vegetative mycelium as vertically growing aerial tubes with stem-like features (stipes). Stipe can be one cell or can be septate and branch into diverse cell types. A configuration of conidiophore and its different branching patterns are illustrated schematically in Figure 1. The terminal cells that generate conidia are called phialides. The branches bearing phialides are termed as metulae. The subjacent branches are named rami.

Figure 1. Conidiophore patterns in Penicillium species (A) Conidiophores with solitary phialides. (B) Monoverticillate. (C) Divaricate. (D, E) Biverticillate. (F) Terverticillate. (G) Quaterverticillate. Scale bar = 10 μ. From Visagie et al. (2014).

In different species, conidiophores can vary from being unbranched or showing multiple levels of branching causing symmetrical or asymmetrical configurations. Conidiophores can have primary (monoverticillate), secondary (biverticillate), tertiary (terverticillate), or quaternary (quaterverticillate) levels of branching. In species with simple conidiophores, phialides are formed solitary at the tip of stipes (Raper and Thom 1949) (Figure 1A). In some species, monoverticillate conidiophores are developed with a terminal whorl of phialides at the end of septate stipes (Figure 1B). Divaricate conidiophores show deviating branching patterns in different parts of the conidiophore. For example, one part could be simple, and another part could have metulae and rami (Figure 1C). Biverticillate conidiophores are formed by a whorl of three or more metulae, between the end of the stipe and the phialides (Figure 1D and E). In terverticillate conidiophores, another level of branching between the stipe and the metulae, the I. Introduction 3 rami, takes place (Figure 1F). Quaterverticillate conidiophores can be seen by only a few species and have one extra level of branching beyond the terverticillate pattern and develop stipe branching (Figure 1G). Terverticillate and quaterverticillate conidiophores tend to be asymmetrical (Visagie et al. 2014).

In addition to conidiophores, some species produce compact mycelial structures, the sclerotia. Sclerotia facilitate the species to persist the stress or environmental extreme conditions. They can form conidiophores or fruiting bodies under appropriate conditions, these structures then produce spores that are distributed into the environment and germinate at the inception of suitable growth conditions and grow into a new mycelium (Raper and Thom 1949).

1.1.2 Sexual reproduction The occurrence of a sexually reproductive (teleomorphic) state in Penicillium species was first described by Berfeld in 1874. He characterized the teleomorphic state by formation of sclerotioid fruiting bodies (cleistothecia) and asci (Pitt 1979). Since then, two sexual breeding systems have been described for this genus, (i) homothallism meaning self-breeding and (ii) heterothallism, which indicates mating between two opposite-sex partners (Dyer and Kück 2017). Remarkably, from 100 teleomorphic Pencillia, just a few heterothallic species have been identified. derixii (Penicillium derixii) was the first described heterothallic species (Takada and Udagawa 1988). About 25 years later, the existence of a heterothallic sexual cycle in and Penicillium roqueforti by the formation of cleistothecia and ascospores was proven (Böhm et al. 2013; Ropars et al. 2014). Despite the report of a sexual cycle in heterothallic species, the different stages within the sexual life cycle leading to the mature fruiting body were described only in some homothallic species by Emmons (1935). Sexual reproduction is mediated by either the development of sexual organs (gametangia) e.g. in Penicillium vermiculatum or, without the formation of gametangia and just by fusion of compatible hyphae (somatogamy) e.g. in Penicillium egyptiacum. In P. vertmiculatum, a female gametangium (ascogonium) develops from vegetative mycelium as a uninucleate and unicellular areal structure. It generates 16-32 nuclei by repetitive miotic divisions. Simultaneously, an adjacent hypha gives rise to a uninucleate male gametangium (antheridium) which coils around the ascogonium. The contact of the bent antheridium tip with the ascogonium leads to the fusion of two cells by the dissolution of their common cell walls. In P. vermiculatum, cell fusion stimulates the pairing of the female nuclei in the ascogonium. The ascogonium then splits up by septation into 8-16 binucleate cells, termed as dikaryons. In some species e.g. Penicillium stipitatum, the dikaryons are produced by pairing of antheridium and I. Introduction 4 ascogonium nuclei. Some of the dikaryons grow laterally and develop to ascus mother cells, with or without the formation of a crozier, a hook-like structure used to maintain the dikaryotic state in daughter hyphae. Meanwhile, the nuclei of ascus mother cells undergo karyogamy and a diploid cell is formed. Following first and second meiosis and subsequent mitosis, 8 haploid ascospores are formed. During these developmental steps, several sterile hyphae grow around the gametangia, attach to each other, and from ball-shaped, fruiting bodies (ascocarps). Later, ascospores discharge from the asci and germinate on an appropriate substrate and form the mycelium (Emmons 1935; Raper and Thom 1949). In heterothallic Penicillium species, cleistothecia are the first visible reproductive, structures. However, in P. roqueforti, formation of ascogonia has been reported as well (Ropars et al. 2014).

Both homothallic and heterothallic breeding systems are governed by mating type (MAT) genes located at MAT loci. In heterothallic species, mating strains carry opposite MAT loci, which are constituted of dissimilar DNA sequences but are located at the same locus on the chromosome. Thus, they represent idiomorphs. The strains with opposite MAT idiomorphs in Penicillia are termed as MAT1-1 and MAT1-2 strains. On the contrary, homothallic species possess both opposite MAT loci, occupying either the same locus or loci on different chromosomes. The MAT loci in heterothallic Penicillium species typically contain a single functional open reading frame. They encode for transcription factors (TFs), which have either an alpha- or a high mobility group (HMG)-DNA-binding domain and are designated as MAT1-1-1 or MAT1-2-1 (Dyer and Kück 2017). The assumed life cycle of heterothallic Penicillium species is depicted in Figure 2. The vegetative mycelium of MAT1-1 and MAT1-2 strains can undergo an asexual cycle by the formation of conidiophores and conidia release. Conidia can germinate and produce new vegetative mycelia. In the sexual cycle, compatible vegetative mycelia from strains of opposite mating type give rise to ascogonium and antheridium. Antheridium coils around ascogonium and both cells fuse at their contact zone leading to the production of dikaryons, which form ascus mother cells ensuing the crozier formation. Following karyogamy, meiosis, and mitosis, eight ascospores are produced in asci, while the mycelia surrounding the asci develop to cleistothecia. The ascospores can be released from cleistothecia, germinate, and go through an asexual cycle or a sexual cycle. I. Introduction 5

Figure 2. Life cycle of heterothallic Penicillium species The asexual cycle starts with the development of a conidiophore from vegetative mycelium. Condia are produced on the conidiophore and can be released and germinate on appropriate substrates and grow into vegetative mycelia again. Alternatively, cells from strains of opposite mating type fuse, following ascogonium and antheridium development and undergo a sexual cycle. As a consequence of cell fusion and hyphal segmentation, dikaryon cells are produced, one of which produce crozier. Formation of a hook cell sustains the dikaryotic state. Afterwards, karyogamy takes place and subsequently, diploid nucleus undergoes the meiosis I and II and mitosis, producing 8 ascospores in an ascus. Parallel to the development of asci, the surrounding mycelia give rise to fruiting bodies, namely cleistothecia. Ascospores can be released, germinate, and complete the life cycle by the formation of vegetative mycelia.

1.1.3 Parasexuality I. Introduction 6

Beside heterothallism and homothallism, a parasexual cycle has been observed in some Penicillium species e.g. P. chrysogenum, Penicillium expansum, and Penicillium italicum, under laboratory conditions (Pontecorvo and Sermonti 1954; Garber and Beraha 1965; Barron 2011). In nature, this life cycle has been seen in Penicillium cyclopium (Jinks 1952a). Parasexuality is a nonsexual mechanism caused by the fusion of vegetative hyphae that triggers the genetic exchange and allows genetic recombination, without meiosis or formation of sexual structures. In such a process, the anastomosis (hyphal fusion) of different fungal individuals is followed by plasmogamy and the formation of heterokaryons, whereby genetically diverse nuclei may coexist in a shared cytoplasm. The consequent karyogamy can lead to mitotic crossing over events leading to recombination. The diploid nucleus afterward produces haploid progeny by haploidization (Pontecorvo 1956; Anderson and Kohn 1998). It is believed that parasexuality rarely occurs in fungi and the frequency of nuclei fusion and recombination in parasexuality is significantly lower in comparison to sexual reproduction (Pontecorvo 1956; Anderson and Kohn 1998). However, this type of genetic recombination seems to be evolutionarily more steady and can develop quicker than the sexual cycle (Pontecorvo 1956; Eagle 2009). Moreover, even if plasmogamy does not lead to nuclei fusion in the parasexual cycle, heterokaryons could have an advantage over the parental haploid homokaryons, regarding the survival ability in a changing environment. In Penicillium species, heterokaryons have been shown to have a higher growth rate than homokaryons (Jinks 1952b; Eagle 2009). However, it needs to be highlighted that no ascospores are produced via a parasexual cycle, hence the vegetative heterokaryons are not resistant to different environmental stress conditions, as ascospores are. Despite the apparent profits of filamentous fungi when they form heterokaryons, there is a genetic mechanism that limits heterokaryon formation between two individuals that possess genetic diversity at het (heterokaryon incompatibility) loci. Heterokaryosis of those individuals leads to compartmentalization and cell lysis of heterokaryotic fusion cells. This phenomenon is called heterokaryon- or vegetative incompatibility (Glass et al. 2000). Thus, a prerequisite of a parasexual cycle is the availability of genetically compatible isolates. Mating type genes could mediate vegetative compatibility in some fungi; although different other genes and strategies mediating vegetative compatibility have been also reported (Leslie 1993).

1.2 Taxonomy Taxonomy is classically categorized into three domains. (i) Classification: orderly assortment of groups; (ii) nomenclature: designation of classified groups and (iii) identification of unidentified organisms, which is the process of determining whether an organism belongs to a I. Introduction 7 described group (Houbraken et al. 2014a). Taxonomy of Penicillia has been eternally challenging since the genus contains various species separated by very diminutive features (Samson and Pitt 2000).

1.2.1 Classification and phylogeny

1.2.1.1 Classical classification Until the end of the 19th century, the anamorph (asexual) and teleomorph (sexual) states of Penicillium were used for the taxonomy of this genus. Therefore, physiological and phenotypic characteristics such as conidiophore, conidia, and fruiting body morphology as well as colony size on agar media were of great interest. Up to the discovery of a teleomorph state, the classification of Penicillium was based on the conidiophore branching pattern (Visagie et al. 2014). In 1874, Berfeld initially demonstrated a link between anamorph and teleomorph stages of Penicillia (Houbraken et al. 2014a). He described the first Penicillium species with a sexual state as Penicillium crustaceum. This strain was able to produce sclerotioid cleistothecia. Ludwig (1892) introduced the name Eupenicillium for the teleomorphic Penicillium species. Later on, Pitt (1979) used the type of fruiting bodies for the classification of anamorphic states. He described Eupenicillium cleistothecia as yellow-brown to yellow-orange fruiting bodies, with a thick cell wall made of pseudoparenchymatic cells. These can be more than 100 µm in size and mature after 10-14 days. The asci in cleistothecia can be scattered (Pitt 1979; Houbraken et al. 2014a). Pitt (1979) explained that fruiting bodies of Talaromyces, namely gymnothecia, are composed of an interwoven network of hyphae, which encompasses asci. They are yellow-white and possess no defined cell wall. The morphology of asci and ascospores are similar to Eupenicillium (Pitt 1979; Pöggeler et al. 2011).

1.2.1.2 Generic classification Following Pitt's classification (1979), different novel methods have been established to elucidate the taxonomy of Penicillium. Such approaches include extracellular enzyme production pattern, secondary metabolite (extrolite) profiling, structure of ubiquinone systems, and DNA sequencing-based techniques. Recently, a polyphasic taxonomy, using a combination of different approaches has been applied for species classification. However, in the modern taxonomy, the DNA based methods play a major role (Frisvad 1981; Pitt and Cruickshank 1990; Smedsgaard and Frisvad 1996; Peterson 2000; Samson et al. 2004; Houbraken et al. 2014b).

Taxonomically, Penicillium belongs to the phylum Ascomycota, order , and family . Trichocomaceae include only teleomorph genera, however, the genera I. Introduction 8

Penicillium and Aspergillus are related to Trichocomaceae, due to their phialide structures (Houbraken and Samson 2011). Sequencing data of four genes showed that Trichocomaceae is divided into three subfamilies Aspergillaceae, Thermoascaceace, and Trichoconaceae. Penicillium species are placed in Aspergillaceae and Trichoconaceae clades. The Aspergillaceae are characterized by the formation of asci within cleistothecia or stromata, whose ascospores are normally oblate to ellipsoidal and have wrinkles or slits. Trichoconaceae are phenotypically distinguishable by the production of asci inside a cluster of aerial hyphae and ascospores without wrinkles or slits (Houbraken and Samson 2011).

Several phylogenetic studies on Penicillium are in agreement with the notion that this genus is polyphyletic, and it could be split into two distinct clades. One clade is designated as Penicillium sensu stricto and addresses the Eupenicillium species, as well as most species previously classified into the subgenera Aspergilloides, Furcatum, and Penicillium in the taxonomic organization of Pitt (1980). Talaromyces and Penicillium species formerly placed in the subgenus Biverticillium generate the second clade, namely Talaromyces sensu stricto (Berbee et al. 1995; Peterson 2000; Houbraken and Samson 2011). The division of Penicillium into two genera could also be signified in morphological, physiological and extrolite production-based classifications. Penicillium s. str. form flask-shaped or cylindrical phialides. In contrast, Talaromyces s. str. develop symmetrically branched conidiophores with lanceolate phialides. An additional phenotypic difference between these two clades is the type of ascomata they form (described above). Compared to Penicillium s. str. species, Talaromycess. str species have a slower growth rate on the agar medium G25N (Pitt 1979). While most Penicillium s. str species comprise Q9 ubiquinone systems, almost all Talaromyces s. str. have Q10(H2) (Peterson 1998). Ubiquinones are terpenoid soluble lipids located in the eukaryotic mitochondrial inner membrane and plasma membrane. They contribute to electron transport, oxidative phosphorylation and active transport. They are applicable for taxonomic classification of filamentous fungi, since structural alternatives could be detected between different taxa. Such variation is based on the length of isoprenoid chains and the degree of saturation (Peterson 1998). Some extrolites such as duclauxin, mitorubrins, rugulosin, skyrin, and gluconic acid are just specific for Talaromyces (Frisvad 1998). Penicillium s. str. are phylogenetically closer to Aspergillus species than Penicillium species reside in Talaromyces clade.

1.2.1.3 Infrageneric classification The first infrageneric classification of the genus Penicillium was proposed by Dierckx (1901). He divided this genus into three subgenera Aspergilloides, Birverticillium, and Eupenicillium. I. Introduction 9

Biourge introduced the sections, series, and subsections for this genus. Later on, Thom (1930) and Raper and Thom (1949) introduced 4 subgenera, 12 sections and 18 subsections for this genus (Raper and Thom 1949; Houbraken et al. 2014a). The system which was presented by Pitt (1979) and considered a combination of colony features and conidiophore phenotypes, achieved much recognition. He divided the genus Penicillium into four subgenera, Aspergilloides, Biverticillium, Furcatum, and Penicillium, and treated Eupenicillium distinctly (Pitt 1979). This classification lost its legitimacy with the advent of DNA sequencing (Berbee et al. 1995). Phylogenetic analysis based on four protein-coding genes revealed a distinct two subgenera, Aspergilliodes and Penicillium, and 25 sections for Penicillium s. str (Houbraken and Samson 2011). The subgenus Aspergilliodes is subdivided into 14 sections and includes the type species of Aspergilliodes (Penicillium glabrum) and Penicillium furcatum (Penicillium citrinum). The majority of the species residing in this subgenus have monoverticillate and biverticillate conidiophores with flask-shaped phialides. Eleven sections including the type species of subgenus Penicillium (P. expansum), belong to the subgenus Penicillium, most of which show a terverticillate or quaterverticillate conidiophore phenotype.

1.2.2 Nomenclature In 1910, the dual nomenclature was established in the International Code of Botanical Nomenclature (ICBN). The article 59 of ICBN directed the possibility to use two names for a single pleomorphic taxon; One name for the anamorphic state of a genus and one for the teleomorphic state (Houbraken et al. 2014a). However, most monographs published after that time for Penicillium were still based on asexual morphology and branching configurations of conidiophores (Thom 1910; Raper and Thom 1949). According to this concept, all species with an anamorphic state were grouped in the Penicillium genus, whether they also reproduced sexually or not. Despite the tendency for the use of anamorphic names of Penicillium species at that time, the nomenclature legislation pushed mycologists to apply the dual nomenclature system for Penicillium. Respecting this rule, Benjamin segregated Penicillium species, which produced a modest form of fruiting bodies, into the genus Talaromyces (Benjamin 1955). Talaromyces vermiculatus (P. vermiculatum), whose sexual cycle is described in detail in section 1.1, is the type species of this genus. Pitt (1979) also treated the Penicillium genus as the anamorphic state of the teleomorphs Eupenicillium and Talaromyces.

In 2011, the article 59 of the ICBN was revised and the principle of “One : one name“ was accepted for pleomorphic fungus. This article gives the anamorphic and teleomorph names the same precedence. Due to this article, if the anamorph genus is the oldest and the most I. Introduction 10 commonly used name, then it is applicable for both morphs (Hawksworth et al. 2011; Norvell 2011). In this regard, Penicillium would have the priority over its teleomorph names. So, both Eupenicillium and Talaromyces species are located in the genus Penicillium. This scenario is approved by different phylogenetic investigations, indicating that anamorphs are intermingled with teleomorph species (LoBuglio et al. 1993; Peterson 2000; Houbraken and Samson 2011; Houbraken et al. 2014a).

1.2.3 Identification Following the recent changes in nomenclature rules, some identified strains are believed to have been sorted into the wrong group. The introduction of new species while using invalid nomenclature also increases the possibility of strain misidentification. (Houbraken et al. 2014a). One example of misidentification is Fleming's original producing P. chrysogenum that was re-classified by extrolites production (Houbraken et al. 2011) and DNA sequencing data (Houbraken et al. 2012) as Penicillium rubens. Another example is P. egyptiacum strain CBS 244.32 that was named as Eupencillium crustaceum until 2013; this name was synonymous for Eupenicillium egyptiacum. After the application of single name nomenclature, this strain started bearing its current name P. egyptiacum (Houbraken et al. 2012). Initially, phenotypical and physiological traits were used for strain identification. Identification based on these traits is very challenging and may lead to misidentification of strains with very similar characteristics. Therefore, DNA-sequencing is nowadays used for strain identification. For this aim, different molecular barcodes are available for Penicillium species Protein-coding genes e.g. β-tubulin and calmodulin sequences are commonly used for interspecific identification (Samson et al. 2004; Visagie et al. 2014). Despite the description of various molecular markers for the identification of Penicillium, the introduction of novel markers for a simple, clear, accurate and immutable identification is of great interest. Identification of strains is critical for understanding the connections between various investigations from different points of view and misidentification of species or strains makes the comparison of studies difficult and can cause confusion and wrong interpretation of results (Houbraken et al. 2014a).

To conclude, the new insights in the taxonomy of Penicillium can serve as a tool for interpretation of the results regarding the evolution of, for example, enzymes, mating type loci, virulence genes, and secondary metabolite biosynthetic gene clusters in this genus (Houbraken et al. 2014a).

1.3 Penicillia as producers of industrially relevant secondary metabolites and elaborator of food products I. Introduction 11

Up to now, more than 350 species have been accepted for the genus Penicillium whose ubiquitous distribution reflects their diverse habitats and lifestyles (Visagie et al. 2014). They can grow on either living or dead substances and can be saprophytic, plant pathogenic, or pathogenic for humans with a compromised immune system (Rabha and Jha 2018). Some Penicillia are able to tolerate extreme environments. Examples include the various Penicillium species that populate deep-sea sediments (Gautschi et al. 2004; Du et al. 2009) and frigid zones (McRae et al. 1999; Sonjak et al. 2006), Penicillium funiculosum, which grows at extremely low pH (Xu et al. 2014b) and the true thermophilic species Penicillium duponti (Hashimoto et al. 1972). The ability of Penicillia to grow in a vast variety of substrates and conditions, as well as their interaction with other organisms, is associated with a range of various and adaptable metabolic strategies. These strategies encompass a secondary metabolism dedicated to the production of diverse low- weight biomolecules which are non-essential metabolites and their biosynthesis is limited to specific fungal taxa (Fox and Howlett 2008; Keller 2019). Fungi produce secondary metabolites to gain a range of ecological benefits, for example, they produce secondary metabolites for defense against predation or environmental stress, or as a means for interaction with other organisms, such as communication, competition, toxicity against bacteria or other fungi, and pathogenicity (Derntl et al. 2017). Penicillium species are considered as producers of various secondary metabolites of different chemical classes, including non- ribosomal peptides (e.g. penicillin), polyketides (e.g., griseofulvin, statins), terpenes (e.g. roquefortin C), or hybrids of two classes (e.g. mycophenolic acid) (Frisvad et al. 2004; Grijseels et al. 2017; Nielsen et al. 2017; Rabha and Jha 2018). These metabolites are synthesized from initial building blocks of primary metabolites. While polyketides and terpenes are derived from acyl-CoAs, non-ribosomal peptides are synthesized from amino acids (Keller 2019). Several of these secondary metabolites possess bioactive properties with industrial relevance and the capability of Penicillia for their biosynthesis makes various species potential microbial cell factories for industrial production. Remarkably, they are not only producers of endogenous products, but also could be used for the manufacturing of heterologous products. Examples of endogenous secondary metabolites with medical applications include different antibiotics, antifungal agents, immunosuppressant compounds, and statins. Exopolysaccharides (EPS), organic acids, and different enzymes produced by Penicillium species are also commercially applied or could be potentially used in different industrial areas (Rabha and Jha 2018), as outlined below.

The antibiotic penicillin has been probably the most outstanding and beneficial secondary metabolite produced by Penicillium species. This antibiotic which is a non-ribosomal peptide I. Introduction 12 is industrially biosynthesized by P. rubens (often introduced as P. chrysogenum) as the most applicable antibiotic for the treatment of microbial infections. It belongs to one of the commercially most advantageous medications for the pharmaceutical industry, with around 8 billion US$ annual worldwide sales (Böhm et al. 2013). The antifungal griseofulvin is another important pharmaceutical compound produced by fermentation of Penicillium griseofulvum (Oxford et al. 1939; Frisvad et al. 2004). The immunosuppressant mycophenolic acid (MPA) is another representative of Penicillium secondary metabolites in the pharmaceutical industry. Penicillium brevicompactum is the main producer of this polyketide on the industrial scale. The derivatives of MPA with the trade names CellCept® (mycophenolate mofetil; Roche) and Myfortic® (mycophenolate sodium; Novartis) are used for prevention of organ rejection in transplantation patients (Bird and Campbell 1982; Siebert et al. 2017). Statins are also fungal secondary metabolites, which can be applied as cholesterol-lowering drugs for preventing cardiovascular diseases. One statin currently in clinical use is mevastatin (compactin), which is fully biosynthesized by P. citrinum (Manzoni and Rollini 2002). These examples demonstrate the immense impact of Penicillium species in the pharmaceutical industry.

In the last two decades, the production of fungal EPS has been one main focus of industrial research, as an alternative to algal and plant polysaccharides. They can be applied in food, pharmaceutical, agriculture and cosmetic industries. The advantage of fungal EPS is not only their facilitated and higher production rate in shorter time, but also some novel applicability. (Mahapatra and Banerjee 2013). Different saprophytic Penicillium species including Penicillium bilaia, P. giseofulvum, Penicillium radicum, and Penicillium simplicissimus are producers of EPS. EPS can mediate the formation of fungal biofilms and facilitate the colonization of soil particles, seeds, and roots systems. Therefore, these species are used as a fungal component of biofertilizers. (Wakelin et al. 2004; Osinska-Jaroszuk et al. 2015). Besides polysaccharides, the potential of a fatty acid produced by a halotolerant P. chrysogenum strain in the pharmaceutical industry has been reported. This unsaturated C-19 fatty acid named Chrysogesid has shown antimicrobial activity against Enterobacter aerogenes (Peng et al. 2011). Various hydrolyzing enzymes involved in the primary metabolism of Penicillium species are also of great industrial importance. Examples include amylases and cellulases which are applicable in food, pharmaceutical, textile, paper, and pulp industries as well as in detergents (Pandey et al. 2000; Balkan and Ertan 2005; Rabha and Jha 2018). Cellulase is commonly produced in industrial scale by the filamentous fungus Trichoderma reesei. However, different studies have pointed to a higher efficiency of cellulases from several Penicillium species like P. funciculosum and Penicillium oxalicum. The cellulase from P. oxalicum also has a I. Introduction 13 significantly higher yield of glucose and greater monosaccharide tolerance during fermentation (Jørgensen and Olsson 2006; Maeda et al. 2013). In China, Penicillium decumbens has been served as a producer of cellulolytic enzymes in industrial scale since 1996 (Fang et al. 2010). Pectinases, which facilitate the degradation of lignocellulosic material, are other enzymes that could be produced by different Penicillium species e.g. P. brevicompactum, Penicillium cupsulatum, P. echinulatum, Penicillium griseoroseum, and P. italicum (Pereira et al. 2002; Schneider et al. 2016; Rabha and Jha 2018). The production of lipolytic enzymes such as lipases and esterases by various Penicillium species has been also described. These enzymes are applicable in food, cosmetics, detergents, pharmaceutical, textile, and fine-chemical industries. Glucose oxidase is another enzyme, whose industrial production relies on fungi. Penicillium adametzii is considered to be a widely utilized fungus for this purpose. Glucose oxidase is used in baking industry for making a stronger dough, and in the food industry for flavor, aroma and increasing food stability (Raveendran et al. 2018). Naringinase is a debittering enzyme used in food processing, and can also be synthesized by Penicillium species (Raveendran et al. 2018).

Along with the usage of numerous compounds from Penicillia, the mycelia of some species have been directly used to process human food products. These fungi serve as ripening or preservation agents or can provide distinct colors or flavors to food products. Penicillium nalgiovense is used for fermentation of salami. The inoculated asexual spores grow to a thin white layer of mycelia on the salami surface. This layer controls the moisture loss and produces enzymes that contribute to the flavor or protect the sausage against other potentially mycotoxigenic fungal species (Chávez et al. 2011). P. roqueforti and Penicillium camemberti are used for the maturation of blue and Camembert cheese, respectively. P. roqueforti can easily colonize the inner part of the cheese and its proteolytic and lipolytic enzymes are responsible for the organoleptic characteristic of the blue cheese. P. camemberti produces proteases during the ripening of Camembert or Brei cheese causing the casein hydrolysis that gives the product its flavor and texture. The produced lipases hydrolyze the mono- and diglycerols to fatty acids that are responsible for the organoleptic properties (Chávez et al. 2011). A list of the most important substances produced by Penicillium species is shown in Table 1.

I. Introduction 14

Table 1. Selective examples of the substances produced by Penicillium species in industrial scales or potent for industrial application

Substance Organism application Amylase P. expansum 1 Food, textile, paper, pharma industry Cellulose P. decumbens 2 Food, textile, paper, pharma industry Esterase P. funiculosum 3 Food, cosmetic, detergents, textile, paper, pharma industry Galactosidases P. chrysogenum 4 Food and feed industry P. simplicissimum 5 Glucose oxidase P. adametzii 6 Food and baking industry Enzymes Inulinase P. funiculosum 7 Confectionary and in fructose sirup Lipases P. solitum 8 Food, cosmetic detergents, textile, paper, pharma industry Naringinase P. ulaiense 9 Food industry Pectinases P. brevicompactum 10 Textile industry P. griseoroseum proteases P. nalgiovense 11 Food, detergents, pharma industry Xylanase P. purpurogenum 12 Food and baking industry Brevianamides P. brevicompactum 13 Anti-insecticidal Griseofulvin P. griseofulvum 14 Antifungal drug Hadacidin P. camemberti 15 Herbicidal activity Mevastatin P. citrinum 16 Cholesterol-lowering drug Secondary Mycophenolic acid P. brevicompactum 17 Immunosuppressive drug metabolites Paclitaxel (Taxol™) P. aurantiogriseum 18 Anticancer drug Penicillin P. rubens 19 Antibiotic Quinolactacins P. bialowiezense 15 Anti-larvae activity Zosteropenillines A–L P. thomii 20 Anticancer activity Biotransformation- P. glabrum 21 Pharmaceutical industry derived steroids P. notatum 21 Exopolysaccharides P. bilaiae 22 Used in biofertilizer Others Fatty acid chrysogesid P. chrysogenum 23 Antibacterial activity Gold nanoparticles P. aurantiogriseum 24 Anti-HIV, anti-angiogenesis, anti- P. citrinum 24 malarial, anti-arthritic, and anticancer P. waksmanii 24 activity 1 Doyle et al. (1989); 2 Fang et al. (2010); 3 Kroon et al. (2000); 4 Aleksieva et al. (2010) 5 Cruz et al. (1999); 6 Eryomin et al. (2006); 7 Danial et al. (2015); 8 Chinaglia et al. (2014); 9 Rajal et al. (2009); 10 Pereira et al. (2002); 11 Papagianni and Sergelidis (2014); 12 Echeverría and Eyzaguirre (2019); 13 Paterson et al. (1990); 14 Oxford et al. (1939); 15 Frisvad et al. (2004); 16 Manzoni and Rollini (2002); 17 Regueira et al. (2011); 18 Yang et al. (2014); 19 Houbraken et al. (2011); 20 Afiyatullov et al. (2017); 21 Nassiri-Koopaei and Faramarzi (2015); 22 Wakelin et al. (2004); 23 Peng et al. (2011); 24 Honary et al. (2013)

All of these examples demonstrate the wide range of Penicillium species applications for the industrial production of beneficial compounds. Moreover, the growing number of genome sequences available for different Penicillium species has pointed to the tremendous yet unexploited potential of this genus for the production of other secondary metabolites (Nielsen et al. 2017). Therefore, the development of different genetic manipulation strategies for strain improvement is of great interest. I. Introduction 15

2. Genetic modification of Penicillium species as a tool for functional analysis and strain improvement Two different strategies can be trailed for discovery of a valuable compound and its industrial biosynthesis; (i) identification or manipulation of the best native producer and (ii) heterologous expression of metabolite genes or gene clusters (Grijseels et al. 2017). Most commonly, the yield by wild type native producers is too low to accomplish an economically beneficial production. Therefore, strain improvement programs to enhance their production and/ or lowering fermentation costs are crucial. Such strategies include the usage of inexpensive raw materials, increasing the production rate, or optimization of purification methods for the desired product. For this purpose, genetic modification can be applied as a useful tool. The heterologous expression strategy is suitable if the native producer is not appropriate for industrial fermentation or its genetic modification is obscure. Nevertheless, heterologous production in a commercial titer is usually challenging and needs further considerable fine-tuning processes (Grijseels et al. 2017). In this concern, the establishment of genetic manipulation systems for a native producer is of great significance. Furthermore, the characterization of the promising gene functions is typically feasible in native producers.

Genetic manipulation of Penicillium species can be divided into two approaches, conventional and directed genetic modifications.

2.1 Conventional generation of recombinant strains For a conventional generation of strains with beneficial attributes, random mutagenesis- selection, protoplast fusion, and genetic recombination by sexual reproduction can be used. Random mutagenesis can be mediated by different mutagens e.g. ultraviolet and X-ray radiation, nitrogen mustard gas, nitroso-methyl guanidine, and ethidium bromide. These mutagens induce single or double-strand DNA breaks. Subsequent DNA repair can cause different changes within the genome, from changing individual bases or small insertions or deletion to translocation or deletion of entire chromosomal areas. By screening of mutants, the strains with improved properties are selected for further rounds of undirected mutagenesis. The advantage of this method is that it is useful despite the lack of information about the organism, its metabolic pathways, and its genome. However, different undesirable and unknown changes like reduced growth rate or genetic instability could also occur besides the increase of favorable production. Moreover, since these modifications are typically irreversible, the later changes of the production protocols e.g. for low-cost fermentation can be restricted (Saunders and Saunders 1987; Parekh et al. 2000). Since the discovery of penicillin by , different mutagenesis- selection strategies have been applied for P. chrysogenum (now I. Introduction 16 identified as P. rubens) to select the strains with enhanced penicillin production levels. (Guzman-Chavez et al. 2018). This strain improvement program has been useful for other Penicillium species as well; like Penicillium griseoroseum, Penicillium occitanis, and P. oxalicum (Agrawal et al. 1999; Trigui-Lahiani et al. 2008; Yao et al. 2015; Lima et al. 2017).

About a decade after the discovery of penicillin by Fleming (1929), P. chrysogenum strain NRRL 1249B2 (Flemings strain) was used for the isolation of penicillin as a medication for the treatment of bacterial infections during world war II (Keller et al. 2005). However, the penicillin level of that strain was so low that searching for a higher producer was started. In 1943, the U.S. Department of Agriculture Northern Regional Research Laboratory (NRRL) in Peoria, Illinois isolated the strain NRRL1951 from melon that showed high penicillin productivity. Afterward, several conventional mutagenesis programs led to the generation of strains with increased penicillium productivity. The strain Q176 was created by UV and X-ray mutagenesis. At the University of Wisconsin, the pigment less strain BL3-D1 was generated by UV mutagenesis of Q176. This phenotype was valuable since extraction of yellow pigment produced naturally by P. chrysogenum is associated with penicillin losses. Subsequently, several rounds of nitrogen mustard mutagenesis followed by one step of UV irradiation was used to obtain the Wisconsin 54–1255 strain that became the laboratory reference strain (Barreiro et al. 2012). P2 was another independently Q176- derived lineage generated by Nippon Kayaku Co., Ltd and served as recipient strain to generate the nitrate reductase-deficient mutant P2niaD18 (Specht et al. 2014; Terfehr et al. 2017).

Another non-specific recombination technique for strain improvement is protoplast fusion. This method involves enzymatic cell wall digestion of two strains, followed by chemofusion and electrofusion of their cell membranes which leads to the formation of heterokaryotic hybrids and nuclear fusion followed by diploidization and haploidization. Fusion can be induced between either wild type or mutated strains and can generate hybrid strains with positive characteristics of both parental strains (Hamlyn et al. 1981; Muralidhar and Panda 2000; Adrio and Demain 2006). Protoplast fusion has been used for strain improvement of P. chrysogenum (Adrio and Demain 2006). Beside the intra-specific application, this technique is also applicable for generation of inter-specific hybrids. For example, protoplasts fusion between P. chrysogenum and P. roqueforti was shown (Anné et al. 1976). Fusion of P. chrysogenum with other Penillium species, e.g. P. cyclopium and Penicillium patulum (syn. P. griseofulvum) resulted in some hybrid strains with changed penicillin titer (Anné 1982). Additionally, increased pectinase production was demonstrated in hybrids of P. expansum and P. griseoroseum (Varavallo et al. 2007). The disadvantages of the protoplast fusion are occurrence of adverse random genetic alteration, genetic instability, and aneuploidy. The discovery of a sexual cycle in supposedly asexual Penicillium species like P. chrysogenum and I. Introduction 17

P. roqueforti expands the possibilities for genetics-mediated strain improvement of those species (Böhm et al. 2013; Ropars et al. 2014). One benefit of sexual reproduction is the occurrence of recombination in whole genomes of mating partners that produce progeny with different characteristics. Thus, progeny with unexpected advantageous traits could be generated (Dyer and Kück 2017). Since sexual propagation is a natural process, no selection markers or genetic transformation system is required for genetic alteration. Hence, it is a suitable tool for strain improvement of species for which no genetic manipulative technique is available. Moreover, since the progeny generated by mating or natural recombination is not considered as genetically modified organisms, further investigations and production lines do not require authorization. However, this method is less efficient and slower than other genetic alteration techniques (Dyer and Kück 2017). Difficulty of using sexual reproduction for strain improvement could be that most industrial strains have undergone accumulated mutagenesis, which can affect the genes involved in sexual reproduction. This can lead to infertility of those strains (Dyer and O'Gorman 2011). Nonetheless, sexual recombination between wild type and industrial strains have been reported for P. chrysogenum (Böhm et al. 2015). It is important to note that karyotype incompatibility of industrial strains could also decrease the fertility (Dahlmann et al. 2015).

2.2 Directed genetic modification To overcome the deleterious effects of undirected genetic modification, targeted genetic alteration can be applied for strain improvement. The specific genetic alterations comprise deletion, insertion or substitution of DNA sequences. To guarantee a successful genetic transformation of fungi, different factors need to be considered. A crucial prerequisite is the knowledge of the genome sequence (Kluge et al. 2018). Another requirement is an efficient transformation system. Different fungal species and strains may require a specific transformation protocol due to the putative distinct cell wall structures (Li et al. 2017a). Development and establishment of constitutive or inducible promoter systems for a species is a key to control the gene expression in recombinant strains (Kluge et al. 2018). Additionally, successful genome manipulation relies on appropriate dominant selection markers (Ruiz-Díez 2002). Considering all of these factors, various DNA- and RNA-based strategies have been developed for directed transformation of filamentous fungi (Ruiz-Díez 2002; Meyer 2008; Kück and Hoff 2010; Li et al. 2017b).

Two different DNA repair mechanisms mediate the integration of a foreign DNA during the transformation of the filamentous fungi; (i) homologous recombination which corresponds to I. Introduction 18 the repair of double-strand DNA breaks directed by homologous DNA sequences and (ii) non- homologous end joining (NHEJ) that ligates the DNA ends, independent of sequence homology (Kück and Hoff 2010). Homologous recombination can be used for site-specific DNA integration. However, in filamentous fungi the NHEJ mechanism is more prominent, directing the ectopic integration of foreign DNA in the genome (Ninomiya et al. 2004). To overcome this obstacle, different approaches have been established for industrially relevant Penicillium species

Split-marker technology is one of the techniques in which the selectable marker gene is divided into two fragments with overlapping sequences and used for deletion or substitution of a DNA sequence. Expression of the functional selectable marker gene is only possible after three crossing overs, two for integration of flanking regions of the genetic locus and one within the selection marker. In this way, the ectopic integration of fragments does not generate the selectable transformants (Kück and Hoff 2010). Along with the split marker technique, impairing the NHEJ system via deletion of the genes responsible for this process, explicitly ku70, ku80, and lig4, tremendously increases the probability of homologous integration. The corresponding mutants have been very effective for site-specific genetic recombination in different Penicillium species (Hoff et al. 2010b; Li et al. 2010; Bugeja et al. 2012; Xu et al. 2014a).

The integrative transformation of protoplasts by polyethylene glycol and calcium chloride (Fincham 1989) or electroporation (Chakraborty et al. 1991), Agrobacterium-mediated transformation (de Groot et al. 1998; Michielse et al. 2005), and gene-particle bombardment (Klein et al. 1987) are predominantly used methods for filamentous fungi. A typical procedure of protoplast- and Agrobacterium-mediated transformation is illustrated in Figure 3. For protoplast-based transformation, the grown mycelia are filtered and treated with digestive enzymes to prepare protoplasts. Protoplast preparation is followed by the uptake of exogenous DNA using polyethylene glycol and calcium chloride. An Agrobacterium-based method is accomplished by co-cultivation of bacterial and fungal cells and selection of transformants. The transformation efficiency and copy number of integrated DNA in these techniques are different. While protoplast transformation results in high copy numbers of integrated DNA, Agrobacterium-mediated methods lead to a single genome insertion. Thus, protoplast transformation is less controlled and transformation efficiency by Agrobacterium is three to six fold higher (Michielse et al. 2005). I. Introduction 19

Figure 3. Schematic workflow diagram of the transformation procedure of filamentous fungi (A) Transformation of protoplasts (B) Agrobacterium-mediated transformation. Modified from Li et al. (2017a).

RNA-based methods are applied for knock-down of a target gene and are usually used, when the target gene is essential or when multiple copies of the gene exist in the genome (Kück and Hoff 2010). RNA-based strategies do not depend on targeted genome integration. Hence, for its application, no NHEJ-impaired strains are required. RNA interference (RNAi) is the most widely used RNA-based strategy in filamentous fungi (Torres-Martinez and Ruiz-Vazquez 2017). In this method, small non-coding RNAs are used and its mode of action is mediated by three core components, Dicer proteins, RNA-induced silencing complex (RISC) and Argonaut proteins. RNAi can be induced in filamentous fungi by two different small RNAs, miRNA and siRNA. For the generation of miRNA, firstly the single strand non-coding RNA (ssRNA) is constructed, which forms a hairpin structure and serves as primary transcripts of miRNAs (pri- miRNAs). Following the processing of pri-miRNAs by ribonucleases, subsequent precursor miRNA (pre-miRNA) is transported into the cytoplasm where the Dicer proteins cleave the hairpin of pre-miRNA. Afterward, the mature miRNAs are bound to the RISC via Argonaut I. Introduction 20 proteins. RISC dissolves the RNA double strands and triggers the binding of the remaining ssRNA to the target mRNA, causing either the degradation of mRNA or its translation repression.

Figure 4. Mechanism of RNAi for gene silencing. In miRNA-mediated silencing, first, the transcribed pri-miRNA with a hairpin structure is processed by ribonucleases to generate pre-miRNA. Afterward, Exportin-5 mediates pre-miRNA transport into the cytoplasm, where pre-miRNA is cleaved by Dicer at the hairpin stem and binds to Argonaut protein-associated RISC leading to the generation of ssRNA. The binding of ssRNA to the target mRNA, leads to the degradation of the target mRNA or the repression of its translation. In siRNA-mediated silencing, dsRNA is synthesized from ssRNA by RNA- dependent RNA polymerases (RdRP) and transported to the cytoplasm. In the cytoplasm, Dicer cleaves the dsRNA into several shorter RNA fragments. Consequence association of Argonaut proteins and RISC to the dsRNA induces the generation of ssRNA and ensuing mRNA degradation. Modified from Xu et al. (2018). I. Introduction 21

Note that miRNAs can target mRNAs that are not fully complementary to it. In siRNA- mediated silencing, an exogenous double-stranded RNA (dsRNA) is used. Such a process requires RNA-dependent RNA polymerases (RdRPs) to synthesize dsRNA from ssRNA. In the cytoplasm, the synthesized dsRNA is cleaved into a large number of different short RNA fragments by Dicer. Following the association of Argonaut proteins and RISC, dsRNA is cleaved to a ssRNA which is complementary to the target mRNA. This leads to the cleavage of the mRNA and translation suppression. siRNAs commonly fully match their mRNA targets (Carthew and Sontheimer 2009; Dang et al. 2011; Xu et al. 2018). A model for the molecular pathway of RNAi is shown in Figure 4. RNAi -mediated gene silencing has been demonstrated in the penicillin producer P. chrysogenum (Janus et al. 2009), in the cheese ripening P. roqueforti (Del-Cid et al. 2016) and in the human pathogen Penicillium marneffei (Sun et al. 2014).

One of the newest genetic manipulative techniques is the clustered regularly interspaced palindromic repeats (CRISPR) system that was initially discovered in prokaryotic microorganism as an immune system against viruses and phages. This method requires different components, a CRISPR associated endonuclease (e.g. Cas proteins or Cpf1 nucleases), guide RNA (sgRNA), which is responsible for the recognition of a target sequence, and one trans- activating RNA (tracr-RNA), in the case of the CRISPR/Cas system. For the application of this method, the sgRNA is designed to consist of a constant part of sgRNA and a sequence complementary to the target sequence. sgRNA builds a complex with tracr-RNA and nuclease which in turn binds to the sequence of interest and induces a double or single-strand DNA break (depending on the nuclease) 3-5 base pair upstream of a protospacer adjacent motif (PAM) sequence. Subsequently, DNA repair mechanisms, NHEJ, and homologous recombination can be used for targeted genome manipulation (Figure 5).

The PAM sequence is a 2-6 bp DNA sequence beside the target sequence, in which the complex of Cas9, tracr and sgRNA is bound. PAM is essential for initial DNA binding and, without PAM, nucleases like Cas9 are not able to recognize the target sequences, even when these sequences are fully complementary to the sgRNA (Doudna and Charpentier 2014).

So far, P. chrysogenum (Pohl et al. 2016) and Penicillium subrubescens (Salazar-Cerezo et al. 2020) are the only Penicillium species, for which the CRISPR/Cas9 system has been successfully applied. I. Introduction 22

Figure 5. Schematic illustration of the application of CRISPR/cas9 technique for genome modifications of filamentous fungi. A combination of transactivating RNA (tracr-RNA) and guide RNA is expressed together with Cas9. The recognition of the target sequence according to the complementary sequence within the guide RNA recruits tracr-RNA and Cas9 to the DNA sequence. This complex induces a double-strand break, 3-5 base pair upstream of a protospacer adjacent motif (PAM) sequence. The subsequent double-strand break can be consequently repaired either by non- homologous end joining (NHEJ) or by homologous recombination Ghosh et al. (2019).

Selection marker recycling methods and autonomously replicating vectors can be used as alternative techniques for the genetic engineering of species, for which few selection markers are accessible. Cre/loxp and FLP/FRT are two site-specific recombination systems used for marker recycling in filamentous fungi (Wang et al. 2017). In the Cre/loxp method, recombinase Cre derived from bacteriophage P1 recognizes the loxp sites and induces recombination (Kühn and Torres 2002). The recombination by FLP/FRT is based on the FLP recombinase from S. cerevisiae and its FRT recognition site. To recycle a marker gene, a two-step strategy is used for both methods (Chen and Rice 2003). First, the selection marker cassette used in transformation is constructed in a way that is flanked by two loxp or FRT13 bp palindromic sequences with 8bp spacer regions and integrated into the genome. Afterward, a recombinase is expressed, which binds to its recognition sites and breaks the DNA within the spacer regions. This generates 8-bp overhanging ends of the two loxp or FRT fragments. Consequently, the complementary overhanging ends come together leading to a recombinant loxp or FRT sequence, thereby ensuing marker deletion (Kühn and Torres 2002; Wang et al. 2017). The examples of the usage of these methods include Cre/loxp for Aspergillus species (Krappmann et al. 2005; Forment et al. 2006; Mizutani et al. 2012) and FLP/FRT for P. chrysogenum (Kopke et al. 2010). I. Introduction 23

For a functional gene analysis, using autonomously replicating vectors could be a good choice. This can result in a high transformation efficiency and mitotic unstable fungal transformants. Therefore, facilitating the recovery of the recombinant DNA from transgenic fungal strains. Although such vectors have been developed for some filamentous fungi e.g. Aspergillus nidulans (Gems et al. 1991), P. chrysogenum (Fierro et al. 1996) and P. nalgiovense, (Fierro et al. 2004), the usage of these vectors has rarely been reported. For this reason, various DNA integrative techniques are more frequently used (Kück and Hoff 2010).

3. Summary The ubiquitous genus Penicillium comprises more than 350 species, of which many are of great industrial significance, as they are the producers of various chemicals, secondary metabolites, pharmaceuticals, and enzymes (Rabha and Jha 2018). Concerning their importance, Penicillia have always been in the limelight of taxonomists and different strategies have been applied for their taxonomic classification, since the beginning of 18th century. Although the primary classification was based on morphological concepts, the application of those methods has been restricted with the advent of DNA sequencing techniques. Nowadays, nearly all classifications and identification of Penicillium species can be addressed by DNA sequencing-based methods (Visagie et al. 2014).

Due to the industrial application of Penicillia, different genetic manipulation strategies have been exploited for strain improvement. Besides the conventional strategies, site-specific genetic engineering strategies are currently used in modern biotechnological industries (Kück and Hoff 2010) and the development of new methods is of great interest. Moreover, induction of a sexual cycle provides a suitable tool for strain optimization. The most industrially relevant species however, are believed to lack a sexual cycle. Interestingly, most of them have functional sex- associated genes such as mating type genes indicating the possible occurrence of sexuality in nature (Dyer and Kück 2017). Thus, the identification of those genes can be used for the discovery of cryptic sexuality in supposedly asexual species. II. Scope of Thesis 24

II. Scope of Thesis P. brevicompactum is one of the Penicillium species with great meaning for the pharmaceutical industry. In 1893 the Italian physician Bartolomeo Gosio discovered that this fungus is the producer of a metabolite with antibacterial properties against Bacillus anthracis. This active compound was classified as a mycophenolic acid (MPA). MPA was the first antibiotic that was isolated and crystallized (Bentley 2000). Later on, the antifungal, antiviral and antitumor properties of MPA were also reported (Regueira et al. 2011). In 1969, scientists also discovered the immunosuppressant activities of MPA (Bentley 2000). In a chain of events, derivatives of MPA, the ester mycophenolate mofetil (MMF, trade name CellCept®) and the salt mycophenolate sodium (EC-MPS, trade name Myfortic®) have been extensively used as immunosuppressive drugs after kidney, heart, and liver transplantation (Bentley 2000; Regueira et al. 2011; van Gelder and Hesselink 2015). Currently, MPA is produced biotechnologically in a large industrial scale by fermentation of P. brevicompactum. In 2014 the sales of 543 million US$ were reported for Myfortic®, and the worth of 938 million US$ of CellCept® was sold in 2013 (Patel et al. 2016). Different studies have demonstrated the ability of P. brevicompactum for production of other secondary metabolites with bioactive properties including asperphenamate, brevianamide A & B (Bird et al. 1981; Bird and Campbell 1982; Frisvad and Samson 2004), and adenophostin A and B (Takahashi et al. 1993), although in smaller amounts. Asperphenamate possesses antitumor activity and its derivatives can be used as autophagy triggers (Yuan et al. 2012). Brevianamide A & B are indole alkaloids with antifeedant properties against the larvae of the insect pests Spodoptera frugiperda and Heliothis virescens (Paterson et al. 1990). Adenophostin A and B can modulate calcium-regulated intracellular signals, since they are agonists of inositol-l,4,5-trisphosphate (InsP3) receptors. It has been proven that several cellular processes like neural functions, muscle contraction, secretion reactions, and cellular growth and differentiation depend on calcium signaling, and drugs or compounds selectively regulating signaling systems are of great therapeutic potential (Takahashi et al. 1993). Accordingly, one can assume diverse therapeutic applications for adenophostin A and B, which showed binding affinity to the InsP3 receptor higher than InsP3 itself (Takahashi et al. 1993). Besides already characterized secondary metabolites, genome sequencing of a P. brevicompactum strain revealed around 73 putative biosynthetic gene clusters (Li et al. 2018). One study pointed to the application of P. brevicompactum for biosynthesis of gold nanoparticles and the effect of those particles on cancer cells (Mishra et al. 2011). Due to the high relevance of P. brevicompactum in industry, there is a permanent commercial need to optimize the production strains and the biosynthesis processes. Concerning II. Scope of Thesis 25 these demands, directed genetic manipulation and induction of a sexual cycle are of greater value for genetic engineering of industrial production strains compared to the conventional strain improvement approaches.

Despite the importance of P. brevicompactum in the biotechnological and pharmaceutical industry and its potential for the production of yet uncharacterized compounds, and besides decade-long research efforts on this fungus, only limited studies have described the genetic engineering of this fungus and few selection markers are available. Consequently, most regulators of secondary metabolism and morphogenesis in P. brevicompactum have not been identified or functionally characterized. Thus, proposing simple genetically manipulative techniques for this fungus is of beneficial relevance. Moreover, since only asexual reproduction has been described for P. brevicompactum, the discovery of a sexual cycle under laboratory conditions could be of great advantage for the generation of strains with novel traits.

Breeding by sexual means can be very beneficial for filamentous fungi. The crossover and recombination events during meiosis can promote genetic diversity in the progeny and thereby fitness of future generations to the environmental changes (Barton 2009). Recombination within the homologous chromosomes during meiosis can also restore some genetic or epigenetic adverse damages or can recover lethal mutations (Normark et al. 2003). Additionally, some favorable mutations can be accumulated in the population. Genome evolution can also be affected by sexual recombination. One of the greatest advantages of sexual propagation for fungi is the production of fruiting bodies and sexual spores that are more resistant to the stress conditions compared to the asexual spores. This promotes the survival of the offspring in the harsh environment (Dyer and Kück 2017). Regarding the application of a sexual reproduction system in laboratory conditions, there are also various advantages. As mentioned above such a system provide a powerful tool for strain improvement of industrial lines (Pöggeler 2001; Böhm et al. 2013). Furthermore, a sexual cycle can be used for classical genetic studies including gene mapping, functional analysis of a gene, and detection of genes of interest via methods such as bulk segregant analysis, and the investigation of mono- or polygenic basis of a trait (Ashton and Dyer 2016). Finally, characterizing a sexual cycle offers essential knowledge about the evolutionary capacity of species (McDonald and Linde 2002; Dyer and Kück 2017). Notwithstanding that the induction of a sexual cycle has many practical applications, its usage for Penicillium had been ignored for decades because the vast majority of industrially relevant species are believed to reproduce only asexually. It has been proposed by Dyer and O'Gorman (2012) that even though no sexual state has been observed in a given species, this is not implicitly a prove for lack of sexuality, and this species may show cryptic II. Scope of Thesis 26 sexuality. Indeed, a sexual life cycle has been discovered recently in some supposedly asexual Penicillium species including P. chrysogenum and P. roqueforti (Böhm et al. 2013; Ropars et al. 2014). Already before this discovery, different studies had proposed the occurrence of sexuality in these fungi. Besides population genetics studies, the identification and verification of transcriptional expression of the genes contributing to sexual reproduction in fungi (MAT genes) was the breakthrough, which led to further attempts to induce a sexual cycle in these industrial species (Hoff et al. 2008; Ropars et al. 2012). In both P. chrysogenum and P. roqueforti heterothallism was observed. Nowadays, increasing numbers of sequencing data can pave the way for the identification of MAT loci and verification of heterothallism in more Penicillium species described as asexual.

The aim of this study is to investigate the existence of putative cryptic sexuality in P. brevicompactum by identification and molecular analysis of mating type genes. In this concern, the genome sequences of two P. brevicompactum strains, available in the online database Joint Genome Institute (JGI) (https://genome.jgi.doe.gov/portal/) and the information of MAT locus organization in other Penicillium species will be used. To determine the type of a putative breeding system (heterothallism or homothallism) a large number of P. brevicompactum strains with different geographical origins will be screened for the mating type genes, and targeted crossings between isolates of opposite mating type will be tested for the induction of a sexual cycle to identify compatible mating partners. Various strategies e.g. different inoculation and incubation conditions. will be tested, since a very specific condition may be the prerequisite for sexual cycle in P. brevicompactum, as described e.g. for P. chrysogenum. Additionally, MAT genes will be used in this thesis for phylogenetic analysis of P. brevicompactum and other Penicillium species These genes have been used as a molecular marker for the analysis of phylogenetic relationships for various ascomycetes, because they apparently evolve faster than other regions of the genome (López-Villavicencio et al. 2010; Pöggeler et al. 2011). The phylogenetic analysis will be completed by comparison of MAT phylogenetic trees with the commonly used molecular marker β-tubulin.

Previous studies have demonstrated that MAT-encoded TFs not only mediate sex determination and sexual reproduction, but also control the activity of many other genes responsible for different traits of biotechnological relevance, including secondary metabolite profile, hyphal morphology, and formation of conidiospores (Böhm et al. 2013; Böhm et al. 2015). Comparative functional analysis of MAT genes from P. brevicompactum and P. chrysogenum in this thesis will widen our knowledge about the regulatory roles of these genes in the genus Penicillium. For this purpose, MAT1-1-1 and MAT1-2-1 knock out and complementation strains II. Scope of Thesis 27 of P. brevicompactum will be generated and functionally characterized concerning pellet morphology and conidia formation. For a more detailed analysis of the pellet phenotype, microscopic analyses will be performed to examine the germination rate. The observed phenotypes from transgenic strains will be compared to the corresponding wild type strains.

Another aim of this thesis is to increase the means of directed genetic manipulation of P. brevicompactum. For this purpose, the application of alternative dominant marker genes for this fungus will be investigated. Therefore, two commercially available antibiotic resistance genes were selected which are commonly used for transformation of filamentous fungi. These markers are the phleomycin resistance gene (ble), which has been shown to be a suitable marker for transformation of some Aspergillus (Austin et al. 1990; He et al. 2007) and Penicillium species (Kolar et al. 1988; Durand et al. 1991), and the nourseothricin resistance gene (nat1) gene from Streptomyces nourseii, which encodes a nourseothricin acetyltransferase. This gene is an appropriate drug-resistance marker for the selection of transgenic P. chrysogenum strains (Kopke et al. 2010). Positively tested selection markers will be used for site-specific DNA- mediated deletion and/ or insertion of genes involved in fungal development, namely MAT1-1- 1, MAT1-2-1, and flbA genes. The transgenic strains will be used for further functional analysis. While MAT1-1-1 and MAT1-2-1 encode the TFs which govern the sexual life cycle in P. chrysogenum (Böhm et al. 2013; Böhm et al. 2015), flbA (fluffy low brlA expression) encodes one of the key regulators of asexual sporulation and mycelial proliferation in Aspergillus species The flbA deletion mutants in these species showed an abolished sporulation phenotype (Lee and Adams 1994; Yu et al. 1996; Shin et al. 2013; Aerts et al. 2018). This phenotypic effect should facilitate the selection of deletion mutants in P. brevicompactum, since one could assume the same non-sporulating phenotype. III. Mahmoudjanlou et al. 2019 28

III. Mahmoudjanlou et al. 2019

Construction of a codon-adapted nourseotricin-resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum

Mahmoudjanlou Y, Hoff B and Kück U (2019)

Journal of Fungi (Basel) 10;5(4). pii: E96. doi: 10.3390/jof5040096. Journal of Fungi

Article Construction of a Codon-Adapted Nourseotricin-Resistance Marker Gene for Efficient Targeted Gene Deletion in the Mycophenolic Acid Producer Penicillium brevicompactum

† Yasaman Mahmoudjanlou, Birgit Hoff and Ulrich Kück * Allgemeine & Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany; [email protected] (Y.M.); birgit.hoff@basf.com (B.H.) * Correspondence: [email protected] † Present address: BASF SE, Carl-Bosch Straße 38, 67056 Ludwigshafen am Rhein, Germany.

 Received: 3 September 2019; Accepted: 6 October 2019; Published: 10 October 2019 

Abstract: Penicillium brevicompactum is a filamentous ascomycete used in the pharmaceutical industry to produce mycophenolic acid, an immunosuppressant agent. To extend options for genetic engineering of this fungus, we have tested two resistance markers that have not previously been applied to P. brevicompactum. Although a generally available phleomycin resistance marker (ble) was successfully used in DNA-mediated transformation experiments, we were not able to use a commonly applicable nourseothricin resistance cassette (nat1). To circumvent this failure, we constructed a new nat gene, considering the codon bias for P. brevicompactum. We then used this modified nat gene in subsequent transformation experiments for the targeted disruption of two nuclear genes, MAT1-2-1 and flbA. For MAT1-2-1, we obtained deletion strains with a frequency of about 10%. In the case of flbA, the frequency was about 4%, and this disruption strain also showed reduced conidiospore formation. To confirm the deletion, we used ble to reintroduce the wild-type genes. This step restored the wild-type phenotype in the flbA deletion strain, which had a sporulation defect. The successful transformation system described here substantially extends options for genetically manipulating the biotechnologically relevant fungus P. brevicompactum.

Keywords: Penicillium brevicompactum; codon-adapted nourseothricin resistance gene; homologous recombination; MAT1-2-1; flbA

1. Introduction Before the advent of DNA-mediated transformation systems in filamentous fungi, random mutagenesis and strain screening programs were the only genetic approaches to strain improvement in biotechnologically relevant fungi. However, these mutagenesis methods often resulted in strains that had deleterious effects on fungal growth, sporulation, and genome stability. In the past 40 years or so, different directed transformation systems have been established to overcome these problems [1]. Such systems deciphered diverse gene functions, based on targeted gene disruption or overexpression experiments [2]. Previously, advanced transformation strategies were developed for filamentous fungi, such as CaCl2/polyethylene glycol, electroporation, particle bombardment, and Agrobacterium tumefaciens–mediated transformation [3–5]. Moreover, RNA interference and CRISPR/Cas9 systems have been described as suitable approaches to genetic manipulation of fungi [6–8]. An efficient transformation strategy requires a suitable screening system and effective selectable markers. Antibiotic resistance genes of bacterial origin are the most frequently used markers in filamentous fungi because no mutant recipient strains are needed to select transformants. In contrast,

J. Fungi 2019, 5, 96; doi:10.3390/jof5040096 www.mdpi.com/journal/jof J. Fungi 2019, 5, 96 2 of 11 auxotrophic marker systems require construction of appropriate recipients. The hygromycin B resistance (hph) gene is an applicable selection marker for most fungi. However, because some Aspergillus species are relatively resistant to this antibiotic, the phleomycin resistance gene (ble) has been described as an alternative dominant selectable marker for these species [2,9]. Application of antibiotics neomycin, carboxin, fludioxonil, and blasticidin for the selection of transformants also has been suggested for genetic engineering of filamentous fungi [3,5,10]. Here, we have tested different bacterial resistance markers for the DNA-mediated transformation of the filamentous fungus Penicillium brevicompactum. In 1893, this filamentous ascomycete was discovered to be the first-known producer with antibacterial activity [11]. Since then, diverse P. brevicompactum strains have been described as producing a large range of bioactive compounds. These products, including secondary metabolites, are of significant biotechnological and medical importance because of their antiviral, antifungal, antibacterial, antitumor, and antipsoriasis activities [12]. Currently, the pharmaceutical industry uses P. brevicompactum for the large-scale biosynthesis of mycophenolic acid, an immunosuppressant agent with derivatives that are applied for autoimmune conditions as well as for kidney, heart, and liver transplantation patients [13]. Because of the limited number of selection markers, few studies have demonstrated genetic engineering of P. brevicompactum. In one report, the nitrate reductase gene from Fusarium oxysporum was used as a selection maker in an appropriate nitrate reductase-deficient recipient [14]. Successful A. tumefaciens–mediated transformation of P. brevicompactum has been described, as well, in which hph was used as a selectable marker under the control of the gpdA promoter from Aspergillus nidulans [15]. Here, we used ble and the nourseothricin-resistance marker nat1 for gene deletion analysis, followed by complementation studies. Although the previously applied ble [16,17] worked properly in the complementation studies, the commercially available nat1 could not successfully be used for selection of antibiotic-resistant P. brevicompactum transformants, although other studies have applied this resistance marker successfully in filamentous fungi [18,19]. To circumvent this difficulty, we developed a novel codon-adapted nat1 selection marker that is suitable for high-frequency transformation of P. brevicompactum. Additionally, we demonstrated that this newly constructed gene together with ble is suitable for site-specific gene deletion experiments. Our findings extend the options for genetic manipulation of this important biotechnological fungus, which is the subject of metabolic engineering experiments, to optimize the production of mycophenolic acid and its analogues [13].

2. Material and Methods

2.1. Strains, Plasmids and Culture Conditions All wild type and recombinant strains from Penicillium brevicompactum used in this investigation are shown in Table 1. The strains Dutch Centraalbureau voor Schimmelcultures CBS 257.29 and CBS 110068 were chosen for transformation and isolation of genomic DNA. To prepare the spore suspension, ◦ all P. brevicompactum strains were grown on Czapek Yeast Agar (CYA) [20] for 7 days at 27 C. Medium M334, containing 2% glucose monohydrate, 2% tryptone, 0.3% potassium dihydrogen phosphate and 0.1% magnesium sulphate heptahydrate, was used as a minimal liquid growth medium. For sensitivity tests against antibiotics, we used a complete culture medium (CCM) [21]. For plasmid construction we used Escherichia coli (E. coli), K12 XL1-Blue. A standard cloning protocol was carried out for electroporation of E. coli cells [22].

2.2. Construction of Transformation Vectors The custom-synthetized Pbnat1 gene is codon-adapted and carries restriction sites for NcoI and BamHI that flank the gene. Prior to ligation, both Pbnat1 and the vector pPtrpCnat1 were digested with NcoI and BamHI. The resulting plasmid, pPtrpC-Pbnat1, served for the construction of deletion vectors. To ensure high homologous recombination efficiency, we used about 1 kb of the 5’ and 3’ flanking regions of MAT1-2-1 and flbA. The MAT1-2-1- 5’ flanking region was amplified by PCR using the oligonucleotides J. Fungi 2019, 5, 96 3 of 11

5´-Pb-MAT1-2-1-MluI-for and 5’-Pb-MAT1-2-1-EcoRI-rv. Restriction with corresponding enzymes and N-terminal fusion to PtrpC-Pbnat1 were followed by C-terminal fusion of the 3’ flanking regions, which had been amplified by PCR using oligonucleotides 3’-Pb-MAT1-2-1-NotI-for/ 3’-Pb-MAT1-2-1-NotI-rv. For ligation, the amplicon was digested with NotI. We used the same strategy for the construction of the plasmid pPb-flbA-KO. The downstream fragment was generated by PCR using 5’-Pb-flbA-PstI-for/ 5´-Pb-flbA-PstI-rv as primers, restricted with PstI, and fused N terminally to PtrpC-Pbnat vector. It then was amplified by PCR with primer pairs 3’-PbflbA-NotI-for/ 3’-flbA-NotI-rv and ligated into the NotI restriction site, which terminates the 3´end of Pbnat1.

Table 1. Fungal strains used in this investigation.

Strain Characteristics, Genotype Source Wild type, neotype of CBS 257.29 (1) P. brevicompactum Dierckx CBS 317.59 Wild type (1) CBS 110068 Wild type (1) CBS 110070 Wild type (1) CBS 110071 Wild type (1) IBT 23078 Wild type (2) [13] ∆PbMAT1-2-1 MAT1-2-1∆::ptrpC::Pbnat1; recipient: CBS 110068 This study flbA∆::ptrpC::Pbnat1; ∆PbflbA This study recipient: CBS 257.29 ∆PbMAT1-2-1::PbMAT1-2-1 pgpd::egfp::PbMAT1-2-1:ptrpC::phle This study ∆PbflbA::PbflbA pgpd::egfp::PbflbA:ptrpC::phle This study 1. CBS-KNAW Collections—Westerdijk, Fungal Biodiversity Institute, Utrecht, Netherlands. 2. IBT, Culture Collection of Fungi, Mycology Group, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark.

For complementation of the corresponding deletion strains, MAT1-2-1 was amplified from genomic DNA of CBS 110068 using oligonucleotides MAT1-2-1-OE-EcoRI-for/ MAT1-2-1_EcoRI-OE-rv. To amplify flbA, we used as template genomic DNA from CBS 257.29, using oligonucleotides Pb-flbA-BglII-OE-for/ Pb-flbA-BamHI-OE-rv. Both genes were fused to the 3´-end of Pgpd-egfp and subsequently introduced separately into pN-EGFP [23]. nat1 in pN-EGFP was further substituted by ble. The sequences of all oligonucleotides are given in Table S3, and plasmids in Table S4.

2.3. Bioinformatics and Programs We obtained the genome sequences of AgRF18 strains from online database Joint Genome Institute (JGI) (https://genome.jgi.doe.gov/portal/). The JGI local blast tool was used to receive the sequences of MAT1-2-1 and flbA genes. To generate the codon usage table, we used the Bioperl based fascodon program. The sequence of the synthetic Pbnat1 gene was designed, using the online tool GENEius-The Tuning Tool (https://www.eurofinsgenomics.eu/en/gene-synthesis-molecular-biology/ geneius/). Custom gene synthesis was done by GenScript Corporation, 120 Centennial Ave., Piscataway, NJ 08854, USA (https://www.genscript.com/). For calculation of CAI, we used the online CAIcal algorithm local version 1.3 (http://genomes.urv.es/CAIcal/)[24]. For in silico cloning strategies, we used SnapGene version 5.0, GSL Biotech LLC program (https://www.snapgene.com/).

2.4. Transformation of P. brevicompactum Strains DNA-mediated transformation of P. brevicompactum strains was performed with some slight modifications, as previously described by Bull et al. (1988) [25]. Briefly, strains were grown in M344 ◦ shaking liquid medium (230 rpm) at 25 C for 24 h. Three grams of filtered mycelia were used for protoplast preparation. Protoplast preparation involved shaking the fungal mycelia at 110 rpm and ◦ 25 C for 2h in 35 mL 0.9 M NaCl buffer containing 40 mg/mL Vinotaste®Pro enzyme. A total of × 7 1 10 protoplasts in 100 µL transformation buffer (0.9M NaCl + 0.9M CaCl2) was transformed with 10 µg linear or circular DNA by means of a second transformation buffer (50% PEG 6000, 50 J. Fungi 2019, 5, 96 4 of 11

mM CaCl2, 10 mM Tris; pH 5.0). For selection of transformants on solid complete culture medium supplemented with 2% glucose, we used 200 µg/mL nourseothricin or 100 µg/mL phleomycin. To obtain purified transformants from single colonies, we streaked spores on CCM medium containing selection antibiotics. Molecular analysis of transgenic fungal strains by PCR and Southern blotting and hybridization was done as described previously [19,22].

3. Results and Discussion The aim of this study was to develop an alternative transformation system for targeted gene disruption in different strains of the biotechnologically relevant fungus P. brevicompactum.

3.1. Test for Sensitivity Against Antibiotics and DNA Transformation To use bacterial resistance markers for DNA-mediated transformation, we tested the sensitivity of six type culture collection strains from P. brevicompactum against different concentrations of nourseothricin and phleomycin. Our results indicated that all wild-type strains (Table 1) are sensitive to even low concentrations of nourseothricin (Figure 1). Indeed, we found a rather high sensitivity in all strains tested, and even a low antibiotic concentration of 10 µg/mL resulted in growth reduction. This exposure is much lower than the 150 µg/mL previously used to select transformants of the related P. chrysogenum [26]. However, a higher phleomycin concentration is required for growth inhibition of CBS 110068 and IBT 23078. Thus, a concentration of 100 µg/mL is necessary for reliable selection of transformants on phleomycin-containing medium.

Figure 1. Test for sensitivity against different concentrations of antibiotics as given for six P. brevicompactum wild type strains. (A) CBS 257.29; (B) CBS 110068; (C) CBS 110070; (D) CBS110071; (E) CBS 317.59; (F) IBT 27083. Antibiotic concentrations (µg/mL) are indicated in white on each plate. For further transformation experiments, CBS 257.29 and CBS 110068 were selected.

J. Fungi 2019, 5, 96 5 of 11

To examine the suitability of ble as well as nat1 for P. brevicompactum transformation, we used two transformation vectors that previously had been successfully applied for Penicillium chrysogenum: p1783-1 and pDrive/PtrpC-Tn5Phleo [21,27]. For hosts, we applied two strains carrying MAT1-1-1 (CBS 257.29) or MAT1-2-1 (CBS 110068). Both show a high production of 3.1 and 1.8 g mycophenolic acid per kilogram of mycelia, respectively, and thus can be considered as high producers of this immunosuppressant [28]. Using the vector pDrive/PtrpC-Tn5Phleo for transformation, we found a rather good frequency of transformants (about 6 per 10 µg DNA) for both recipient strains on phleomycin-containing plates. In contrast, the nat1 gene (p1783-1) yielded no transformants in 15 individual transformation experiments. This gene originates from the prokaryote Streptomyces nourseii, and its codon usage is not adapted to the expression machinery of eukaryotic host systems. To optimize expression of nat1, we decided to synthesize it with a codon bias adapted to P. brevicompactum.

3.2. Construction of a Codon-Adapted nat1 Gene The successful expression of codon-optimized genes in diverse hosts from filamentous fungi has previously been reported. For example, a completely synthetized codon-optimized flp recombinase gene from yeast Saccharomyces cerevisiae allowed for successful establishment of a marker recycling system in P. chrysogenum and Sordaria macrospora [19]. Moreover, construction of a codon-adapted luciferase gene for expression in Neurospora crassa provided a novel reporter assay system for this fungus [29]. Furthermore, the efficient expression of a codon-optimized Derf7 gene encoding a mite allergen in Aspergillus oryzae suggested that fungi can serve as hosts for the synthesis of recombinant allergen used in immunotherapy [30]. To improve the expression efficiency of nat1 in P. brevicompactum, we determined the codon bias for the fungus. For this purpose, we accessed the available genome sequences of the P. brevicompactum strain AgRF18 (online database of the Joint Genome Institute: https://genome.jgi.doe.gov/portal/) to generate a codon usage table (Supplementary Table S1). Table 2 gives the corresponding frequencies of codon usages. We selected 12,343 protein-coding sequences (CDS) and 5,661,200 codons from P. brevicompactum and compared them with the codon usage of native nat1 from S. noursei. This comparison yielded significant differences between the codon biases. For instance, AGT is the preferential codon for the amino acid aspartate in P. brevicompactum (51.8%) but is used at a frequency of only 4.4% in the native nat1 gene, which shows a bias toward using AGC to encode aspartate (Table 2). Moreover, the GC content is quite different between nat1 (71.2%) and the P. brevicompactum genome (52.8%). Because of these differences, we designed a novel nat1 gene with a codon bias adapted to the codon-preferred bias of P. brevicompactum. To obtain the most suitable codon, we generated the codon-optimized gene in silico, as described in the materials and methods section. This modified sequence is given in Figure 2, and served for the customized gene synthesis. We designated the corresponding gene as Pbnat1. This novel Pbnat1 gene with 88.4% DNA homology to nat1 has a GC content of 63.7%, which is more similar to the overall GC content of the P. brevicompactum genome (52.8%). This modification is important because GC content is a main mediator of codon and amino acid usage and thus the most significant factor determining codon bias [31]. In total, in Pbnat1, we changed 7.5% of the first (GC1) and 23.2% of the third (GC3) codon positions (Supplementary Table S2).

Figure 2. Comparative sequence alignment of codon adapted Pbnat1 gene with the commercially available nat1 gene. 82 nucleotide changes are marked in light blue.

J. Fungi 2019, 5, 96 6 of 11

Table 2. Comparison of the codon usage of nat1 and Pbnat1 with the genomic codon usage of P. brevicompactum (Pb) (AgRF18), (https://genome.jgi.doe.gov/portal/). Numbers give the usage bias of each codon for each amino acid in percent. Preferred amino acid codons are labelled in red. First column in the left indicate the first base of triplets, upper row the second base and last column the third base.

TCAG Pb Pb Pb Pb nat1 Pb nat1 Pb nat1 Pb nat1 P.b nat1 nat1 nat1 nat1 T Phe 0 37.5 32.47 Ser 0 33.3 19 Tyr 0 28.6 40.7 Cys 0 0 43.2 T Phe 100 62.5 67.53 Ser 33.3 44.5 21.6 Tyr 100 71.4 59.3 Cys 100 100 56.8 C Leu 0 0 4.9 Ser 0 0 15.2 Stop 0 0 31.8 Stop 0 0 45.5 A Leu 7.1 0 19.4 Ser 44.4 0 14.9 Stop 0 0 22.7 Trp 100 100 100 G C Leu 0 0 19.7 Pro 0 30 26.1 His 0 25 44.2 Arg 0 15.4 18.6 T Leu 38.4 57.1 25.5 Pro 40 70 30 His 100 75 55.8 Arg 38.5 0 29.7 C Leu 0 0 8.4 Pro 0 0 25.3 Gln 0 0 45.8 Arg 0 0 19 A Leu 61.6 42.9 22.1 Pro 60 0 18.6 Gln 100 100 54.2 Arg 61.5 0 13.6 G A Ile 0 33.3 36.5 Thr 0 0 25.1 Asn 0 33,3 40.5 Ser 11.15 0 11.7 T Ile 100 66.7 52.7 Thr 82.4 70.6 35.7 Asn 100 66,7 59.5 Ser 11.15 22.3 17.6 C Ile 0 0 10.8 Thr 0 0 23.7 Lys 0 0 34.1 Arg 0 0 11.3 A Met 100 100 100 Thr 17.6 29.4 15.5 Lys 100 100 65.9 Arg 0 0 7.8 G G Val 0 33.3 27.2 Ala 5.3 31.6 27.5 Asp 4.4 47.8 51.8 Gly 0 30 29 T Val 73.3 53.3 37.1 Ala 47.5 68.4 32.8 Asp 95.6 52.2 48.2 Gly 45 70 32.7 C Val 0 0 8.6 Ala 10.4 0 22 Glu 15.4 30.7 42.8 Gly 0 0 24.8 A Val 26.7 13.4 27.1 Ala 36.8 0 17.7 Glu 84.6 69.3 57.2 Gly 52.4 0 13.5 G

To examine the compatibility Pbnat1 with P. brevicompactum codon bias, we calculated the codon adaptation index (CAI) for both nat1 and Pbnat1. For this purpose, we used the online CAIcal algorithm (http://genomes.urv.es/CAIcal/)[24]. The CAI for a specific gene is defined by comparing its codon usage rate and frequency in a reference set of genes. Assigned values are between 0 and 1. With CAI values closer to 1, the expression level of the targeted gene is expected to be better in a heterologous host system [19,32,33]. Changing about 43% of all codons shifted the CAI value for Pbnat1 from 0.83 to 0.91. This result led us to predict that Pbnat1 compared with nat1 would have an optimized and more accurate expression in P. brevicompactum.

3.3. Use of the Codon-Optimized Pbnat1 Gene for Site-Specific Deletion of Two Nuclear Genes To improve the transformation efficiency in P. brevicompactum, we developed a reliable procedure based on the use of protoplasts. For this purpose, we tested two buffers: potassium phosphate [14] and 0.9 M sodium chloride [25] for CBS 257.29 and CBS 110068. Based on our results, we concluded that 0.9 M sodium chloride containing 40 mg/mL Vinotaste®Pro digestive enzyme was the only effective buffer for protoplasting of P. brevicompactum strains. After establishing the transformation method (see the material and methods section), we investigated the efficiency of the newly constructed Pbnat1 gene in P. brevicompactum using the vector pPtrpC-Pbnat1. This plasmid contains the Pbnat1 gene under the control of the trpC promoter from A. nidulans. Application of Pbnat1 led to successful transformation events at a frequency of about 10 nourseothricin-resistant transformants per 10 µg of circular DNA. All transformants were genetically stable on selective media and were propagated for more than 12 months. In the next step, we applied the Pbnat1 gene containing the vector pPtrpC-Pbnat1 for site-specific deletion of two genes inferred to be involved in fungal development: flbA and MAT1-2-1. flbA encodes a regulator of G-protein signaling that is involved in asexual sporulation and mycelial proliferation, and MAT1-2-1 encodes a transcription factor that controls sexual development [17,34,35]. To generate deletion mutants by homologous recombination, we transformed the strains with a linear deletion cassette containing 5‘-and 3’- flanking regions derived from the target gene and each about 1 kb long (Figures S1A and S2A). The corresponding cassettes were obtained from plasmids, shown in J. Fungi 2019, 5, 96 7 of 11

Figure 3A,B. CBS 257.29 and CBS 110068 served as the recipient strains for disruption of flbA and MAT1-2-1, respectively. With linear DNA for deletion of MAT1-2-1, we observed a frequency of 10–12 transformants per 10 µg of linear DNA. The transformation frequency was considerably less when we used the flbA deletion cassette (pPb-flbA-KO), with about 2–4 transformants per 10 µg of DNA.

Figure 3. Maps of vectors for DNA mediated transformation of P. brevicompactum.(A,B) Plasmids pPb-MAT1-2-1-KO (A) and pPb-flbA-KO (B) for site-specific deletion of MAT1-2-1 and flbA genes, carrying the codon adapted pbnat1 gene under the transcriptional control of the PtrpC promoter; (C,D) Plasmids pPb-MAT1-2-1-comp (C) and pPb-flbA-comp (D) for complementation of the corresponding deletion strains. Both plasmids carry the egfp-tagged gene of interest under the constitutive gpd promoter from A. nidulans.

To verify that homologous recombination had occurred, we performed PCR amplification and Southern hybridizations with a probe that was homologous to the 5’-flanking region of the target gene (Supplementary Figures S1B, S1C, S2B, S3F). Our data confirmed the presence of homokaryotic deletion strains. While the MAT1-2-1 deletion strains showed no detectable phenotype, the flbA deletion J. Fungi 2019, 5, 96 8 of 11 strains were distinct from the wild type because of a delayed and reduced sporulation phenotype (Figure 4). This developmental feature is similar to previously reported phenotypes that resulted when the homologous flbA gene was deleted in different Aspergillus species [36,37]. Verification of 60 putative MAT1-2-1 strains revealed a frequency of 10% for deletion of MAT1-2-1 gene. In contrast, only 1 of 24 putative flbA deletion strains was identified as a homokaryotic deletion strain.

Figure 4. Phenotypic analysis of flbA deletion and corresponding complementation strains after 7 days ◦ of growth on CCM medium at 27 C, under different light condition. (A) light, (B) dark. CBS 257.29: wild type; PT 40-6: deletion strain; PT 55-3: complementation strain.

In the next step, we conducted complementation experiments to confirm the successful deletion of the target genes. For complementation, we used the plasmids pPb-MAT1-2-1-comp and pPb-flbA-comp to transform the appropriate deletion strains (Figure 3C, 3D). PCR analysis and Southern hybridization with a target gene–specific probe served as confirmation of the successful ectopic integration of the wild-type genes (Figure S3). The ectopic integration of flbA into the deletion strains activated a wild type–like sporulation phenotype (Figure 4). In our experiments we observed a sufficiently high frequency of homologous recombination in P. brevicompactum. Therefore, it is not necessary to use mutants in which non-homologous end joining (NHEJ) is abolished by mutation of the NHEJ repair system. Neurospora crassa was the first filamentous ascomycete in which homologous recombination was enhanced by disrupting genes for the catalytic subunit (DNA-PKcs) or the regulatory DNA-binding subunits (Ku70/80 heterodimer) [38,39]. Later, strains from diverse ascomycetes were generated to serve as recipients for the targeted integration of foreign genes [6,40]. However, several reports showed that non-homologous end joining–deficient strains accumulate random mutations and thus are less suitable for long-term experiments. Furthermore, these recipient strains show an elevated sensitivity to different chemicals such as bleomycin, methyl methanesulfonate, and ethyl methanesulfonate (for review, see [6,40]). Thus, some investigators have avoided using specific recipients for homologous recombination because the natural homologous recombination frequency was similar to that described here [41–43]. In conclusion, we have extended the molecular tools for genetic manipulation of the biotechnologically relevant fungus P. brevicompactum. The opportunity to generate deletion and complementation strains by the use of different marker genes opens up future avenues to

J. Fungi 2019, 5, 96 9 of 11 research to identify factors that control or regulate secondary metabolism in this mycophenolic acid–producing fungus.

Supplementary Materials: The following are available online at http://www.mdpi.com/2309-608X/5/4/96/s1, Figure S1: Construction of a MAT1-2-1 deletion mutant; Figure S2: Construction of a flbA deletion strains; Figure S3: Complementation of MAT1-2-1 and flbA deletion strains; Table S1: Codon usage in P. brevicompactum. The genomic sequences of strain AgRF18 was used for calculating frequency of preferred amino acid codons; Table S2: Comparison of CAI and GC-content of nat1 and Pbnat1; Table S3: List of oligonucleotides used in this study; Table S4. List of plasmids, used in this study. Author Contributions: Y.M., B.H., and U.K. designed the experimental strategy, Y.M. performed the experiments; Y.M. and U.K. analyzed data; Y.M., B.H., U.K. wrote the manuscript. Funding: The work of the authors was funded by the Christian Doppler Society (Vienna, Austria) and a grand from the German Research Foundation (DFG KU 517/15-1). Acknowledgement: The authors are thankful to Fabian Becker for conducting some of the experiments during his Bachelor thesis and T. Dahlmann for help in bioinformatics applications. We would like to acknowledge the technical advice of I. Godehardt, and the fruitful discussion with T. Dahlmann (RUB, Bochum), J. Finke (Janssen-Cilag GmbH, Neuss, D), H. Hecker (Novartis, Kundl, A), H. Kürnsteiner (Novartis, Kundl, A), and I. Zadra (Novartis, Kundl, A). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Kluge, J.; Terfehr, D.; Kück, U. Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi. Appl. Microbiol. Biotechnol. 2018, 102, 6357–6372. [CrossRef][PubMed] 2. He, Z.M.; Price, M.S.; Obrian, G.R.; Georgianna, D.R.; Payne, G.A. Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus. BMC Microbiol. 2007, 7, 104. [CrossRef][PubMed] 3. Ruiz-Diez, B. Strategies for the transformation of filamentous fungi. J. Appl. Microbiol. 2002, 92, 189–195. [CrossRef][PubMed] 4. Michielse, C.B.; Arentshorst, M.; Ram, A.F.; van den Hondel, C.A. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genet. Biol. 2005, 42, 9–19. [CrossRef][PubMed] 5. Weld, R.J.; Plummer, K.M.; Carpenter, M.A.; Ridgway, H.J. Approaches to functional genomics in filamentous fungi. Cell Res. 2006, 16, 31–44. [CrossRef][PubMed] 6. Kück, U.; Hoff, B. New tools for the genetic manipulation of filamentous fungi. Appl. Microbiol. Biotechnol. 2010, 86, 51–62. [CrossRef] 7. Krappmann, S. CRISPR-Cas9, the new kid on the block of fungal molecular biology. Med. Mycol. 2017, 55, 16–23. [CrossRef] 8. Shi, T.Q.; Liu, G.N.; Ji, R.Y.; Shi, K.; Song, P.; Ren, L.J.; Huang, H.; Ji, X.J. CRISPR/Cas9-based genome editing of the filamentous fungi: The state of the art. Appl. Microbiol. Biotechnol. 2017, 101, 7435–7443. [CrossRef] 9. Austin, B.; Hall, R.M.; Tyler, B.M. Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance. Gene 1990, 93, 157–162. [CrossRef] 10. Jiang, D.; Zhu, W.; Wang, Y.; Sun, C.; Zhang, K.Q.; Yang, J. Molecular tools for functional genomics in filamentous fungi: Recent advances and new strategies. Biotechnol. Adv. 2013, 31, 1562–1574. [CrossRef] 11. Bentley, R. Mycophenolic Acid: A one hundred year odyssey from antibiotic to immunosuppressant. Chem. Rev. 2000, 100, 3801–3826. [CrossRef][PubMed] 12. Zhang, W.; Cao, S.; Qiu, L.; Qi, F.; Li, Z.; Yang, Y.; Huang, S.; Bai, F.; Liu, C.; Wan, X.; et al. Functional characterization of MpaG’, the O-methyltransferase involved in the biosynthesis of mycophenolic acid. Chembiochem 2015, 16, 565–569. [CrossRef][PubMed] 13. Regueira, T.B.; Kildegaard, K.R.; Hansen, B.G.; Mortensen, U.H.; Hertweck, C.; Nielsen, J. Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum. Appl. Environ. Microbiol. 2011, 77, 3035–3043. [CrossRef][PubMed] 14. Varavallo, M.A.; de Queiroz, M.V.; Pereira, J.F.; Ribeiro, R.A.; Soares, M.A.; Ribeiro, J.B.; de Araujo, E.F. Development of a transformation system for Penicillium brevicompactum based on the Fusarium oxysporum nitrate reductase gene. Braz. J. Microbiol. 2005, 36, 184–189. [CrossRef] J. Fungi 2019, 5, 96 10 of 11

15. Dong, Y.; Zhang, J.; Xu, R.; Lv, X.; Wang, L.; Sun, A.; Wei, D. Insertion mutation in HMG-CoA lyase increases the production yield of MPA through Agrobacterium tumefaciens-mediated transformation. J. Microbiol. Biotechnol. 2016, 26, 1924–1932. [CrossRef][PubMed] 16. Kolar, M.; Punt, P.J.; Vandenhondel, C.A.M.J.J.; Schwab, H. Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli LacZ fusion gene. Gene 1988, 62, 127–134. [CrossRef] 17. Böhm, J.; Dahlmann, T.A.; Gümüser, H.; Kück, U. A MAT1-2 wild-type strain from Penicillium chrysogenum: Functional mating-type locus characterization, genome sequencing and mating with an industrial penicillin-producing strain. Mol. Microbiol. 2015, 95, 859–874. [CrossRef] 18. Kück, U.; Hoff, B. Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi. Fungal Genet. Newsl. 2006, 53, 9–11. [CrossRef] 19. Kopke, K.; Hoff, B.; Kück, U. Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes. Appl. Environ. Microbiol. 2010, 76, 4664–4674. [CrossRef] 20. Visagie, C.M.; Houbraken, J.; Frisvad, J.C.; Hong, S.B.; Klaassen, C.H.; Perrone, G.; Seifert, K.A.; Varga, J.; Yaguchi, T.; Samson, R.A. Identification and nomenclature of the genus Penicillium. Stud. Mycol. 2014, 78, 343–371. [CrossRef] 21. Böhm, J.; Hoff, B.; O’Gorman, C.M.; Wolfers, S.; Klix, V.; Binger, D.; Zadra, I.; Kurnsteiner, H.; Poggeler, S.; Dyer, P.S.; et al. Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. Proc. Natl. Acad. Sci. USA 2013, 110, 1476–1481. [CrossRef][PubMed] 22. Sambrook, J.R.; Green, M.R. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, USA, 2001. 23. Kück, U.; Pöggeler, S.; Nowrousian, M.; Nolting, N.; Engh, I. Sordaria macrospora, a model system for fungal development. In Physiology and Genetics: Selected Basic and Applied Aspects; Anke, T., Weber, D., Eds.; Springer: Berlin/Heidelberg, Germany, 2009; pp. 17–39. 24. Puigbò, P.; Bravo, I.G.; Garcia-Vallve, S. CAIcal: A combined set of tools to assess codon usage adaptation. Biol. Direct 2008, 3, 38. [CrossRef][PubMed] 25. Bull, J.H.; Smith, D.J.; Turner, G. Transformation of Penicillium chrysogenum with a dominant selectable marker. Curr. Genet. 1988, 13, 377–382. [CrossRef][PubMed] 26. Hoff, B.; Kamerewerd, J.; Sigl, C.; Zadra, I.; Kück, U. Homologous recombination in the antibiotic producer Penicillium chrysogenum: Strain DeltaPcku70 shows up-regulation of genes from the HOG pathway. Appl. Microbiol. Biotechnol. 2010, 85, 1081–1094. [CrossRef][PubMed] 27. Gesing, S.; Schindler, D.; Franzel, B.; Wolters, D.; Nowrousian, M. The histone chaperone ASF1 is essential for sexual development in the filamentous fungus Sordaria macrospora. Mol. Microbiol. 2012, 84, 748–765. [CrossRef][PubMed] 28. Mahmoudjanlou, Y.; Dahlmann, T.; Kück, U. Molecular analysis of mating type genes in the mycophenolic acid producer Penicillium brevicompactum. 2019; in preparation. 29. Gooch, V.D.; Mehra, A.; Larrondo, L.F.; Fox, J.; Touroutoutoudis, M.; Loros, J.J.; Dunlap, J.C. Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock. Eukaryot. Cell 2008, 7, 28–37. [CrossRef][PubMed] 30. Tokuoka, M.; Tanaka, M.; Ono, K.; Takagi, S.; Shintani, T.; Gomi, K. Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Appl. Environ. Microbiol. 2008, 74, 6538–6546. [CrossRef][PubMed] 31. Zhou, Z.; Dang, Y.; Zhou, M.; Li, L.; Yu, C.H.; Fu, J.; Chen, S.; Liu, Y. Codon usage is an important determinant of gene expression levels largely through its effects on transcription. Proc. Natl. Acad. Sci. USA 2016, 113, E6117–E6125. [CrossRef] 32. Sharp, P.M.; Li, W.H. The codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987, 15, 1281–1295. [CrossRef] 33. Quax, T.E.; Claassens, N.J.; Soll, D.; van der Oost, J. Codon bias as a means to fine-tune gene expression. Mol. Cell 2015, 59, 149–161. [CrossRef] 34. Yu, J.H.; Wieser, J.; Adams, T.H. The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J. 1996, 15, 5184–5190. [CrossRef][PubMed] J. Fungi 2019, 5, 96 11 of 11

35. Becker, K.; Beer, C.; Freitag, M.; Kück, U. Genome-wide identification of target genes of a mating-type-domain transcription factor reveals functions beyond sexual development. Mol. Microbiol. 2015, 96, 1002–1022. [CrossRef][PubMed] 36. Seo, J.A.; Yu, J.H. The phosducin-like protein PhnA is required for Gbetagamma-mediated signaling for vegetative growth, developmental control, and toxin biosynthesis in Aspergillus nidulans. Eukaryot. Cell 2006, 5, 400–410. [CrossRef][PubMed] 37. Aerts, D.; Hauer, E.E.; Ohm, R.A.; Arentshorst, M.; Teertstra, W.R.; Phippen, C.; Ram, A.F.J.; Frisvad, J.C.; Wosten, H.A.B. The FlbA-regulated predicted transcription factor Fum21 of is involved in fumonisin production. Antonie Van Leeuwenhoek 2018, 111, 311–322. [CrossRef][PubMed] 38. Ninomiya, Y.; Suzuki, K.; Ishii, C.; Inoue, H. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc. Natl. Acad. Sci. USA 2004, 101, 12248–12253. [CrossRef] 39. Walker, J.R.; Corpina, R.A.; Goldberg, J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 2001, 412, 607–614. [CrossRef] 40. Krappmann, S. Gene targeting in filamentous fungi: The benefits of impaired repair. Fungal Biol. Rev. 2007, 21, 25–29. [CrossRef] 41. Siewers, V.; Kokkelink, L.; Smedsgaard, J.; Tudzynski, P. Identification of an abscisic acid gene cluster in the grey Botrytis cinerea. Appl. Environ. Microbiol. 2006, 72, 4619–4626. [CrossRef] 42. Lin, C.H.; Chung, K.R. Specialized and shared functions of the histidine kinase- and HOG1 MAP kinase-mediated signaling pathways in Alternaria alternata, a filamentous fungal pathogen of citrus. Fungal Genet. Biol. 2010, 47, 818–827. [CrossRef] 43. Lou, H.; Ye, Z.; Yun, F.; Lin, J.; Guo, L.; Chen, B.; Mu, Z. Targeted gene deletion in Cordyceps militaris using the split-marker approach. Mol. Biotechnol. 2018, 60, 380–385. [CrossRef]

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Table S1. Codon usage in P. brevicompactum. The genomic sequences of strain AgRF18 was used for calculating frequency of preferred amino acid codons.

fields: [triplet] [frequency: per thousand] ([number])

UUU 12.6 (71568) UCU 16.1 (91342) UAU 11.3 (64019) UGU 5.7 (32482)

UUC 26.2 (148291) UCC 18.3 (103490) UAC 16.5 (93591) UGC 7.5 (42450)

UUA 4.4 (24630) UCA 12.9 (73146) UAA 0.7 (3697) UGA 1.0 (5420)

UUG 17.6 (99392) UCG 12.6 (71116) UAG 0.5 (3099) UGG 15.2 (86302)

CUU 17.8 (100530) CCU 15.6 (88584) CAU 11.2 (63312) CGU 11.0 (62065)

CUC 23.1 (130942) CCC 17.9 (101550) CAC 13.7 (77695) CGC 17.5 (99140)

CUA 7.6 (42852) CCA 15.1 (85606) CAA 18.6 (105265) CGA 11.2 (63627)

CUG 20.0 (113406 CCG 11.1 (62688) CAG 22.0 (124620) CGG 8.0 (45184)

AUU 18.7 (105906) ACU 14.8 (83656) AAU 15.1 (85709) AGU 9.9 (56091)

AUC 27.0 (152588) ACC 21.1 (119507) AAC 22.2 (125473 AGC 14.9 (84326)

AUA 5.5 (31032) ACA 14.0 (79116) AAA 15.7 (88997) AGA 6.7 (38023)

AUG 20.2 (114569) ACG 9.2 (52330) AAG 30.3 (171504) AGG 4.6 (26150)

GUU 16.7 (94447) GCU 23.1 (130555) GAU 29.0 (164398) GGU 19.6 (111162)

GUC 22.8 (128878) GCC 27.5 (155664) GAC 27.2 (153773) GGC 22.1 (124973)

GUA 5.3 (29738) GCA 18.5 (104642) GAA 26.0 (146960) GGA 16.7 (94555)

GUG 16.6 (93739) GCG 14.8 (83520) GAG 34.7 (196349) GGG 9.1 (51732)

Table S2. Comparison of CAI and GC-content of nat1 and Pbnat1

Gene CAI Total GC GC1 (%) GC2 (%) GC3 (%) content (%) nat1 0.83 71.2 68.9 48.9 95.8 Pbnat1 0.91 63.7 69.5 48.9 72.6

1

Table. S3. List of oligonucleotides used in this study

Name Sequence (5′–3′) Specificity

5'-Pb-flbA-PstI-for ATACTGCAGCTCATACAGGCGTCCT 5′ flanking region of flbA

CAGCC

5´-Pb-flbA-PstI-rv ATCCTGCAGGAATGGTTTTGAGTCT 5′ flanking region of flbA

TCGGGT

3'-Pb-flbA-NotI-for TATTAAGCGGCCGCGATTCCGACTC 3′ flanking region of flbA

CTCATGA

3'-Pb-flbA-NotI-rv CATTAGCGGCCGCAGTATCTACCAC 3′ flanking region of flbA

CTGAGCAACC

5´-Pb-MAT1-2-1-MluI- ACATAACGCGTCCCTCAACGATGGT 5′ flanking region of MAT1-2-1

for CCGCAC

5'-Pb-MAT1-2-1- ATGTAGAATTCGCACGACGAGGGC 5′ flanking region of MAT1-2-1

EcoRI-rv TCATGGA

3'-Pb-MAT1-2-1-NotI- ATACTGCGGCCGCGCTTTTCATCCC 3′ flanking region of MAT1-2-1

for ATCGTTTCT

3'-Pb-MAT1-2-1-NotI- ATGTAGCGGCCGCGAACCACCAAT 3′ flanking region of MAT1-2-1

rv CATCTCTCT

Pb-FlbA-5'-genome-for GGTCGAGCTAAGGGAAGATA 5′ flanking region of flbA

Pb-FlbA-3'-genome-rv CGCATGCTTTGGCCACAAGA 3′ flanking region of flbA

Pb_flbA-for CCAACTCAACCCGGAAACCA flbA

Pb-flbA-rv GCTCATGCTGCGTTCAGGGAT flbA

4736-f ACTTTCATCTGGGCCAGCGAGTGG apn2

2756-r GCCCGCCAGCGTCTGGGCGAAATG sla2

Pb_spec_MAT1-2-1-f CCTGGAGTTACCACCTACTC MAT1-2-1

Pb_MAT1-2_r TGATGTCCATGTAGTCGGTC MAT1-2-1

PtrpC_seq_r CTCCACTAGCTCCAGCCAAG ptrpC

Pbnat1-s CTCGATGACACGGCTTACCGCTA Pbnat1

Tn5-phleo-BoxI-for GAATAGACTTACGTCCATGGGCGA ble

AATGACCGACC

Tn5-phleo-ApaI-rv GAATTGGGCCCTCATGAGATGCCTG ble

CAAGCA

MAT1-2-1-OE-EcoRI- GATTATGAATTCGAGCCCTCGTCGT MAT1-2-1

for GCCATG

2

MAT1-2-1_EcoRI-OE- GGCGCGGAATTCGGACATTGAGAC MAT1-2-1

rv TGAAGGCAG

Pb-flbA-BglII-OE-for GCGGCTAGATCTATGCCAACTCAAC flbA

CCGGAAA

Pb-flbA-BamHI-OE-rv GATTATGGATCCTTGTCAGGCGCGG flbA

GCTGAAC

Table S4. List of plasmids, used in this study

Name Characteristics source

pDrive/ptpc-Tn5Phleo trpC promoter of Aspergillus nidulans, ble resistance gene of Böhm et

Streptoalloteichus hindustanus al., 2013

P17831-nat1 gpd promoter of A.nidulans, egfp, TtrpC of A. nidulans, trpC Gesing et

promoter of A. nidulans, nat1 resistance gene of S. noursei al. 2012

PN-EGFP gpd promoter of A.nidulans, egfp, TtrpC of A. nidulans, trpC Kück et al.,

promoter of A. nidulans, hph resistance gene of Streptomyces 2009

hygroscopicus

pPtrpC-nat1 trpC promoter of A. nidulans, nat1 resistance gene of S. Kück et al.

noursei, 2009

pPtrpC-Pbnat1 trpC promoter of A. nidulans, codon adapted synthetized This study

Pbnat1 resistance gene

pPb-MAT1-2-1-KO 5′ flanking region of MAT1-2-1 gene, trpC promoter of A. This study

nidulans, Pbnat1 (codon adapted nat1 resistance gene of S.

noursei), 3′ flanking region of MAT1-2-1 gene

pPb-flbA-KO 5′ flanking region of flbA gene, trpC promoter of A. nidulans, This study

Pbnat1(codon adapted nat1 resistance gene from S. noursei),

3′ flanking region of flbA gene

pPb-MAT1-2-1-comp gpd promoter of A.nidulans, egfp, MAT1-2-1 gene from P. This study

brevicompactum TtrpC of A. nidulans

pPb-flbA-comp gpd promoter of A.nidulans, egfp, flbA1 gene from P. This study

brevicompactum TtrpC of A. nidulans

3

Figure S1.: Construction of a MAT1-2-1 deletion mutant. (A) MAT1-2-1 locus in the reference strain and in the deletion mutant ΔMAT1-2-1. Dashed lines indicated the homologous recombination event.

Arrows show the primer pairs used for PCR analyis. Restriction enzyme recognition site used for digestion of genomic DNA and the size of corresponding fragments are indicated. (B) Autoradiograph of a Southern hybridization analysis. 5’ flanking regions served as radioactive labelled probes (C)

Verification of recombinant strains with PCR. Numbers above autoradiogram and gels indicate individual transformants.

4

Figure S2. Construction of a flbA deletion strains. (A) flbA locus in the reference strain and in the deletion mutant ΔflbA. Dashed lines indicated the homologous recombination event (B) Verification of recombinant strains with PCR. The arrows shown in (A) represent the primer pairs used for PCR analysis. CBS 257.29: wild type; PT 40-6: ΔflbA.

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Figure S3. Complementation of MAT1-2-1 and flbA deletion strains. (A) Vector map for integration of the MAT1-2-1 gene into the corresponding deletion strains. (B) PCR analysis for verification of MAT1-

2-1 recombinant strains (C) Evidence for complementation of ΔMAT1-2-1 by Southern hybridization 6 with radioactively labeled probe specific for the MAT1-2-1 gene. (D) Schematic map of construct used for complementation of flbA deletion strains. (E) PCR analysis to prove the genomic integration of flbA gene in deletion strains. CBS 257.29 & CBS 110068 are wild-type strains. (F) Evidence for complementation of ΔflbA by Southern hybridization with radioactively labeled probe specific for the flbA gene. Arrows in A & D show the position of primer pairs used for PCR.

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IV. Mahmoudjanlou et al. 2020

Molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum: Phylogeny and MAT protein characterization suggest a cryptic sexual life cycle

Mahmoudjanlou Y, Dahlmann TA and Kück U (2020)

submitted to Fungal Biology (under review) IV. Mahmoudjanlou et al. 2020 1

Molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum: Phylogeny and MAT protein characterization suggest a cryptic sexual life cycle

Yasaman Mahmoudjanlou, Tim A. Dahlmann, Ulrich Kück*

Allgemeine und Molekulare Botanik, Ruhr-Universität, Bochum, D-44780 Bochum, Germany, *corresponding author: [email protected]

Key words: Penicillium brevicompactum, mating type loci, transcription factor, phylogeny, karyotype

IV. Mahmoudjanlou et al. 2020 2

Abstract The mycophenolic acid producing ascomycete Penicillium brevicompactum is considered to be an anamorphic (asexual) species, for which a sexual cycle was never observed. However, since recent reports of otherwise asexually propagating filamentous fungi have demonstrated a sexual cycle controlled by mating type loci, we carried out a molecular analysis of mating type loci from Penicillium brevicompactum. Using data from extensive DNA sequencing analysis, we determined the mating type loci from 22 strains derived from various type culture collections. We found 8 strains carrying a MAT1-1 locus encoding a 362 amino acid alpha domain transcription factor. The other 14 possessed a MAT1-2 locus encoding a 298 amino acid HMG domain transcription factor. cDNA analysis confirmed that both mating type loci are transcriptionally expressed. The karyotype of six selected strains, determined using contour-clamped homogeneous electric field (CHEF) electrophoresis, demonstrated distinct differences in size and numbers of chromosomes between the strains investigated. Interestingly, our phylogenetic survey of 72 strains from 10 different Penicillium spp. revealed that MAT genes serve as excellent molecular markers to determine phylogenetic relationships among species closely related toP. brevicompactum. Based on our sequencing results, we constructed transformation vectors for site-specific deletion of mating type loci from two selected strains of opposite mating type. Complementation strains were constructed containing both the mating type locus deletion cassette and a MAT-GFP fusion gene. These strains were used for comparative phenotypic analyses between strains containing or lacking the mating type gene. Whereas all MAT1-2 strains were indistinguishable, the MAT1-1 and MAT1-1-1 deletion strains differed distinctly. The MAT1-1-1 deletion strain produced more conidiospores on solid media, but smaller pellets in liquid media. This is probably the consequence of fewer conidial germ tubes than with the wild type mating type strain. Finally, we showed that the MAT-GPF fusion protein is localized to the nuclei and detectable in protein samples by Western analysis. Together, our results suggest that the asexually propagating fungus P. brevicompactum is a heterothallic species with a cryptic sexual life cycle.

IV. Mahmoudjanlou et al. 2020 3

Introduction The genus Penicillium belongs to the phylum Ascomycota and species of this genera are found frequently worldwide (Robert A. Samson & Pitt, 2000). Many species are of great interest for a wide variety of medical and biotechnological applications, since they produce pharmaceutically relevant primary and secondary metabolites. A recent comparison of genomic sequences from 24 different Penicillium species revealed the huge potential of this genus to produce secondary metabolites amounting to around 1,317 putative substances (Nielsen et al., 2017).

Penicillium was taxonomically introduced as the anamorphic state of teleomorphic Eupenicillium and Talaromyces (Pitt, 1979). However, the genus was redefined in a phylogenetic analysis using four molecular markers (J. Houbraken & Samson, 2011). This study generated a monophyletic genus for both anamorphs and teleomorphs as Penicillium sensu stricto and revealed that strict anamorphs were combined with teleomorph species, suggesting a possible sexual state in anamorphic species. The Penicillium subgenus Penicillium comprises six sections, namely Digitata, Coronata, Chrysogena, Penicillium, Roqueforti and Viridicata, and includes some of the most common fungi responsible for food spoilage and indoor contamination, as well as several highly biotechnologically relevant species (Frisvad & Samson 2004).

In ascomycetes, sexual reproduction is determined by either the homothallic or heterothallic breeding system. While homothallism denotes self-breeding of single strains, heterothallism indicates the mating between two partners of opposite sex (Dyer & Kück, 2017; Taylor et al., 1999). In fungi, both breeding systems are controlled by mating type (MAT) genes, located at the MAT locus (Dyer & Kück, 2017; Ni et al., 2011). In heterothallic species, mating strains carry opposite MAT loci, which comprise dissimilar DNA sequences located at the same locus on the chromosome, i.e. they represent idiomorphs. In contrast, homothallic species possess both opposite MAT loci, occupying either the same locus or loci on different chromosomes. The MAT loci in heterothallic Penicillium species typically include just a single functional open reading frame encoding transcription factors, which carry either an alpha or a high mobility group (HMG)-DNA-binding domain, called MAT1-1-1 or MAT1-2-1, respectively (Dyer & Kück, 2017; Turgeon & Yoder, 2000).

Accumulating genome data and the rather conserved sequences adjacent to mating type loci provided early evidence for MAT loci in asexual species. Furthermore, population genetics indicated the presence of cryptic sexuality in otherwise asexual (anamorphic) species, IV. Mahmoudjanlou et al. 2020 4 including representatives from the genus Penicillium. As a consequence, several attempts were successful in inducing a sexual cycle in some apparently asexually propagating species such as Penicillium chrysogenum and Penicillium roqueforti (Böhm et al., 2015; Böhm et al., 2013; Ropars et al., 2014). As an application, sexual reproduction has the potential to generate improved recombinant strains for industrial production lines (Böhm et al., 2013; S. Pöggeler, 2001).

In this study, we present a molecular analysis of the mating type loci from the mycophenolic acid producer P. brevicompactum. DNA sequence analysis provided evidence for the presence of a mating type locus, although P. brevicompactum is supposed to lack a sexual life cycle. DNA sequencing data resulted in the construction of phylogenetic trees based on mating type sequences from more than 70 Penicillium strains. Moreover, biochemical and imaging studies demonstrated the expression and cellular localization of the MAT1-1-1 protein. Finally, gene disruption of the mating type locus provided some clues about the function of the encoded transcription factor. Together, our data indicates that P. brevicompactum undergoes a sexual life cycle in nature.

Material and Methods

Fungal strains and culture conditions All wild type fungal strains used for phylogenetic analyses are given (Supplemental Table 1). All strains were obtained from the CBS-KNAW culture collection, IMI (CABI Genetic Resources Collection, Surrey, UK), Leibniz Institute DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) or were donated by Dr. Martin Kirchmair (Institut für Mikrobiologie Innsbruck University, Austria). P. brevicompactum recombinant strains used in this study are listed in (Supplemental Table 2). For the preparation of spore suspensions and quantification of spore formation, all strains were grown on a solid complete culture medium (CCM) for 168 h at 27°C (Böhm et al., 2013). To carry out the genomic DNA isolation, all fungal strains were grown in CCM liquid medium for 72 h at 27°C, except P. brevicompactum, P. bialowiezense, and P. olsonii, which were grown on Czapek Yeast Autolysate (CYA) medium. For pellet formation and protein extraction, P. brevicompactum strains were grown in shaking flasks (120 rpm) for 72 h at 27°C.

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DNA extraction, amplification, and sequencing Genomic DNA was extracted using the protocol described previously (Lecellier & Silar, 1994). Oligonucleotides used for DNA amplification and sequencing are listed in (Supplemental Table 3). The PCR products were purified by a cleaning kit (Macherey-Nagel™ NucleoSpin™ Gel and PCR Clean-up Kit, Düren, Germany) according to the manufacturer´s recommendations. Sanger sequencing was performed by Eurofins genomics (https://www.eurofinsgenomics.eu/).

Construction of Transformation Vectors The deletion vector was constructed using the plasmid pPtrpC-Pbnat (Mahmoudjanlou et al., 2019), which contains a Pbnat1 gene adapted to the codon usage of P. brevicompactum. The deletion cassette was constructed by ligating about 1 kb of the 5’ and 3’ flanking regions of MAT1-1-1 with the Pbnat1 gene. The genomic DNA of the strain CBS 257.29 served as a template for PCR procedures. The 5’ and 3’ flanking regions of MAT1-1-1 were amplified using oligonucleotides Pbre-MAT1-5'-MluI-f x Pb-MAT1-5'-EcoRI-r, and Pbre-MAT1-3'- NotI-f x Pbre-MAT1-3'-NotI-r. The 5’ flanking region was first integrated into pPtrpC-Pbnat, following restriction digests with MluI and EcoRI enzymes. Next, the 3’ flanking region was ligated into the NotI cleaved product of the previous step, resulting in the construction of pPb- MAT1-1-1-KO. This vector served for site-specific gene substitution of the MAT1-1-1 gene.

To construct a complementation vector, MAT1-1-1 was amplified by the oligonucleotides Pbre- MAT1-BglII-comp-f and Pbre-MAT1-EcoRI-comp-r. The subsequent fragment was fused in frame to the 3´-end of Pgpd-egfp in vector pN-EGFP (Kück et al., 2009). The nat1 gene in pN- EGFP was substituted by the ble gene, resulting in the construction of vector pPb-MAT1-1-1- comp.

Karyotype analysis For karyotype analysis, the intact chromosomal DNA was prepared and separated via contour- clamped homogeneous electric field (CHEF) electrophoresis according to the protocol described previously for Acremonium chrysogenum (Walz & Kück, 1991).

Identification and isolation of genes Mating type genes were identified using a genome sequence of a Penicillium species as a source or by performing PCR with the primers 4736-f and 2756-r, which are located within the homologous sequences normally flanking the MAT genes in ascomycetes. Consequently, a primer-walking strategy was used to sequence the whole ORF of the mating type gene. In cases IV. Mahmoudjanlou et al. 2020 6 where genome sequences were lacking, primers were designed based on mating type sequences from closely related species.

Phylogenetic analysis To re-evaluate the species identification, all strains analyzed in this investigation (Supplemental Table 1) were subjected to molecular analysis, using DNA sequences from the internal transcribed spacer (ITS) DNA (Glass & Donaldson, 1995; Schoch et al., 2012), the ß- tubulin gene (Glass & Donaldson, 1995), and the mating type genes. For phylogenetic analysis, we used the program MEGA7 (Kumar et al., 2016). The protein-coding sequences were aligned by ClustalW software in the MEGA7 package and the phylogenetic trees were constructed using the maximum likelihood method based on the Tamura-Nei model (Tamura & Nei, 1993). Bootstrap values were based on 1000 replicates. For phylogenetic trees of MAT genes, we used the sequence of the MAT gene-encoded transcription factors. The frequency of the taxa clustering was set to 100.

Bioinformatics and programs Different sequences for phylogenetic analysis were obtained from different online databases, including Joint Genome Institute (JGI) (https://genome.jgi.doe.gov/portal/), National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) and Ensembl Genome Browser (https://www.ensembl.org/index.html). For this purpose, the local BLAST tool or whole-genome sequencing (WGS) pipeline was used. For in silico cloning strategies, we used SnapGene version 5.0, GSL Biotech LLC program (https://www.snapgene.com/). Alignment of protein sequences was illustrated by the software jalview (Clamp et al., 2004).

In vitro recombinant technology Escherichia coli K12 XL1-Blue MRF’ cells were used for cloning. Cloning and propagation of recombinant plasmids were performed using standard protocols (Sambrook & Russell, 2001). The plasmids used in this investigation are given (Supplemental Table 4).

DNA-meditated fungal transformation DNA-mediated fungal transformation was performed according to published methods (Mahmoudjanlou et al., 2019). Transgenic fungal strains were verified by PCR analysis, Southern blotting and hybridization, as described previously (Birgit Hoff et al., 2010; Sambrook & Russell, 2001).

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Protein extraction and Western analysis Protein extraction used the method previously described, with slight modifications (Bayram et al., 2008). Briefly, mycelia were filtered and ground in liquid nitrogen to prepare crude extracts in extraction buffer (100 mM Tris-HCl pH 7.6, 250 mM NaCl, 10% glycerol, 0.05% NP-40, 1 mM EDTA, 2 mM DTT) supplemented with protease inhibitor cocktail Set IV (Calbiochem, Merck). The subsequent 30 min centrifugation of mycelia at 11,000 rpm and 4°C allows the release of total proteins into the supernatant. Separation of proteins was performed using SDS- PAGE in the vertical electrophoresis system MiniPROTEAN Tetra Cell (BioRad). Western blotting was performed as described previously (Steffens et al., 2016), using JL-8 anti-GFP monoclonal antibody (JL-8) (Takara), and anti-mouse IgG, HRP-linked second antibody (Cell Signaling Technology) to detect EGFP or EGFP-MAT1-1-1 from P. brevicompactum in total protein extracts.

Pellet formation For the pellet quantification assay, we used the method described recently for P. chrysogenum (Böhm et al., 2013). The pellet size of different strains obtained from two independent experiments was statistically analyzed by R software (https://www.r-project.org/) and ggplot package was used to illustrate the violin plot-based results.

Microscopic investigations For the microscopic investigation, strains were grown on CCM-coated slides at 27 °C and constant light for 20 h. For microscopy of germinating spores, we used the AxioPhot microscope (Zeiss) connected to an AxioCam color and ZEN software to compose images (version 2.3, Zeiss). For localization studies, we performed a differential interference contrast (DIC)- and fluorescence microscopy using an AxioImager microscope (Zeiss) with a Photometrix Cool SnapHQ camera (Roper Scientific) and the software MetaMorph (version 7.7, Universal Imaging). To obtain the fluorescence images, a SPECTRA x 6 LCR SA LED lamp (lumencor) was used for the excitation of fluorophores. DAPI staining detection was carried out by Chroma filter set 31000v2 (excitation filter D350/50, emission filter D460/50, beam splitter 400dclp). Enhanced green fluorescent protein (EGFP) (Cinelli et al., 2000) was detected using the filter set 49002 (excitation filter HQ470/40, emission filter HQ525/50, beam splitter T495LPXR).

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Statistics The violin plot results from pellet quantification were analyzed for the significance of differences between the groups using the ANOVA model in R.

Results

Molecular organization and phylogeny of P. brevicompactum MAT1-1 and MAT1-2 loci Sequences adjacent to MAT loci are highly conserved among ascomycetes and carry genes for cytoskeleton assembly control factor SLA2 and DNA lyase APN2 (Coppin et al., 1997; Metzenberg & Glass, 1990; S. Pöggeler, 1999). To investigate whether MAT genes also exist in P. brevicompactum, we designed PCR primers based on conserved sequences in P. chrysogenum and related Penicillium species. Using genomic DNA from strain CBS 257.29 and CBS 317.59 together with oligonucleotides 4736-f and 2756-r for PCR analysis, we amplified a fragment of about 3.8 kb, carrying either the MAT1-1 or MAT1-2 locus.

Cloning and primer walking DNA sequencing revealed the genomic organization of the MAT1- 1-1 and MAT1-2-1 open reading frames (Fig. 1). The MAT1-1-1 gene has a size of 1,138 bp, carrying an ORF for 362 amino acids, which is interrupted by a 49 bp intron. The intron interrupts the sequence encoding the DNA binding α-domain. The MAT 1-2-1 region is 1000 bp and carries two introns of 52 and 51 bp. 3´ and 5´ flanking sequences showed a similarity of more than 95% between MAT1-1 and MAT1-2 loci. When cDNA was generated using RT- PCR amplification, the amplified fragments were smaller than the PCR products generated with genomic template DNA (Fig. 1). Thus, we not only verified the presence of the predicted introns, but also found evidence for the transcriptional expression of both mating type loci in the corresponding strains.

Sequence alignment of the mating type-encoded transcription factors In ascomycetes, mating type locus-encoded transcription factors control the expression of sex- associated genes as well as genes involved in metabolism and development (Becker et al., 2015). These transcription factors usually carry a DNA binding domain with a conserved motif. While MAT1-1-1 transcription factor contains an alpha domain, MAT1-2-1 proteins have a high mobility group (HMG)-domain (Martin et al., 2010).

To verify the similarity of the whole protein sequence of both transcription factors among Penicillium, we performed multiple alignments of amino acid sequences for 10 species. DNA- binding MATalpha-HMG and MATA-HMG domains were highly conserved in all 10 species (Fig. 2). The MATalpha-HMG domains showed an overall identity of 43% and a similarity of IV. Mahmoudjanlou et al. 2020 9

65%. The identity of the MATA-HMG domains was about 53%. The poorest similarity was observed when P. citrinium sequences were compared with all other domains. Overall, MAT1- 1-1 seems to be more conserved than MAT1-2-1. Both transcription factors show higher similarity at the N-terminus regions of the DNA-binding domains. For MAT1-1-1 we observed a highly conserved region of 86 amino acid residues at the N-terminus.

We calculated the sequence identities and similarities using P. brevicompactum as a reference. Although the C-terminal regions of all investigated MAT1-1-1 proteins display similarities, the corresponding region of MAT1-2-1-encoded transcription factors is less conserved. N-terminal regions of MAT1-1-1 displayed identities ranging from 73% for P. bialowiezense to 24% for P. citrinum. The identity of the C-terminal region was 64% for P. bialowiezense and 13% for P. citrinum. For MAT1-2-1 N-terminal regions, we observed the highest identity of 54% for P. bialowiezense and the lowest identity of 20% for P. citrinum. These similarity values were about 10% lower for C-terminal regions. Interestingly, the protein sequences of P. rubens and P. chrysogenum, which are described as two distinct species (Jos Houbraken et al., 2011), showed 100% identity.

The transcription factors encoded by MAT1-1-1 and MAT1-2-1 from most Penicillium species have a rather similar C- and N-terminal region, with similarity values between 100% and 79% for the C- and N-terminal regions of both mating type transcription factors. Exceptions are found when comparing sequences from species of the Penicillium section Coronata and P. citrinum. For example, the C-terminal sequence of MAT1-2-1 from P. brevicompactum shows a low 10% identity and 26% similarity to the corresponding P. chrysogenum sequence.

Phylogenetic and karyotype analysis of Penicillium strains To ensure the correct taxonomical classification of the Penicillium cultures used in this study, wild-type strains received from international type culture collections were tested by PCR and phylogenetic analyses using the widely used markers β-tubulin and internal transcribed spacer (ITS) (R.A. Samson et al., 2004; P. Skouboe et al., 1996). In total, partial β-tubulin sequences of 72 strains were either generated or obtained from databases as indicated in (Supplemental Table 1). Sequence comparison revealed that 7 out of 28 P. brevicompactum strains, obtained from culture collections, were misidentified as P. brevicompactum and actually belong to the closely related species P. bialowiezense (= P. biourgeianum) (four strains), P. olsonii (two strains), or P. chrysogenum (one strain). IV. Mahmoudjanlou et al. 2020 10

The results of the maximum likelihood analysis of β-tubulin (Fig. 3) confirms a previous study, which showed that P. brevicompactum, P. bialowiezense, and P. olsonii form a monophyletic group (74%), representing Penicillium subgenus Penicillium, sect. Coronata (Frisvad & Samson, 2004; Peterson, 2004; Visagie et al., 2014). This section is clearly separated from the other phylogenetic groups with representatives from five different sections of the Penicillium subgenus. Within the sect. Coronata, separation of P. olsonii is supported by a high bootstrap value (93%), while separation of P. brevicompactum and P. bialowiezense is weak. Interestingly, the β-tubulin sequence of the homothallic P. egyptiacum strain CBS 244.32, which is the ex-type strain of this species (Visagie et al., 2014), is incorrectly grouped away from the other members of the section Chrysogena next to P. roqueforti. The β-tubulin alignment sometimes results in low bootstrap values, indicating contradictory data in some cases.

After species identification, the mating-type loci were amplified, and the nucleotide sequences were determined by Sanger sequencing. Phylogenetic trees based on the coding sequence of the MAT1-1-1 and MAT1-2-1 genes generated maximum likelihood trees of 35 MAT1-1-1 sequences obtained from the Penicillium strains (Fig. 4). The various Penicillium subgenus Penicillium sections can be clearly separated from each other due to their high bootstrap values of ≥99%. In particular, the distinction inside the sect. Coronata is now reliable (88-99%) compared to the β-tubulin tree. Remarkably, strains from P. chrysogenum and P. rubens appear as a monophyletic group. Furthermore, strain P. egyptiacum CBS 244.32 is now correctly grouped within the section Chrysogena. Similarly, the MAT1-2-1 maximum likelihood tree calculated based on 37 MAT1-2-1 coding sequences, shows consistent results (Fig. 4). In summary, MAT sequences are appropriate for constructing phylogenetic trees with reliable bootstrap values of more than 80%.

Unexpectedly, all P. brevicompactum strains, even with an identical geographical origin, displayed very different morphological phenotypes (Supplemental Fig. 1). To exemplify the karyotype of P. brevicompactum, six strains from major type culture collections were selected for pulsed-field gel electrophoresis. The electrophoretic conditions were chosen according to our previous experience with successfully separating rather large chromosomes above 8 Mb (Stimberg et al., 1992; Walz & Kück, 1991). The electrophoretic karyotyping was extended further by locating MAT genes on separate chromosomes using Southern hybridization analysis. The morphological heterogeneity of all strains is reflected by the heterogeneous karyotypes (Fig. 5). Strains vary not only in chromosome numbers, but also in genome sizes IV. Mahmoudjanlou et al. 2020 11 between 28 to 35 Mbp. These values are close to the published genome size for P. brevicompactum strain AgRF18 and 1011305, which are 31.68 and 32.11 Mbp, respectively (https://genome.jgi.doe.gov/portal/).

Construction of the MAT1-1-1 and MAT1-2-1 deletion strains Next we investigated the function of the MAT genes in MAT1-1 (CBS 257.29) and MAT1-2 (CBS 110068) strains, which were selected due to their rather high titer of mycophenolic acid (Mahmoudjanlou et al., 2019). We recently reported a successful DNA-mediated transformation system for P. brevicompactum using a codon-optimized nourseothricin resistant gene (Mahmoudjanlou et al. 2019). This modified gene is contained in vector pPtrpC-Pbnat1, which was used for the targeted deletion of genes by homologous recombination. We recently described the construction of a MAT1-2-1 deletion strain (Mahmoudjanlou et al. 2019). Here, we used a similar strategy to construct a MAT1-1-1 deletion strain. In total, out of 80 fungal transformants, we obtained a single strain that had the MAT1-1-1 gene substituted through homologous recombination by the nourseothricin resistance cassette. The site-specific substitution was further verified by PCR analysis and Southern hybridization (Supplemental Fig. 2). As an additional control for our functional analysis, the MAT1-1-1 or MAT1-2-1 deletion strains were complemented with the corresponding wild type MAT genes under the control of a gpd promoter from A. nidulans. In both cases the MAT genes were fused C- terminally to egfp reporter gene (Supplemental Fig. 2).

Functional analysis of mating type loci We used Western analysis to confirm the synthesis of the epitope-tagged proteins EGFP- MAT1-1-1 and EGFP-MAT1-2-1 in crude protein extracts from the corresponding recombinant strains. GFP-specific antibodies detected the protein in the complemented strains, as well as in the control strain expressing the gfp gene only (Fig. 6A). EGFP-MAT1-1-1 was further characterized by fluorescence microscopy of germinating conidiospores. The protein co-localized with the fluorescence dye DAPI that stains the nuclear DNA, as well as mitochondrial DNA in dividing mitochondria (Fig. 6B).

For a functional analysis, we used both mating type deletion strains (ΔMAT1-1-1; ΔMAT1-2- 1) to measure conidiospore formation, pellet sizes in liquid cultures, germ tube formation on solid media, and stress reaction on high salt media. The observed phenotypes from the mutant strains were compared to the corresponding wild-type strains. For the MAT1-2-1 deletion IV. Mahmoudjanlou et al. 2020 12 mutant, we observed no significant differences compared to the wild type (Supplemental Fig. 3). However, in the case of ΔMAT1-1-1, we found significant deviations from the wild type.

The MAT1-1-1 deletion strain merited more detailed statistical analysis of the number of conidiospores on solid media, the size of pellets in liquid cultures and the number of germ tubes in germinating conidia. The corresponding parameters were not only compared to the wild type, but also to two complemented ΔMAT1-1-1::egfp-MAT1-1-1 strains. Quantification of conidiospores from P. brevicompactum showed that sporulation is not light-dependent (Fig. 7; (Supplemental Fig. 4). However, the number of conidiospores increased by about 50% in ΔMAT1-1-1 compared to the reference strains. Re-introduction of the MAT1-1-1 wild-type gene led to wild-type-like sporulation.

Another difference was seen when the strains were grown for three days in liquid media. The deletion strain generated smaller pellets (Ø 1-1.5 mm) than the reference strains (Ø 2 mm) (Fig. 8). This difference is most probably a consequence of delayed germ tube formation of conidiospores after 20 hours growth on solid media: 60% of germinating conidia from wild type and complementation strains generated three or more germ tubes, while the remainder had only two germ tubes (Fig. 9). In contrast, the MAT1-1-1 deletion strain showed considerably fewer conidia with three or more germ tubes (Fig. 9).

Discussion In this study our molecular analysis of mating type loci from 22 Penicillium brevicompactum strains revealed that all strains carried a transcriptionally and translationally expressed MAT1- 1 or MAT1-2 locus encoding the corresponding alpha domain or HMG domain transcription factor. That we observed almost equal numbers of MAT1-1 and MAT1-2 strains suggest a putative sexual cycle in P. brevicompactum. Phenotypic functional analysis of deletion strains confirmed that the MAT1-1-1 transcription factor, plays an important role in developmental processes of P. brevicompactum.

Further, our phylogenetic analysis indicates that mating type sequences are excellent marker molecules for identifying Penicillia at the species level. Traditionally, morphological concepts were used to classify Penicillium species, but DNA sequencing approaches provided multiple evidence for the miss-classification of morphologically similar species (Visagie et al., 2014). Some molecular markers are generally used for species identification such as ITS and β-tubulin (BenA). However, there are some obstacles regarding their usage. ITS is the most commonly used molecular marker for fungi; however, it is not distinct enough to distinguish closely IV. Mahmoudjanlou et al. 2020 13 related species (Pernille Skouboe et al., 1999). Therefore, open-access gene banks include large numbers of miss-identified species (Kõljalg et al., 2013; Santamaria et al., 2012). β-tubulin (BenA) is solid for species identification but contains a huge portion of confusing or conflicting alignment sites, leading to erroneous alignments making it sometimes difficult for phylogenetic analysis (Visagie et al., 2014).

Our approach to use the mating type genes for taxonomic classification is promising since their apparent faster evolution compared to other regions of the genome makes them useful tools for constructing phylogenetic trees. Previous studies have reported high interspecies differences and low intraspecific variations for mating type genes, suggesting their relevance for phylogenetic investigations (López-Villavicencio et al., 2010; S. Pöggeler et al., 2011). Based on MAT and beta-tubulin sequences from 72 strains, here we provide a comparative phylogenetic analysis that allows one to distinguish between highly related Penicillium species.

Our functional analyses of the MAT1-1-1 gene suggest that this transcription factor controls other developmental processes besides sexuality. This agrees with previous functional studies of MAT transcription factors in P. chrysogenum, which revealed that they not only mediate sexual propagation, but also affect different traits of biotechnological importance, including asexual conidiogenesis, pellet morphology, hyphal morphology, and penicillin biosynthesis (Böhm et al., 2013). Our data from conidiospore quantification, pellet size distribution, and germ tube formation are similar to previous observations made in P. chrysogenum. However, the pellet formation data point to significant differences between the two Penicillium species. Whereas in P. chrysogenum deletion and overexpression of MAT1-1-1 resulted in the formation of bigger pellets and an increased number of germ tubes, in P. brevicompactum a smaller pellet size, and reduced germ tube formation were observed. These features may have applied significance, since previously it was shown that aggregation of germinating spore hyphae and smaller pellets contribute to elevated MPA production (Doerfler et al., 1978; Ozaki et al., 1987). Another distinct difference is the lack of any light-dependent conidiospore formation in P. brevicompactum, which was described for a wide range of filamentous fungi (Böhm et al., 2013; B. Hoff et al., 2010; Krijgsheld et al., 2013). These phenotypic descriptions point to similar, but also distinct different functions of homologous mating type transcription factors.

In our study, amino acid sequence alignment of MAT transcription factors revealed a very high conserved region within the DNA-binding domains of both transcription factors; however, adjacent N- and C-terminal regions showed less similarity. Differences were more prominent IV. Mahmoudjanlou et al. 2020 14 at the C-terminus. These deviating amino acid sequences might explain the observed functional differences between MAT transcription factors from P. brevicompactum and P. chrysogenum.

Transcription factors control gene expression by deciphering upstream regulatory regions of a gene, often for a wide range of genes. Their functions depend on sequence-specific DNA recognition. The flexibility of transcription factors and their binding sites can be explained by direct and indirect interactions with other transcription factors and cofactors (Slattery et al., 2014). The DNA binding domains of transcription factors are usually not the only mediators of cooperative protein-protein interactions; the C-terminal domains may also contain protein- interacting sites that are integral for accuracy of transcriptional regulation. For instance, the C- terminal region of the human HMG box containing SOX9 transcription factor contains two transactivation residues, whose interaction with yet unexploited partner proteins controls the efficiency of SOX9 for transcriptional activation. Moreover, SOX9 regulates the transcription of the AMH (anti-Mullerian hormone) gene by forming an associated complex with other transcription factors such as WT1. This association between SOX9 and WT1 is promoted by the C-terminal sequence of HSP70, which interacts with a region of 100 amino acids at the C- terminal domain of SOX9, lacking any structural domain (Harley et al., 2003; Maheswaran et al., 1998). Thus, the C-terminal amino acid differences in MAT transcription factors from P. brevicompactum and P. chrysogenum may contribute to the differential functions of the transcription factors from both species.

When we determined the mating type loci from 22 natural isolates of P. brevicompactum collected at diverse global locations, we found that 8 carry the MAT1-1 locus, and 14 the MAT1- 2 locus. This finding suggests that a putative sexual cycle exists in P. brevicompactum.

Although a few exceptions were reported, when natural populations of asexual ascomycetes were previously investigated for the global geographical distribution of MAT genes, usually a 1:1 distribution was found, providing indirect evidence for a heterothallic breeding system (Dyer and Kück 2017). In some instances, these assumptions were further confirmed by inducing a sexual life cycle in the laboratory (Böhm et al., 2013; O'Gorman et al., 2009).

Since we also observed an almost equal number of MAT1-1 and MAT1-2 strains, and their geographic origin shows a global distribution (Supplemental Fig. 4), this suggests the presence of a heterothallic sexual life cycle for P. brevicompactum in nature. However, our different attempts to induce a sexual cycle in P. brevicompactum have so far not been successful (Mahmoudjanlou, unpublished data). This difficulty could be due to the different karyotypes IV. Mahmoudjanlou et al. 2020 15 and chromosomal arrangements of mating partners, which leads to sexual incompatibility and prevents sexual fertility and meiotic recombination (Dahlmann et al., 2015). Previously, intraspecific infertility phenotypes were observed for Sordaria macrospora strains with heterogeneous karyotypes (Pöggeler et al., 2000). Karyotype heterogeneity and chromosomal rearrangement were also believed to be the barrier to mating of the industrial P. rubens strain P2niaD18 with the MAT1-2 wild type Pc3 (Böhm et al., 2015).

Previously, a screen with 50 Aspergillus fumigatus strains from various geographic regions resulted in the discovery of a highly fertile (super-mater) pair that can complete the sexual cycle in a rather short period of four weeks (Sugui et al., 2011). Therefore, attempts are currently underway to screen more natural isolates from P. brevicompactum in order to detect super-mating type strains for inducing a sexual cycle.

Conclusion Mating type-encoded transcription factors determine the sexual life cycle in all ascomycetes. Here, we isolated and characterized the mating type loci from the biotechnologically relevant filamentous fungus Penicillium brevicompactum. Using the MAT sequences from 22 wild type strains of P. brevicompactum, our phylogenetic analysis indicates that mating type sequences can provide valuable marker molecules as tools for identifying Penicillia at the species level. Further functional analyses revealed that the MAT1-1-1 transcription factor seems to control major developmental processes. Taken together, we can conclude that P. brevicompactum has a potential heterothallic sexual life cycle, which could be a useful tool for constructing recombinant strains in biotechnical applications.

Acknowledgements We thank Ingeborg Godehardt and Susanne Schlewinski for superb technical help. This study was funded by the German Research Foundation (DFG) (Bonn Bad-Godesberg, Germany) (KU 517/ 15-1).

IV. Mahmoudjanlou et al. 2020 16

Tables

Table 1. Chromosome number and size for different P. brevicompactum wild type strains investigated by CHEF gel electrophoresis strain CBS CBS CBS CBS CBS CBS 119375 317.59 257.29 256.31 629.66 110069

Chromosome size 13 11 13 11 13 13 (Mpb) 8 9.5 8 9.5 8 9.5 4,5 4.5 5.7 4.5 5 4.5 3,5 3 4.5 3.5 3.5 4 3.5 3 3,5 Genome size 29 28 34.7 28.5 32.5 34.5 Number of 4 4 5 4 5 5 chromosomes

IV. Mahmoudjanlou et al. 2020 17

Figures

Fig. 1. Mating type locus organization and transcriptional expression of MAT loci from P. brevicompactum. A.) Physical map of the MAT1-1 and MAT1-2 loci. Both are flanked by almost identical genomic regions carrying the genes for APN2 and SLA2. B.) PCR amplification using genomic DNA “gDNA”, and cDNA “+”, received by reverse transcription from total RNA as a template. “-“ indicates control samples, where no template cDNA was added. IV. Mahmoudjanlou et al. 2020 18

Fig. 2. Alignment of MAT protein sequences from 10 Penicillium species. The darkness of the grey color corresponds to the sequence similarity. The Conserved DNA binding alpha domain is shown in blue. DNA binding HMG-domain is illustrated in red. All species show a high homology in the alpha domain box and N-terminus regions. Less similarity was observed at the C-terminus.

IV. Mahmoudjanlou et al. 2020 19

Fig. 3. Identification of 22 P. brevicompactum strains based on β-tubulin sequences. The mating types of P. brevicompactum strains are indicated in blue (MAT1-1) and red (MAT1-2). This tree shows three distinct clades for 10 Penicillium species. The first clade compromises P. brevicompactum, P. bialowiezense and P. olsonii. β-tubulin sequences of P. citrinum distributed in a monophyletic clade. The other Penicillium species built a common group. IV. Mahmoudjanlou et al. 2020 20

Fig. 4. Phylogenetic trees of MAT genes from 10 Penicillium species. 1000 bootstrap replicates were used. All Penicillium species are clustered in three clades. P. brevicompactum, P. bialowiezense, and P. olsonii reside in the same clade. P. citrinum is located in a distinct clade from other species. The MAT genes from 6 other species show a common ancestry relationship.

IV. Mahmoudjanlou et al. 2020 21

Fig. 5. CHEF gel electrophoresis to separate chromosomal DNA from P. brevicompactum strains as indicated. The MAT1-1-1 (blue stars), and MAT1-2-1 (red stars) probes hybridized as indicated.

IV. Mahmoudjanlou et al. 2020 22

Fig. 6. Translational expression and cellular localization of mating type gene encoded transcription factor MAT1-1-1. (A) Separation of crude protein extracts from recombinant P. brevicompactum strains. Transformation of CBS 257.29 with pN-EGFP results in recombinants strains, indicated with the designation “PT51”. Protein from wild type (CBS 257.29) serves as a control. Complementation of ΔMAT1-1-1 with pPbMAT1-1-1-comp generates transformants designated “PT52”. For protein detection, a GFP specific antibody was used in western blot analysis. (B) Co-localization of the GFP-labeled MAT1-1-1 protein and the DAPI stained nuclei (white arrows) in germ tubes. In addition to nuclei, DAPI stains dividing mitochondria in fast-growing cells. The bar represents 20 μm.

IV. Mahmoudjanlou et al. 2020 23

Fig. 7. Quantification of conidiospore formation by parental and recombinant MAT1-1 strains after 168 h growth on CCM medium in the dark and light. Error bars show mean ± SD (n=3) from three independent experiments. The statistical significance was tested by t-test. Three asterisks represent the P≤ 0.001.

IV. Mahmoudjanlou et al. 2020 24

Fig. 8. Measurement of pellet size in liquid media (CCM), grown for 72 h. CBS 257-29 (A) schematic illustration of pellet formation (B) statistical demonstration of pellet size. The box inside the violin demonstrates 50% of measured data and the horizontal line in the box represents the median. The violin plot shows the complete distribution of the data and illustrates their density at diverse values. IV. Mahmoudjanlou et al. 2020 25

Fig 9. Germ tube formation of conidia after growth on solid CCM media for 20 h. The number of germ tubes from 300-350 germinating conidia was counted.

References Bayram, O., Krappmann, S., Ni, M., Bok, J. W., Helmstaedt, K., Valerius, O., Braus- Stromeyer, S., Kwon, N. J., Keller, N. P., Yu, J. H., & Braus, G. H. (2008). VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science, 320(5882), 1504-1506. doi:10.1126/science.1155888 Becker, K., Beer, C., Freitag, M., & Kück, U. (2015). Genome-wide identification of target genes of a mating-type alpha-domain transcription factor reveals functions beyond sexual development. Mol Microbiol, 96(5), 1002-1022. doi:10.1111/mmi.12987 Böhm, J., Dahlmann, T. A., Gümüser, H., & Kück, U. (2015). A MAT1-2 wild-type strain from Penicillium chrysogenum: functional mating-type locus characterization, genome sequencing and mating with an industrial penicillin-producing strain. Mol Microbiol, 95(5), 859-874. doi:10.1111/mmi.12909 Böhm, J., Hoff, B., O'Gorman, C. M., Wolfers, S., Klix, V., Binger, D., Zadra, I., Kürnsteiner, H., Pöggeler, S., Dyer, P. S., & Kück, U. (2013). Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. Proc Natl Acad Sci U S A, 110(4), 1476-1481. doi:10.1073/pnas.1217943110 Cinelli, R. A., Ferrari, A., Pellegrini, V., Tyagi, M., Giacca, M., & Beltram, F. (2000). The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and IV. Mahmoudjanlou et al. 2020 26

localization: local fluorescence study at the single-molecule level. Photochem Photobiol, 71(6), 771-776. doi:10.1562/0031-8655(2000)071<0771:tegfpa>2.0.co;2 Clamp, M., Cuff, J., Searle, S. M., & Barton, G. J. (2004). The Jalview Java alignment editor. Bioinformatics, 20(3), 426-427. doi:10.1093/bioinformatics/btg430 Coppin, E., Debuchy, R., Arnaise, S., & Picard, M. (1997). Mating types and sexual development in filamentous ascomycetes. Microbiol Mol Biol Rev, 61(4), 411-428. Dahlmann, T. A., Böhm, J., Becker, K., & Kück, U. (2015). Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains. Curr Genet, 61(4), 679- 683. doi:10.1007/s00294-015-0497-7 Doerfler, D. L., Nulton, C. P., Bartman, C. D., Gottlieb, F. J., & Campbell, I. M. (1978). Spore germination, colony development, and secondary metabolism in Penicillium brevicompactum: a radiogas chromatographic and morphological study. Can J Microbiol, 24(12), 1490-1501. doi:10.1139/m78-239 Dyer, P. S., & Kück, U. (2017). Sex and the imperfect fungi. Microbiol Spectr, 5(3). doi:doi:10.1128/microbiolspec.FUNK-0043-2017 Frisvad, J. C., & Samson, R. A. (2004). Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol, 49, 1-174. Glass, N. L., & Donaldson, G. C. (1995). Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol, 61(4), 1323-1330. Harley, V. R., Clarkson, M. J., & Argentaro, A. (2003). The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocr Rev, 24(4), 466- 487. doi:10.1210/er.2002-0025 Hoff, B., Kamerewerd, J., Sigl, C., Mitterbauer, R., Zadra, I., Kürnsteiner, H., & Kück, U. (2010). Two components of a velvet-like complex control hyphal morphogenesis, conidiophore development, and penicillin biosynthesis in Penicillium chrysogenum. Eukaryot Cell, 9(8), 1236-1250. doi:10.1128/EC.00077-10 Hoff, B., Kamerewerd, J., Sigl, C., Zadra, I., & Kück, U. (2010). Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway. Appl Microbiol Biotechnol, 85(4), 1081-1094. doi:10.1007/s00253-009-2168-4 Houbraken, J., Frisvad, J. C., & Samson, R. A. (2011). Fleming's penicillin producing strain is not Penicillium chrysogenum but P. rubens. IMA Fungus, 2(1), 87-95. doi:10.5598/imafungus.2011.02.01.12 Houbraken, J., & Samson, R. A. (2011). Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Stud Mycol, 70(1), 1-51. doi:10.3114/sim.2011.70.01 IV. Mahmoudjanlou et al. 2020 27

Kõljalg, U., Nilsson, R. H., Abarenkov, K., Tedersoo, L., Taylor, A. F. S., Bahram, M., Bates, S. T., Bruns, T. D., Bengtsson-Palme, J., Callaghan, T. M., Douglas, B., Drenkhan, T., Eberhardt, U., Dueñas, M., Grebenc, T., Griffith, G. W., Hartmann, M., Kirk, P. M., Kohout, P., Larsson, E., Lindahl, B. D., Lücking, R., Martín, M. P., Matheny, P. B., Nguyen, N. H., Niskanen, T., Oja, J., Peay, K. G., Peintner, U., Peterson, M., Põldmaa, K., Saag, L., Saar, I., Schüßler, A., Scott, J. A., Senés, C., Smith, M. E., Suija, A., Taylor, D. L., Telleria, M. T., Weiss, M., & Larsson, K.-H. (2013). Towards a unified paradigm for sequence-based identification of fungi. Mol Ecol, 22(21), 5271-5277. doi:10.1111/mec.12481 Krijgsheld, P., Bleichrodt, R., van Veluw, G. J., Wang, F., Müller, W. H., Dijksterhuis, J., & Wösten, H. A. (2013). Development in Aspergillus. Stud Mycol, 74(1), 1-29. doi:10.3114/sim0006 Kück, U., Pöggeler, S., Nowrousian, M., Nolting, N., & Engh, I. (2009). Sordaria macrospora, a model system for fungal development. In W. D. e. P. a. G. Anke T. (Ed.), The Mycota XV (A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research) (Vol. 15, pp. 17-39). Heidelberg, New York, Tokyo: Springer. Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol, 33(7), 1870-1874. doi:10.1093/molbev/msw054 Lecellier, G., & Silar, P. (1994). Rapid methods for nucleic acids extraction from Petri dish- grown mycelia. Curr Genet, 25(2), 122-123. doi:10.1007/BF00309536 López-Villavicencio, M., Aguileta, G., Giraud, T., de Vienne, D. M., Lacoste, S., Couloux, A., & Dupont, J. (2010). Sex in Penicillium: Combined phylogenetic and experimental approaches. Fungal Genetics and Biology, 47(8), 693-706. doi:https://doi.org/10.1016/j.fgb.2010.05.002 Maheswaran, S., Englert, C., Zheng, G., Lee, S. B., Wong, J., Harkin, D. P., Bean, J., Ezzell, R., Garvin, A. J., McCluskey, R. T., DeCaprio, J. A., & Haber, D. A. (1998). Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70. Genes Dev, 12(8), 1108-1120. doi:10.1101/gad.12.8.1108 Mahmoudjanlou, Y., Hoff, B., & Kück, U. (2019). Construction of a codon-adapted nourseotricin-resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum. J Fungi (Basel), 5(4). doi:10.3390/jof5040096 Martin, T., Lu, S. W., van Tilbeurgh, H., Ripoll, D. R., Dixelius, C., Turgeon, B. G., & Debuchy, R. (2010). Tracing the origin of the fungal alpha1 domain places its ancestor in the HMG-box superfamily: implication for fungal mating-type evolution. PLoS One, 5(12), e15199. doi:10.1371/journal.pone.0015199 IV. Mahmoudjanlou et al. 2020 28

Metzenberg, R. L., & Glass, N. L. (1990). Mating type and mating strategies in Neurospora. Bioessays, 12(2), 53-59. doi:10.1002/bies.950120202 Ni, M., Feretzaki, M., Sun, S., Wang, X., & Heitman, J. (2011). Sex in fungi. Annu Rev Genet, 45, 405-430. doi:10.1146/annurev-genet-110410-132536 Nielsen, J. C., Grijseels, S., Prigent, S., Ji, B., Dainat, J., Nielsen, K. F., Frisvad, J. C., Workman, M., & Nielsen, J. (2017). Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species. Nature Microbiology, 2, 17044. doi:10.1038/nmicrobiol.2017.44 O'Gorman, C. M., Fuller, H., & Dyer, P. S. (2009). Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature, 457(7228), 471-474. doi:10.1038/nature07528 Ozaki, H., Kubota, K., & Takahashi, H. (1987). Effects various adsorbents on mycelium formation and mycophenolic acid production by Penicillium brevicompactum. Agricultural and Biological Chemistry, 51, 2503-2508. doi:10.1080/00021369.1987.10868426 Peterson, S. W. (2004). Multilocus DNA sequence analysis shows that Penicillium biourgeianum is a distinct species closely related to P. brevicompactum and P. olsonii. Mycol Res, 108(Pt 4), 434-440. doi:10.1017/s0953756204009761 Pitt, J. I. (1979). The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London ; New York: Academic Press. Pöggeler, Masloff, Jacobsen, & Kück. (2000). Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora. Journal of Evolutionary Biology, 13(2), 281-289. doi:10.1046/j.1420-9101.2000.00174.x Pöggeler, S. (1999). Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes. Curr Genet, 36(4), 222-231. doi:10.1007/s002940050494 Pöggeler, S. (2001). Mating-type genes for classical strain improvements of ascomycetes. Appl Microbiol Biotechnol, 56(5-6), 589-601. doi:10.1007/s002530100721 Pöggeler, S., O'Gorman, C. M., Hoff, B., & Kück, U. (2011). Molecular organization of the mating-type loci in the homothallic Ascomycete Eupenicillium crustaceum. Fungal Biol, 115(7), 615-624. doi:10.1016/j.funbio.2011.03.003 Ropars, J., Lopez-Villavicencio, M., Dupont, J., Snirc, A., Gillot, G., Coton, M., Jany, J. L., Coton, E., & Giraud, T. (2014). Induction of sexual reproduction and genetic diversity in the cheese fungus Penicillium roqueforti. Evol Appl, 7(4), 433-441. doi:10.1111/eva.12140 Sambrook, J., & Russell, D. W. (2001). Molecular cloning: a laboratory manual (3 ed. Vol. 1). Cold Spring Harbour, New York: Cold Spring Harbour Laboratory Press. Samson, R. A., & Pitt, J. I. (2000). Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Australia: Harwood Academicn Publishers. IV. Mahmoudjanlou et al. 2020 29

Samson, R. A., Seifert, K. A., Kuijpers, A. F. A., Houbraken, J. A. M. P., & Frisvad, J. C. (2004). Phylogenetic analysis of Penicillium subgenus Penicillium using partial ß- tubulin sequences. Stud Mycol, 49, 175-200. Santamaria, M., Fosso, B., Consiglio, A., De Caro, G., Grillo, G., Licciulli, F., Liuni, S., Marzano, M., Alonso-Alemany, D., Valiente, G., & Pesole, G. (2012). Reference databases for taxonomic assignment in metagenomics. Brief Bioinform, 13(6), 682- 695. doi:10.1093/bib/bbs036 Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., Chen, W., Fungal Barcoding, C., & Fungal Barcoding Consortium Author, L. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A, 109(16), 6241-6246. doi:10.1073/pnas.1117018109 Skouboe, P., Boysen, M., Pedersen, L. H., & Frisvad, J. C. (1996). Identification of Penicillium species using the internal transcribed spacer (ITS). In L. Rossen, V. Rubio, M. T. Dawson, & J. C. Frisvad (Eds.), Fungal identification techniques (pp. 160-164). Brussels, Belgium: European Commission. Skouboe, P., Frisvad, J. C., Taylor, J. W., Lauritsen, D., Boysen, M., & Rossen, L. (1999). Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate Penicillium species. Mycol Res, 103(7), 873-881. doi:10.1017/S0953756298007904 Slattery, M., Zhou, T., Yang, L., Dantas Machado, A. C., Gordan, R., & Rohs, R. (2014). Absence of a simple code: how transcription factors read the genome. Trends Biochem Sci, 39(9), 381-399. doi:10.1016/j.tibs.2014.07.002 Steffens, E. K., Becker, K., Krevet, S., Teichert, I., & Kück, U. (2016). Transcription factor PRO1 targets genes encoding conserved components of fungal developmental signaling pathways. Mol Microbiol, 102(5), 792-809. doi:10.1111/mmi.13491 Stimberg, N., Walz, M., Schörgendorfer, K., & Kück, U. (1992). Elecrtrophoretic karyotyping from Tolypocladium inflatum and six related strains allows differentiation of morphologically similar species. Appl Microbiol Biotechnol, 37, 485-489. Sugui, J. A., Losada, L., Wang, W., Varga, J., Ngamskulrungroj, P., Abu-Asab, M., Chang, Y. C., O'Gorman, C. M., Wickes, B. L., Nierman, W. C., Dyer, P. S., & Kwon-Chung, K. J. (2011). Identification and characterization of an Aspergillus fumigatus "supermater" pair. mBio, 2(6):e00234-11. doi:10.1128/mBio.00234-11 Tamura, K., & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees Mol Biol Evol, 103, 512-526. doi:10.1093/oxfordjournals.molbev.a040023 Taylor, J., Jacobson, D., & Fisher, M. (1999). The evolution of asexual fungi: Reproduction, Speciation and Classification. Annu Rev Phytopathol, 37, 197-246. doi:10.1146/annurev.phyto.37.1.197 IV. Mahmoudjanlou et al. 2020 30

Turgeon, B. G., & Yoder, O. C. (2000). Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genet Biol, 31(1), 1-5. doi:10.1006/fgbi.2000.1227 Visagie, C. M., Houbraken, J., Frisvad, J. C., Hong, S. B., Klaassen, C. H., Perrone, G., Seifert, K. A., Varga, J., Yaguchi, T., & Samson, R. A. (2014). Identification and nomenclature of the genus Penicillium. Stud Mycol, 78, 343-371. doi:10.1016/j.simyco.2014.09.001 Walz, M., & Kück, U. (1991). Polymorphic karyotypes in related Acremonium strains. Curr Genet, 19(2), 73-76. doi:10.1007/bf00326285 IV. Mahmoudjanlou et al. 2020 31

Supplementary material

Supplementary tables

Supplemental Table 1. List of 72 Penicillium wild type strains from 10 different Penicillium spp. used in this study. Aspergillus fumigatus served as a reference strain. Underlined were those strains, which were characterized by pulsed-field electrophoreses.

Species strain Reference species Reference of supplier origin mating type ß-tubulin

A. fumigatus AF293 Aspergillus fumigatus Nierman et al. (2005) Shrewsbury, UK, MAT1-2 Nierman et al. (2005)

P. bialowiezense CBS 108958 P. brevicompactum J.C. Frisvad Denmark MAT1-2 this study

P. bialowiezense CBS 112882 P. bialowiezense J.C. Frisvad (Samson et al., 2004) Denmark MAT1-2 (this study) this study

P. bialowiezense CBS 288.97 P. brevicompactum J.C. Frisvad Sweden MAT1-2 (this study) this study

P. bialowiezense CBS 671.95 P. brevicompactum H.A. van der Aa Netherlands MAT1-1 (this study) this study

P. bialowiezense IMI 380334 P. brevicompactum Paterson (2004) Great Britain MAT1-1 (this study) this study

P. brevicompactum Penbr2 (1011305) P. brevicompactum Ndagijimana et al. (2008) Italy MAT1-2 (JGI) JGI

P. brevicompactum AgRF18 P. brevicompactum JGI unknown MAT1-2 (JGI) JGI

P. brevicompactum CBS 110067 P. brevicompactum J.C. Frisvad Samson et al. (2004) Madird, New Mexico MAT1-2 (this study) this study

P. brevicompactum CBS 110068 P. brevicompactum M. Christensen, deposited by Wyoming MAT1-2 (this study) this study J.C. Frisvad Samson et al. (2004)

P. brevicompactum CBS 112476 P. brevicompactum J.C. Frisvad unknown MAT1-2 (this study) this study

P. brevicompactum CBS 119375 P. brevicompactum C. Trautmann Germany MAT1-2 (this study) this study

P. brevicompactum CBS 287.53 P. brevicompactum H.J. Swart South Africa MAT1-2 (this study) this study IV. Mahmoudjanlou et al. 2020 32

P. brevicompactum CBS 317.59 P. brevicompactum S. Abe (Frisvad & Samson, 2004) Japan MAT1-2 (this study) this study

P. brevicompactum CBS 480.84 P. brevicompactum J.C. Frisvad (Samson et al., 2004) Lyngby, Denmark MAT1-2 (this study) this study

P. brevicompactum CBS 629.66 P. brevicompactum unknown Connecticut MAT1-2 (this study) this study

P. brevicompactum DSM 62828 P. brevicompactum Bredemeier Germany MAT1-2 (this study) this study

P. brevicompactum IMI 380329 P. brevicompactum Paterson (2004) Great Britain MAT1-2 (this study) this study

P. brevicompactum IMI 380346 P. brevicompactum Paterson (2004) Great Britain MAT1-2 (this study) this study

P. brevicompactum IMI 380348 P. brevicompactum Paterson (2004) Great Britain MAT1-2 (this study) this study

P. brevicompactum CBS 256.31 P. brevicompactum C. Thom (Frisvad & Samson, 2004) Connecticut MAT1-1 (this study) this study

P. brevicompactum CBS 257.29 P. brevicompactum P. Biourge (Samson et al., 2004) Belgium MAT1-1 (this study) this study

P. brevicompactum CBS 110069 P. brevicompactum J.I. Pitt, deposited J.C. Frisvad Sydney, New South MAT1-1 (this study) this study (Samson et al., 2004) Wales

P. brevicompactum CBS 110070 P. brevicompactum B. Andersen, deposited by J.C. Frisvad Brazil MAT1-1 (this study) this study

P. brevicompactum CBS 110071 P. brevicompactum unknown unknown MAT1-1 (this study) this study

P. brevicompactum CBS 110072 P. brevicompactum P. Biourge, desposited by J.C. Frisvad Belgium MAT1-1 (this study) this study

P. brevicompactum CBS 118854 P. brevicompactum S.B. Hong DaeJeon, Korea MAT1-1 (this study) this study

P. brevicompactum DSM 62871 P. brevicompactum R. Schneider Germany MAT1-1 (this study) this study

P. chrysogenum CBS 776.95 P. chrysogenum Houbraken et al. (2012) New Mexico, USA - Houbraken et al. (2012)

P. chrysogenum IB 1.59/12 P. solitum M. Kirchmair Inssbruck, Austria MAT1-2 (this study) this study

P. chrysogenum IB 23/2 16E42 P. solitum M. Kirchmair Inssbruck, Austria MAT1-2 (this study) this study

P. chrysogenum IB 2B/06 2.53/7 P. citrinum M. Kirchmair Inssbruck, Austria MAT1-2 (this study) this study

P. chrysogenum IB 3.24/2 P. brevicompactum M. Kirchmair Inssbruck, Austria MAT1-2 (this study) this study IV. Mahmoudjanlou et al. 2020 33

P. chrysogenum IS-1 P. chrysogenum E. Nevo Dead see, Israel MAT1-2 (this study) this study

P. chrysogenum IS-3 P. chrysogenum E. Nevo Dead see, Israel MAT1-2 (this study) this study

P. chrysogenum PC08105C P. chrysogenum Henk et al. (2011) unknown MAT1-1 (this study) Henk et al. (2011)

P. chrysogenum 2M2A P. chrysogenum M. Kirchmair Inssbruck, Austria MAT1-1 (this study) this study

P. chrysogenum DAOM 193710 P. chrysogenum Scott et al. (2004) Connecticut, USA MAT1-1 (this study) this study

P. chrysogenum IB 08/922 P. chrysogenum M. Kirchmair Inssbruck, Austria MAT1-1 (this study) this study

P. citrinum CBS 117.64 P. citrinum H.J. Hueck Netherlands MAT1-2 (this study) NCBI

P. citrinum CBS 309.48 P. citrinum G. Sabet Cairo, Egypt MAT1-2 (this study) this study

P. citrinum DSM 1179 P. citrinum F.T. Brooks unknown MAT1-2 (this study) this study

P. citrinum CBS 139.45 P. citrinum C. Thom unknown MAT1-1 (this study) this study

P. citrinum CBS 341.61 P. citrinum C.A. Ghillini Italy MAT1-1 (this study) this study

P. citrinum DSM 1997 P. citrinum L. Leistner Germany MAT1-1 (this study) this study

P. citrinum DSM 62830 P. citrinum Bredemeier Germany MAT1-1 (this study) this study

P. citrinum JCM 22607 P. citrinum unknown Netherlands MAT1-1 (this study) this study

P. egyptiacum CBS 244.32 P. egyptiacum Y.S. Sabet (Pöggeler et al., 2011) Egypt MAT1-1 and MAT1-2 Houbraken et al. (2012)

P. expansum ATCC 24692 P. expansum JGI West Virginia MAT1-2 (JGI) JGI (this study)

P. expansum T01 P. expansum Li et al. (2015) unknown MAT1-2 Li et al. (2015) Li et al. (2015)

P. expansum Pd1/PEXP P. expansum Ballester et al. (2015) Israel MAT1-1, Ballester et al. (2015) Ballester et al. (2015)

P. expansum MD-8/PEX2 P. expansum Ballester et al. (2015) USA MAT1-1 Ballester et al. (2015) Ballester et al. (2015)

P. expansum CMP1/PEX1 P. expansum Ballester et al. (2015) Spain MAT1-2 Ballester et al. (2015) Ballester et al. (2015) IV. Mahmoudjanlou et al. 2020 34

P. italicum PHI-1/PITC P. italicum Ballester et al. (2015) Spain MAT1-2 Ballester et al. (2015) Ballester et al. (2015)

P. italicum GL-Gan1 P. italicum WGS, NCBI Guangzhou, China MAT1-1 WGS

P. olsonii CBS 245.32 P. brevicompactum unknown unknown MAT1-1 (this study) this study

P. olsonii DSM 21173 P. brevicompactum A. Rabenstein Germany MAT1-2 (this study) this study

P. rubens CBS 319.59 P. rubens Houbraken et al. (2012) Japan - Houbraken et al. (2012)

P. rubens NRRL 1249.B21 P. chrysogenum Hoff et al. (2008) unknown MAT1-2 (this study) Hoff et al. (2008)

P. rubens Pc3 (IB 08/921) P. chrysogenum M. Kirchmair Böhm et al. (2015) Inssbruck, Austria MAT1-2 (this study) Böhm et al. (2015)

P. rubens PC0820A P. chrysogenum Eagle (2009) France MAT1-1 (this study) Henk et al. (2011)

P. rubens PC088B P. chrysogenum Henk et al. (2011) unknown MAT1-1 (this study) Henk et al. (2011)

P. rubens DAOM 155627 P. chrysogenum Scott et al. (2004) Ottawa, Canada MAT1-1 (this study) Scott et al. (2004)

P. rubens NRRL 1951 P. chrysogenum Henk et al. (2011) Illinois MAT1-1 (this study) Henket al. (2011)

P. rubens P2niaD18 P. chrysogenum Specht et al. (2014) Illinois MAT1-1 (this study) Specht et al. (2014)

P. rubens IB 16A2/12 P. solitum M. Kirchmair Inssbruck,Austria MAT1-2 (this study) this study

P. roqueforti FM 164 P. roqueforti An et al. (2009) Japan MAT1-2 An et al. (2009) An et al. (2009)

P. roqueforti JCM 22842 P. roqueforti An et al. (2009) Japan MAT1-1 An et al. (2009) An et al. (2009)

P. solitum ASM95277 (RS1) P. solitum Yu et al. (2016) Oregon MAT1-2 Yu et al. (2016) Yu et al. (2016)

P. solitum ATCC 20606 P. citrinum Merck & Co. Inc. unknown MAT1-2 (this study) this study

P. solitum MJCB01.1 (NJ1) P. solitum Yin et al. (2016) Illinois MAT1-1 Yin et al. (2016) Yin et al. (2016)

P. solitum CBS 109827 P. solitum G. Fischer Germany MAT1-1 (this study) this study

P. solitum CBS 109828 P. solitum J.C. Frisvad Denmark MAT1-1 (this study) this study IV. Mahmoudjanlou et al. 2020 35

P. solitum CBS 146.86 P. solitum J.C. Frisvad Denmark MAT1-1 (this study) this study

P. solitum CBS 424.89 P. solitum R. Westling Germany MAT1-1 (this study) this study

IV. Mahmoudjanlou et al. 2020 36

Supplemental Table 2. List of recombinant P. brevicompactum strains used in this study

Strain name Recipient strain genotype source

PT43-10 CBS 257.29 ΔMAT1-1-1 This study

PT52-2 PT43-10 ΔMAT1-1-1:: ΔMAT1-1-1 This study

PT52-5 PT43-10 ΔMAT1-1-1:: ΔMAT1-1-1 This study

PT45-36 CBS 110068 ΔMAT1-2-1 Mahmoudjanlou et al. (2019)

PT45-55 CBS 110068 ΔMAT1-2-1 Mahmoudjanlou et al. (2019)

PT45-58 CBS 110068 ΔMAT1-2-1 Mahmoudjanlou et al. (2019)

Supplementary Table 3. Oligonucleotides used in this study

Oligonucleotide Sequence (5’-3’) Specificity usage

ITS-1 TCCGTAGGTGAACCTGCGG ITS, flanking 5.8 S rRNA PCR sequencing

ITS-4 TCCTCCGCTTATTGATATGC ITS, flanking 5.8 S rRNA PCR sequencing

Bt2A-f GGTAACCAAATCGGTGCTGCTTTC β-tubulin (BenA) PCR sequencing

Btb-r ACCCTCAGTGTAGTGACCCTTGGC β-tubulin (BenA) PCR sequencing

4736-f ACTTTCATCTGGGCCAGCGAGTGG APN2 PCR

2756-r GCCCGCCAGCGTCTGGGCGAAATG SLA2 PCR

APN2-II-f AGGCCTCATTCATTGCTCTG APN2 sequencing

APN2-Pcit-f GATGCTCGACGTTCATTTGG APN2 from P. citrinum sequencing

TFIID-II-r GGTATGCAGCATCACAGAC TFIID sequencing

Pcit-TFIID-Pcit-r AAGATGACTGCCATTCAGGC TFIID PCR sequencing

Pchr-SLA2-r TGTATCCTGCCCATGTACCG SLA2 from P. chrysogenum PCR sequencing

Pcit-MAT1-I-f ATGGAAGCATTACCAATGCC MAT1-1-1 from P. citrinum, sequencing

Pcit-MAT1-II-f CAACAAACGAGATCGTCGAG MAT1-1-1 from P. citrinum sequencing

Pcit-MAT1-I-r GACGTGGAAAGTCACAAAGG 3’ flanking region of MAT1-1-1 sequencing and MAT1-2-1 from P. citrinum

Pcit-spec-MAT1-f GCTCTACTTCGATAGGTCCC MAT1-1-1 from P. citrinum, sequencing P. citrinum specific

Pcit-MAT1-r CAATGGTACGGGTTCGATGC MAT1-1-1 from P. citrinum sequencing

Pcit-MAT2-f CCACCGAGGATCAAACAGAC 5’ flanking region of sequencing MAT1-2-1 from P. citrinum

Pcit-MAT2-I-f ATTGGCGTCGAAGTCTTCAG MAT1-2-1 from P. citrinum sequencing IV. Mahmoudjanlou et al. 2020 37

Pcit-spec-MAT2-f CAGGGCATGAATCTCTATCC MAT1-2-1 from P. citrinum, sequencing P. citrinum specific

Pcit-MAT2-r AACCGAGTCGCTTGCTGTTC MAT1-2-1 from P. citrinum sequencing

Psol-MAT1-I-f GATCTGCAGTGCCACGTG 5’ flanking region of MAT1-1-1 PCR and MAT 1-2-1 from P. solitum sequencing

Psol-MAT1-I-r GCATCTCACTGTGAGTAGGAG MAT1-1-1 from P. solitum sequencing

Psol-MAT1-II-f GACCAAGTTGTGAAGCACTG MAT 1-1-1 from P. solitum PCR sequencing

Psol-MAT1-II-r CCTGAGAACTTGTCATGAACG 3’ flanking region of MAT1-1-1 PCR and MAT 1-2-1 from sequencing P. solitum,

Psol-MAT1-III-f CACTCTGAACCCTTACTACATTGTTC MAT1-1-1 from P. solitum PCR sequencing

Psol-MAT1-III-r CTCATAGGTGACCGGTCAGC 3’ flanking region of MAT1-1-1 PCR from P. solitum sequencing

Psol-MAT1-IV-for AACAGCAGTATGCAGCAGAC 5’ flanking region of MAT1-1-1 sequencing from P. solitum

Psol-MAT2-f AAGGGGATATCGCTCGGTAG 5’ flanking region of MAT1-1-1 sequencing and MAT1-2-1 from P. solitum

Psol-MAT2-I-r TGGTGGGCATGAGAATGTCC MAT1-2-1 from P. solitum sequencing

Psol-MAT2-II-r CATGTGTGTTTCGCTCTT GG 3’ flanking region of MAT1-2-1 sequencing from P. solitum

Psol-MAT2-III-r AATGCGACCGTATCCTCGAG 3’ flanking region of MAT1-2-1 sequencing from P. solitum

Psol-spec-MAT2-f ATTCTTACCACTCCGGCTTC MAT1-2-1 from P. solitum PCR P. solitum specific

Psol-MAT1-I-r AAGAAAGCCAGTCAGCTCAG MAT1-2-1 from P. solitum PCR

Pchr-MAT1-I-f ACAATCGGAATCAGACTCGG 5’ flanking region of MAT1-1-1 sequencing and MAT1-2-1 from P. chrysogenum

Pchr-spec-MAT1-f GGAGCTGAAGCGTATTGATC MAT1-1-1 from PCR P. chrysogenum P. chrysogenum specific

Pchr-MAT1-r ATTGAGAGGGCTGGGTAACG 3’ flanking region of MAT1-1-1 PCR from P. chrysogenum sequencing

Pchr-MAT1-II-f ATGTCTACCTCTCTTGATGC MAT1-1-1 from PCR P. chrysogenum

Pchr-MAT1-II-r GGTGTCGACATCGAAGT MAT1-1-1 from PCR P. chrysogenum sequencing

Pchr-MAT2-I-f ATGATGGCGAAAACCCTCTTG MAT1-2-1 from PCR P. chrysogenum sequencing

Pchr-spec-MAT2-f CTGGCGATTCTGTCAAGATC MAT1-2-1 from PCR P. chrysogenum, sequencing P. chrysogenum specific

Pchr-MAT2-r AGTCGGCAGAAAGATTCAGC 3’ flanking region of MAT1-2-1 PCR from P. chrysogenum sequencing

Pchr-MAT2-I-r AGTCGGCAGAAAGATTCAGC 3’ flanking region of MAT1-2-1 sequencing from P. chrysogenum

IV. Mahmoudjanlou et al. 2020 38

Pbre-MAT1-cDNA-f CCTTGAGATGTTGCTTTTCCG MAT1-1-1 from PCR P. brevicompactum and sequencing P. bialowiezense RT-PCR

Pbre-MAT1-cDNA-r GTGTATGCCTTGGCCAGGA MAT1-1-1 from PCR for P. brevicompactum and cDNA P. bialowiezense PCR sequencing

Pbre-MAT1-II-r CCGAAAGCGCAAGTGAGGAC 3’ flanking region of MAT1-1-1 PCR and MAT1-2-1 from sequencing P. brevicompactum

Pbre-spec-MAT1-f TTCTCCGTCGCGTCACTAAG MAT1-1-1 from PCR P. brevicompactum, sequencing

P. brevicompactum specific

Pbre-MAT1-r TCAGAGCTGTCCAGTGAGAG MAT1-1-1 from PCR P. brevicompactum sequencing

Pbre-MAT2-I-f CTGTGGAACCGCAGAATA AG 5’ flanking region of MAT1-1-1 PCR and MAT1-2-1 from sequencing P. brevicompactum

Pbre-MAT2-II-f GCTGCATCCATGATTGGTGC 5’ flanking region of MAT1-1-1 PCR and MAT1-2-1 from sequencing P. brevicompactum

Pbre MAT2-I-r TGACAACCTCGTGCTTGGCC MAT1-2-1 from PCR P. brevicompactum

Pbre-MAT2-II-r TACGGAGTAAGAGCAAGTCG 3’ flanking region of MAT1-1-1 PCR and MAT1-2-1 from P. sequencing brevicompactum

Pbre-spec-MAT2-f CCTGGAGTTACCACCTACTC MAT1-2-1 from PCR P. brevicompactum sequencing

Pbre-MAT2-r TGATGTCCATGTAGTCGGTC MAT1-2-1 from PCR P. brevicompactum sequencing

Pbre-MAT2-cDNA-f ATGGGTTTTGACAACGTGAACG MAT1-2-1 from RT-PCR P. brevicompactum

Pbre-MAT2-cDNA-r CATCCTCCACAGCGAGATCGGCAT MAT1-2-1 from RT-PCR P. brevicompactum

Pbial-MAT1-f CAAGTCTAGCGGCCCTACTG MAT1-1-1 from sequencing P. bialowiezense

Pbial-MAT1-I-f GACGAGATGTACTCCGACAT MAT1-1-1 from PCR P. bialowiezense sequencing

Pbial-MAT2-f CCCAACTGCTTCATTCTTTAC CG MAT1-2-1 from PCR P. bialowiezense sequencing

Pbial-MAT2-r GCGGCGGGGAGCATACTGATA A MAT1-2-1 from PCR P. bialowiezense sequencing

Pols-MAT1-I-r CTTCGAACTCCTCCGTGGGCCA MAT1-1-1 from P. olsonii PCR sequencing

Pols-MAT1-II-r CGATCTGCAGCTAGTGATCGCGGATGATTGTG MAT1-1-1 from P. olsonii sequencing TAAGCCTT

Pols-MAT1 -f GTCTTGCATGGCCTCCTCCGAGGAC MAT1-1-1 from P. olsonii sequencing

Pols-MAT2-f_ GGCATTGATTCCATTGGCTT MAT1-2-1 from P. olsonii sequencing

Pols-MAT2-I-f GGATTGAGATTAGTAGACAG MAT1-2-1 from P. olsonii sequencing

Pols-MAT2-II-f CCTTCCCACCTACACTTCCT MAT1-2-1 from P. olsonii sequencing IV. Mahmoudjanlou et al. 2020 39

Pols-MAT2-III-f ACCATGATTACGCCAAGCTC MAT1-2-1 from P. olsonii sequencing

T7 TAATACGACTCACTATAGGG T7 promoter sequencing SP 6 CATTTAGGTGACACTATAG SP 6 promoter sequencing

Pbre-MAT1-5'-MluI-f ACATAACGCGTCCCTCAACGATGGTCCGCAC 5’ flank region of MAT1-1-1 Cloning from P. brevicompactum

Pb-MAT1-5'-EcoRI-r TACTAGAATTCCCATGGCGACCAGGCAATTG 5’ flank region of MAT1-1-1 Cloning from P. brevicompactum

Pbre-MAT1-3'-NotI-f ATACTGCGGCCGCGCTTTTCATCCCATCGTTT 3’ flank region of MAT1-1-1 Cloning CT from P. brevicompactum

Pbre-MAT1-3'-NotI-r ATCTAGCGGCCGCGAGCACACACTGCATACC 3’ flank region of MAT1-1-1 Cloning C from P. brevicompactum

Tn5-phleo-BoxI-f GAATAGACTTACGTCCATGGGCGAAATGACC ble Cloning GACC

Tn5-phleo-ApaI-r GAATTGGGCCCTCATGAGATGCCTGCAAGCA ble Cloning

Pbre-MAT1-BglII- CGCGCAGATCTCAATGTCTACTCTAGTTGGCG MAT1-1-1 from Cloning comp-f P. brevicompactum

Pbre-MAT1-EcoRI- CCATAGAATTCATCAGAGCTGTCCAGTGAGA MAT1-1-1 from Cloning comp-r P. brevicompactum

Supplemental Table 4. Plasmids used in this study

Name Characteristics source pDrive cloning vector T7 RNA polymerase promoter: MCS: SP6 RNA polymerase Qiagen, Hilden, promoter Germany pDrive/ptrpc-Tn5Phleo trpC promoter of A. nidulans, ble resistance gene of Böhm et al. (2013) Streptoalloteichus hindustanus PN-EGFP gpd promoter of A. nidulans, egfp, TtrpC of A. nidulans, trpC Kück et al. (2009) promoter of A. nidulans, hph resistance gene of Streptomyces hygroscopicus pPtrpC-Pbnat1 trpC promoter of A. nidulans, codon adapted synthetized Pbnat1 Mahmoudjanlou et al. resistance gene (2019) pPb-MAT1-1-1-KO 5′ flanking region of MAT1-1-1 gene, trpC promoter of This study A. nidulans, Pbnat1 (codon adapted nat1 resistance gene of S. noursei), 3′ flanking region of MAT1-1-1 gene pPb-MAT1-1-1-comp gpd promoter of A. nidulans, egfp, MAT1-1-1 gene from P. This study brevicompactum, TtrpC of A. nidulans

IV. Mahmoudjanlou et al. 2020 40

Supplementary figures

Supplementary Figure 1. Phenotypic analysis of different P. brevicompactum wild-type strains with different geographical origins. All strains were grown on CYA medium at 27 °C for 4 days. The MAT1-1 strains are shown in blue and MAT1-2 in red.

IV. Mahmoudjanlou et al. 2020 41

Supplementary Figure 2. Construction of a MAT1-1-1 deletion strain and complementation of ΔMAT1-1-1 transgenic strain (A) MAT1-1-1 locus in the recipient strain and in the deletion mutant ΔMAT1-1-1. Dashed lines indicate the homologous recombination event (B) Vector map for integration of the MAT1-1-1 gene into the corresponding deletion strains. (C) Verification of recombinant strains with PCR. The arrows shown in (A) represent the primer pairs used for PCR analysis. (D) Evidence for deletion of MAT1-1-1 by Southern hybridization with a radioactively labeled probe specific for the 5’ flanking region of MAT1-1-1 gene.

IV. Mahmoudjanlou et al. 2020 42

Supplementary Figure 3. Characterization of MAT1-2-1 deletion strains. (A) Quantification of spore formation by parental and MAT1-2-1 deletion stains grown on CCM medium for 168 h in the light and dark. Error bars show mean ± SD (n=3) from three independent experiments. (B) Pellet formation in shaking liquid medium (CCM) after 72 h. (C) The number of germ tubes formed by each germination conidia after 20 h of growth on the CCM-coated slides. About 300 germinating conidia were investigated for the number of germ tubes.

IV. Mahmoudjanlou et al. 2020 43

Supplementary Figure 4. Geographic distribution of 19 MAT1-1 and MAT1-2 P. brevicompactum strains. The strains were obtained from different culture collections as given in Supplemental Table 2.

References (Supplementary material): An, K. D., Kiyuna, T., Kigawa, R., Sano, C., Miura, S., & Sugiyama, J. (2009). The identity of Penicillium sp. 1, a major contaminant of the stone chambers in the Takamatsuzuka and Kitora Tumuli in Japan, is Penicillium paneum. Antonie Van Leeuwenhoek, 96(4), WT 579-592. doi:10.1007/s10482-009-9373-0 Ballester, A. R., Marcet-Houben, M., Levin, E., Sela, N., Selma-Lazaro, C., Carmona, L., Wisniewski, M., Droby, S., Gonzalez-Candelas, L., & Gabaldon, T. (2015). Genome, transcriptome, and functional analyses of Penicillium expansum provide new insights into secondary metabolism and pathogenicity. Mol Plant Microbe Interact, 28(3), 232-248. doi:10.1094/MPMI-09-14-0261-FI Böhm, J., Dahlmann, T. A., Gümüser, H., & Kück, U. (2015). A MAT1-2 wild-type strain from Penicillium chrysogenum: functional mating-type locus characterization, genome sequencing and mating with an industrial penicillin-producing strain. Mol Microbiol, 95(5), 859-874. doi:10.1111/mmi.12909 Böhm, J., Hoff, B., O'Gorman, C. M., Wolfers, S., Klix, V., Binger, D., Zadra, I., Kürnsteiner, H., Pöggeler, S., Dyer, P. S., & Kück, U. (2013). Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. Proc Natl Acad Sci U S A, 110(4), 1476-1481. doi:10.1073/pnas.1217943110 Eagle, C. (2009). Mating-type genes and sexual potential in the ascomycete genera Aspergillus and Penicillium. PhD thesis, University of Nottingham, England, UK. Frisvad, J. C., & Samson, R. A. (2004). Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Stud Mycol, 49, 1-174. IV. Mahmoudjanlou et al. 2020 44 Henk, D. A., Eagle, C. E., Brown, K., Van Den Berg, M. A., Dyer, P. S., Peterson, S. W., & Fisher, M. C. (2011). Speciation despite globally overlapping distributions in Penicillium chrysogenum: the population genetics of Alexander Fleming's lucky fungus. Mol Ecol, 20(20), 4288-4301. doi:10.1111/j.1365-294X.2011.05244.x Hoff, B., Pöggeler, S., & Kück, U. (2008). Eighty years after its discovery, Fleming's Penicillium strain discloses the secret of its sex. Eukaryot Cell, 7(3), 465-470. doi:10.1128/EC.00430-07 Houbraken, J., Frisvad, J. C., Seifert, K. A., Overy, D. P., Tuthill, D. M., Valdez, J. G., & Samson, R. A. (2012). New penicillin-producing Penicillium species and an overview of section Chrysogena. Persoonia, 29, 78-100. doi:10.3767/003158512X660571 Kück, U., Pöggeler, S., Nowrousian, M., Nolting, N., & Engh, I. (2009). Sordaria macrospora, a model system for fungal development. In W. D. e. P. a. G. Anke T. (Ed.), The Mycota XV (A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research) (Vol. 15, pp. 17-39). Heidelberg, New York, Tokyo: Springer. Li, B., Zong, Y., Du, Z., Chen, Y., Zhang, Z., Qin, G., Zhao, W., & Tian, S. (2015). Genomic characterization reveals insights into patulin biosynthesis and pathogenicity in Penicillium species. Molecular Plant-Microbe Interactions, 28(6), 635-647. doi:10.1094/MPMI-12-14-0398-FI Mahmoudjanlou, Y., Hoff, B., & Kück, U. (2019). Construction of a codon-adapted nourseotricin-resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum. J Fungi (Basel), 5(4). doi:10.3390/jof5040096 Ndagijimana, M., Chaves-López, C., Corsetti, A., Tofalo, R., Sergi, M., Paparella, A., Guerzoni, M. E., & Suzzi, G. (2008). Growth and metabolites production by Penicillium brevicompactum in yoghurt. Int J Food Microbiol, 127(3), 276-283. doi:10.1016/j.ijfoodmicro.2008.07.019 Nierman, W. C., Pain, A., Anderson, M. J., Wortman, J. R., Kim, H. S., Arroyo, J., Berriman, M., Abe, K., Archer, D. B., Bermejo, C., Bennett, J., Bowyer, P., Chen, D., Collins, M., Coulsen, R., Davies, R., Dyer, P. S., Farman, M., Fedorova, N., Fedorova, N., Feldblyum, T. V., Fischer, R., Fosker, N., Fraser, A., García, J. L., García, M. J., Goble, A., Goldman, G. H., Gomi, K., Griffith-Jones, S., Gwilliam, R., Haas, B., Haas, H., Harris, D., Horiuchi, H., Huang, J., Humphray, S., Jiménez, J., Keller, N., Khouri, H., Kitamoto, K., Kobayashi, T., Konzack, S., Kulkarni, R., Kumagai, T., Lafon, A., Latgé, J.-P., Li, W., Lord, A., Lu, C., Majoros, W. H., May, G. S., Miller, B. L., Mohamoud, Y., Molina, M., Monod, M., Mouyna, I., Mulligan, S., Murphy, L., O'Neil, S., Paulsen, I., Peñalva, M. A., Pertea, M., Price, C., Pritchard, B. L., Quail, M. A., Rabbinowitsch, E., Rawlins, N., Rajandream, M.-A., Reichard, U., Renauld, H., Robson, G. D., Rodriguez de Córdoba, S., Rodríguez-Peña, J. M., Ronning, C. M., Rutter, S., Salzberg, S. L., Sanchez, M., Sánchez-Ferrero, J. C., Saunders, D., Seeger, K., Squares, R., Squares, S., Takeuchi, M., Tekaia, F., Turner, G., Vazquez de Aldana, C. R., Weidman, J., IV. Mahmoudjanlou et al. 2020 45 White, O., Woodward, J., Yu, J.-H., Fraser, C., Galagan, J. E., Asai, K., Machida, M., Hall, N., Barrell, B., & Denning, D. W. (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature, 438(7071), 1151-1156. doi:10.1038/nature04332 Paterson, R. R. (2004). The isoepoxydon dehydrogenase gene of patulin biosynthesis in cultures and secondary metabolites as candidate PCR inhibitors. Mycol Res, 108(Pt 12), 1431-1437. doi:10.1017/s095375620400142x Pöggeler, S., O'Gorman, C. M., Hoff, B., & Kück, U. (2011). Molecular organization of the mating-type loci in the homothallic Ascomycete Eupenicillium crustaceum. Fungal Biol, 115(7), 615-624. doi:10.1016/j.funbio.2011.03.003 Samson, R. A., Seifert, K. A., Kuijpers, A. F. A., Houbraken, J. A. M. P., & Frisvad, J. C. (2004). Phylogenetic analysis of Penicillium subgenus Penicillium using partial ß- tubulin sequences. Stud Mycol, 49, 175-200. Scott, J., Untereiner, W. A., Wong, B., Straus, N. A., & Malloch, D. (2004). Genotypic variation in Penicillium chysogenum from indoor environments. Mycologia, 96(5), 1095-1105. Specht, T., Dahlmann, T. A., Zadra, I., Kürnsteiner, H., & Kück, U. (2014). Complete sequencing and chromosome-scale genome assembly of the industrial progenitor strain P2niaD18 from the Penicillin producer Penicillium chrysogenum. Genome Announc, 2(4). doi:10.1128/genomeA.00577-14 Yin, G., Zhang, Y., Pennerman, K. K., Hua, S. S., Yu, J., Guo, A., Liu, Z., & Bennett, J. W. (2016). Draft genome sequence of the fungus Penicillium solitum NJ1. Genome Announc, 4(6). doi:10.1128/genomeA.01176-16 Yu, J., Wu, G., Jurick, W. M., 2nd, Gaskins, V. L., Yin, Y., Yin, G., Bennett, J. W., & Shelton, D. R. (2016). Genome sequence of Penicillium solitum RS1, which causes postharvest apple decay. Genome Announc, 4(3). doi:10.1128/genomeA.00363-16 V. Discussion 30

V. Discussion The modern biotechnology industry applies diverse genetic engineering strategies for strain improvement. An appropriate screening system and effective selectable markers are the prerequisites for applying such strategies. Induction of a sexual cycle can also be used for biotechnological applications and generation of strains with desired traits. Within the scope of this work, a transformation system has been improved for biotechnological relevant P. brevicompactum. Moreover, phylogeny and MAT protein characterization suggested a cryptic sexual life cycle for this fungus. In the following chapter, the results and insights provided by this thesis will be discussed in the context of the data from the current researches.

1. Improvement of a transformation system for P. brevicompactum In the last decades, different transformation systems and suitable selectable markers have been developed as applicable approaches for strain improvement and functional genetic analysis of several biotechnologically relevant Penicillia e.g. P. chrysogenum, P. nalgiovense, Penicillium purpurogenum, and P. roqueforti (Sánchez et al. 1987; Durand et al. 1991; Fierro et al. 2004; de Boer et al. 2013; Kojima et al. 2015). For these species, antibiotic resistance genes of bacterial origin are commonly applied. The advantage of antibiotic resistance markers is that for their applications mutant strains are not required. On the contrary, for the application of auxotrophic marker systems, the generation of appropriate recipients showing an auxotrophy is needed. Besides the hygromycin B phosphotransferase gene (hph), the nat1 gene, encoding the nourseothricin acetyltransferase from Streptomyces nourseii, has been demonstrated as a reliable marker for DNA-mediated transformation of different filamentous fungi e.g. Acremonium chrysogenum and P. chrysogenum (Kück and Hoff 2006; Bloemendal et al. 2014). The use of the phleomycin resistance gene ble from Streptoalloteichus hindustanus has been also described for transformation of Aspergillus species and P. chrysogenum (Kolar et al. 1988; Punt and van den Hondel 1992). Different selection markers used for transformation of Penicillium species are listed in Table 2. Despite the long-standing intensive investigations on P. brevicompactum only a few studies have demonstrated genetic engineering in this fungus. Thus, a limited number of available selection marker genes is the main problem. However, the hph gene controlled by the gpdA promoter from Aspergillus niger is an appropriate selection marker for transformation of P. brevicompactum (Regueira et al. 2011; Dong et al. 2016). The nitrate reductase gene (niaD) from Fusarium oxysporum has also been described to be applicable for this fungus (Varavallo et al. 2005) but requires a niaD mutant as the recipient strain. V. Discussion 31

Table 2. Selection markers used for site-specific transformation of Penicillium species

Transformed Marker product/effect Native organism Penicillium species ble Phleomycin resistance Streptoalloteichus P. brevicompactum 1 A protein that binds to hindustanus P. chrysogenum 2 phleomycin, inhibiting its DNA P. nalgiovense 3 cleavage activity. P. roqueforti 4 ergA Squalene epoxidase Penicillium chrysogenum P. chrysogenum 5 Terbinafine resistance hph Hygromycin Streptomyces P. brevicompactum 6 B phosphotransferase hygroscopicus P. digitatum 7 P. expansum 7 Antibiotic Hygromycin B resistance P. oxalicum 8 resistance P. roqueforti 9 marker P. subrubescens 10 nat1 Nourseothricin acetyltransferase Streptomyces nourseii P. brevicompactum 1 P. chrysogenum 11 Nourseothricin resistance P. citrinum 12 P. decumbens 13 P. digitatum 14 ptrA Pyrithiamine resistance Aspergillus oryzae P. chrysogenum 15 A putative protein involved in P. decumbens 13 biosynthesis of thiamine P. oxalicum 8 amdS Acetamidase Aspergillus nidulans P. chrysogenum 16 Acetamide resistance P. nalgiovense 17 niaD Nitrate reductase Fusarium oxysporum P. brevicompactum 18 Penicillium camemberti P. camemberti 19

Auxotrophy Chlorate resistance Aspergillus niger P. canescens 20 marker Nitrate sensitivity Penicillium chrysogenum P. chrysogenum 21 Fusarium oxysporum P. griseoroseum 22 pyrG Orotidine 5' phosphate Aspergillus nidulans P. camemberti 19 decarboxylase Penicillium chrysogenum P. chrysogenum 23 Uridine auxotrophy Penicillium nalgiovense P. nalgiovense 3 1 this study; 2 Böhm et al. (2013); 3 Fierro et al. (2004); 4 Del-Cid et al. (2016); 5 Sigl et al. (2010); 6 Regueira et al. (2011); 7 Buron-Moles et al. (2012); 8 Jiang et al. (2016); 9 Durand et al. (1991); 10 Salazar-Cerezo et al. (2020); 11 Kopke et al. (2010); 12 Nara et al. (1993); 13 Li et al. (2010); 14 Gandia et al. (2016); 15 Ziemons et al. (2017); 16 Beri and Turner (1987); 17 Geisen and Leistner (1989); 18 Varavallo et al. (2005); 19 Navarrete et al. (2009); 20 Aleksenko et al. (1995); 21 Gouka et al. (1991); 22 Pereira et al. (2004); 23 Casqueiro et al. (1999).

Due to the limited availability of selectable markers for P. brevicompactum, identification and development of additional markers for genetic engineering is of notable interest. In this study, V. Discussion 32 nat1 and ble genes were tested as alternative antibiotic selection markers to hph for the transformation of P. brevicompactum (chapter III-(Mahmoudjanlou et al. 2019)). The effect of different concentrations of hygromycin, nourseothricin and phleomycin on the growth rate of P. brevicompactum demonstrated high sensitivity of this fungus to all three antibiotics, emphasizing the possible usage of these substances as selection markers for DNA-mediated transformation.

One previous study showed that a concentration of 10 µg/ml of phleomycin is sufficient to inhibit the growth of A. nidulans, A. niger, and P. chrysogenum, whereas, A. oryzae showed sensitivity only at a 10-fold higher concentration (Punt and van den Hondel 1992). In our study, we observed a strain-specific sensitivity of P. brevicompactum. While two strains exhibited a similar sensitivity as A. oryzae, four other strains were less resistant to phleomycin. Further, our antibiotic sensitivity assay revealed that protoplasts are more sensitive than asexual spores (Chapter III; Mahmoudjanlou et al. (2019)). This effect could be due to the composition of the media. Indeed, for phleomycin a decreased antibiotic activity in hypertonic media (as used for protoplast regeneration) has been reported (Austin et al. 1990; Punt and van den Hondel 1992). In our investigation, we verified the applicability of ble as well as nat1 for P. brevicompactum transformation using two transformation vectors that previously had been successfully applied for P. chrysogenum. Although transformation with the ble gene led to the generation of transgenic resistant transformants, the application of nat1 gene did not result in the formation of nourseothricin resistant strains, indicating ineffective heterologous expression of nat1 in P. brevicompactum. It is known that there exist some bottlenecks for heterologous expression of proteins in a new host that is taxonomically not related to the native producer (Tanaka et al. 2014). To overcome these limitations, a wide range of strategies has been applied, which contribute to transcriptional gene regulation, including alteration of regulatory regions, multiple integrations of the coding gene and strengthen of RNA stability by modification of mRNA structural elements (Gustafsson et al. 2004; Kopke et al. 2010). Additionally, codon usage, meaning the preferred set of codons used by a certain organism has been described as a considerable factor for the efficiency of heterologous protein expression. Therefore, the introduction of additional tRNA genes for rare codons into a host organism and fine-tuning of the codon usage of the native gene are two strategies used for the improvement of heterologous protein expression. The accessible complete genome sequences of various filamentous fungi, different available algorithms for determination of codon bias and more and more decreasing costs for de novo gene synthesis allow the design and synthesis of recombinant genes, which are adapted to the codon usage of these fungi (Kopke et al. 2010; Quax et al. 2015). To V. Discussion 33 circumvent the difficulty of ineffective expression of nat1 in P. brevicompactum, we used a fully synthesized codon adapted version of nat1, namely Pbnat1. To design a novel nat1 gene codon adapted to P. brevicompactum, we compared the codon bias of P. brevicompactum strain AgRF18, whose genome was sequenced recently (https://genome.jgi.doe.gov/portal/), to the codon usage of nat1 from S. nourseii. This comparison revealed remarkable variations between the codons. For instance, codon AGT is used with a very low frequency for the amino acid aspartate in the native nat1 gene, whereas it is the preferential codon for aspartate in P. brevicompactum. Moreover, we identified CGG and CCG as rare codons in P. brevicompactum for the amino acids arginine and proline, respectively, that are present in nat1. Regarding these significant differences, it was necessary to optimize the nat1 sequence for heterologous expression in P. brevicompactum. In total, 82 nucleotides were substituted and resulted in a variation of 81 codons, representing 43 % of the codons within the gene encoding a 570 amino acid long protein. Commonly, the codon adaptation index (CAI), meaning the ratio of codon usage of a specific gene to the codon usage of a set of highly expressed genes, is used for prediction of expression efficiency of a heterologous gene (Sharp and Li 1987). Possible values range from 0 to 1; the closer the CAI value is to 1, the higher the expression level of the gene can be expected (Sharp and Li 1987). As shown in chapter III, in our study, the codon optimization resulted in a change of CAI value from 0.83 for nat1 to 0.91 for Pbnat1 suggesting higher transcriptional expression (Mahmoudjanlou et al. 2019).

Besides codon usage, GC content of coding sequences correlates with expression level (Quax et al. 2015). In this investigation, a striking difference between the GC content of nat1 from S. nourseii (71.2 %) and protein-coding sequences within the genome of P. brevicompactum (52.8 %) was an indicator of the inefficient expression of nat1 in this fungus. The optimized Pbnat1 with a GC content of 63.2 %, in contrast showed a closer value to the global GC content of P. brevicompactum. Notably, in Neurospora crassa, mainly codon usage and not the GC content of whole protein-coding genes was associated with protein level. However, GC content at the third position of codons (GC3), which connected to codon usage considers being a mediator of both mRNA and protein levels (Zhou et al. 2016). In agreement, in our study, the total change of 7.5 % of first (GC1) and 23.2 % of third (GC3) codon position respectively, led to the successful expression of codon adapted Pbnat1 gene in P. brevicompactum, which was confirmed by the generation of recombinant strains resistant to nourseothricin (Chapter III).

Previous studies demonstrated that codon bias is associated with translation efficiency by regulating translation initiation, translation elongation rate, and posttranslational protein modifications (Quax et al. 2015). An effect of codon usage bias on transcription has been also V. Discussion 34 described in different organisms (Zhou et al. 2016). While codon usage was proposed to correlate with mRNA stability in Saccharomyces cerevisiae and modulation of translation elongation rate (Presnyak et al. 2015), in N. crassa codon optimization regulates gene expression mainly by affecting transcription via reduction of histone H3 lysine 9 trimethylation and largely independent of mRNA translation and mRNA stability (Zhou et al. 2016). Lysine 9 methylation is normally associated with transcriptional repression through the formation of heterochromatin (compact chromatin structure) (Stewart et al. 2005). Increased heterologous gene expression by codon optimization in A. oryzae contributed to enhanced steady-state mRNA levels. This was mediated by a reduction of destructive premature polyadenylation of the non-codon optimized mRNA. It was shown that premature polyadenylation led to the generation of abnormal mRNAs that lack a termination codon. These mRNAs are degraded by mRNA decay pathway (Tokuoka et al. 2008). For Pbnat1, further experiments are required to understand the contribution of codon optimization to transcriptional and/ or translational protein expression efficiency. To this end, RNA-sequencing to identify mRNA and tRNA abundance, ribosome frequency profiling, and proteomics may be used.

Numerous reports have demonstrated an effective expression of codon-optimized genes in different filamentous fungi for various purposes. For instance, a marker recycling strategy was developed for P. chrysogenum and Sordaria macrospora using a fully synthesized codon- adapted flp recombinase gene from S. cerevisiae (Kopke et al. 2010; Teichert et al. 2017). In another study, the efficient expression of a codon-optimized luciferase gene in N. crassa offered a novel reporter assay approach for this fungus (Gooch et al. 2008). Moreover, Der f 7 gene encoding a mite allergen was codon-optimized and successfully expressed in A. oryzae indicating the usage of this fungus for heterologous production of recombinant allergens used in immunotherapy (Tokuoka et al. 2008). Heterologous expression of a codon-adapted xynB gene from the bacterium Dictyoglomus thermophilum in T. reesei led to the high production of a thermostable xylanase enzyme with relevance in pulp and paper industry (Te'o et al. 2000). In this study, we demonstrated that a codon-optimized nat1 gene, Pbnat1, can be used as an additional dominant selection marker for P. brevicompactum.

Although codon optimization has been confirmed to be beneficial for heterologous protein expression, some studies demonstrated that it exacerbates protein expression (Griswold et al. 2003; Klasen and Wabl 2004). For instance, expression of a codon adapted Fusarium solani cutinase in Escherichia coli reduced significantly its expression, even though the native fungal sequence contained many rare E. coli codons (Griswold et al. 2003). In our study, the applicability of Pbnat1 was proven by site-specific gene deletion of three different nuclear V. Discussion 35 genes. Although the gene deletion was successful for all three genes, transformation efficiency differed greatly. Deletion strains were obtained for MAT1-2-1 and flbA with a transformation frequency of about 10% and 4%, respectively (chapter III). This frequency was 1.25 % and thus much lower for MAT1-1-1 (chapter IV). This locus-dependent efficiency has been described for A. nidulans (Bird and Bradshaw 1997) and P. chrysogenum as well (Casqueiro et al. 1999). For yeast, it has been shown that several factors, including the size of the homologous regions and the DNA topology, contribute to gene deletion efficiency (Casqueiro et al. 1999). Since we used almost an equal length of homologous regions for all three genes, most likely DNA topology, which tightly depends on sequence (Chuprina et al. 1991), confers the observed variation in deletion efficiency of different genes.

It is known that the degree of homologous recombination is species dependent. Whereas S. cerevisiae has a very high rate of homologous recombination, most filamentous fungi show a very low recombination frequency, and the NHEJ system is predominantly used for DNA double-strand break repair (Ninomiya et al. 2004). To enhance the homologous recombination efficiency, strains from different filamentous fungi with impaired NHEJ have been generated. A significantly increased rate of homologous recombination efficiency was reported for the first time for strains of N. crassa that contain disruptions of genes encoding for the catalytic subunit (DNA-PKcs) or the regulatory DNA-binding subunits of the NHEJ machinery (Ku70/80 heterodimer) (Walker et al. 2001; Ninomiya et al. 2004). Later, NHEJ-deficient strains from different Pencillium species were generated as recipients for effective targeted DNA transformation. Table 3 summarizes NHEJ deletion approaches and their efficiency for subsequent gene deletion from different Penicillium species A disadvantage of using NHEJ– deficient strains is that these strains accumulate point mutations demonstrating their limited usability for long-term experiments. Moreover, an enhanced sensitivity to some antibiotics and mutagenic chemicals such as bleomycin, methyl methanesulfonate, and ethyl methanesulfonate was reported for these recipients (for review see (Krappmann 2007; Kück and Hoff 2010), and Table 3). In our study, a sufficient frequency of homologous recombination in P. brevicompactum strain was demonstrated. Thus, the generation of NHEJ-deficient mutants was not integral, and the recombinant deletion strains considered to be suitable for long-term functional investigations. However, construction of the NHEJ-deficient strain could facilitate the generation of deletion mutants and functional analysis in P. brevicompactum. However, since no functional studies have been reported for such strains from P. brevicompactum, possible phenotypical effects of deleting genes involved in NHEJ still need to be analyzed in this fungus. V. Discussion 36

Table 3. Examples of gene deletions targeting the NHEJ in various Penicillium species modified from (Qiao et al. 2019)

Targeting Disrupted Efficiency in Penicillium Target efficiency in Effect of NHEJ-impaired NHEJ mutant species locus reference mutant strain gene strain strain hdfA P. chrysogenum niaD 1-1.6 % 47% Less fitness of mutant in a hdfB 56% direct competition 1 ku70 (syn. P. chrysogenum pcbC 0.4 % 33- 66% Higher sensitivity to hdfA) Pcrfx1 osmotic stress 2 PcvelA ku70 P. decumbens creA 33% 100% No phenotypical effect 3 xlnR 91% 100% ku70 P. digitatum PdchsII 0.6% 11% Enhanced temperature PdchsV sensitivity 4 pkuA P. marneffei 10 loci 0.42% 22.2% -66.7% Decreased genetic stability (ku70) over-time 5 lig4 P. chrysogenum mre11 0 25% No phenotypical effect 6 niaD 5% 70% rad50 5% 40% ligD P. oxalicum PoargB 19% 97% Increased sensitivity to PoagaA 73% 90% high concentrations of Podpp4 0% 27% methyl methane sulfonate 7 ligD P. marneffei 8 loci 0.42% 99.3% No phenotypical effect 5 1 Snoek et al. (2009); 2 Hoff et al. (2010b); 3 Li et al. (2010); 4 Gandia et al. (2016); 5 Bugeja et al. (2012); 6 de Boer et al. (2010); 7 Qin et al. (2017).

2. Evidence of a heterothallic sexual state and cryptic sexuality in P. brevicompactum Recently, the phylogenetic analysis of Penicillium species indicated the phylogenetic relationship between species described as anamorph or teleomorph proposing the presence of a sexual state in asexual species (Houbraken and Samson 2011). Moreover, a wide range of population genetics studies has provided evidence for meiotic recombination and the existence of cryptic sexuality in exclusively asexually propagating species, for example in P. chrysogenum (Henk et al. 2011), Penicillium commune (Lund et al. 2003), Penicillium miczynskii (Tuthill 2004), P. roqueforti (Ropars et al. 2012), and Penicillium verrucosum (Frisvad et al. 2005). The discovery of MAT1-1 and MAT1-2 loci in P. chrysogenum and their transcriptional expression (Hoff et al. 2008) supported the hypothesis of the presence of a sexual state in anamorphic species Subsequently, several investigations V. Discussion 37 reported also the presence of MAT loci in otherwise asexual species including P. camemberti (Eagle 2009), Penicillium dipodomyis (Henk and Fisher 2011), P. expansum (Julca et al. 2015), and P. roqueforti (Eagle 2009). Similarly, in the last years, accumulating genome sequencing data revealed that other Penicillium species harbor at least one of both MAT loci (Dyer and Kück 2017). Additionally, the successful induction of a sexual cycle in P. chrysogenum (Böhm et al. 2013) and P. roqueforti (Ropars et al. 2014) proved the idea of cryptic sexuality. Taken together, these studies indicate the potential of more Penicillium species to undergo a sexual life cycle, and future investigations may uncover a teleomorphic state in these species

In this study, P. brevicompactum was investigated for the occurrence of a putative sexual life cycle. Investigation of the genome sequences of two P. brevicompactum strains, available in the online database of the Joint Genome Institute (JGI) (https://genome.jgi.doe.gov/portal/) showed the existence of the MAT1 locus encoding solely MAT1-2-1 for both strains. Moreover, Eagle (2009) reported that all of their six screened P. brevicompactum strains contained MAT1- 2-1. Therefore, this study searched for the possible existence of the MAT1-1-1 gene in a large number of P. brevicompactum strains. The sequences adjacent to the MAT loci, harboring the genes for cytoskeleton assembly control factor SLA2 and DNA lyase APN2, are highly conserved among ascomycetes (Metzenberg and Glass 1990; Coppin et al. 1997; Pöggeler 1999; Mandel et al. 2007; Ramirez-Prado et al. 2008; Wada et al. 2012; Böhm et al. 2013; Tsui et al. 2013). In this study, the SLA2‐APN2 positional strategy revealed the molecular organization of MAT1-1 and MAT1-2 loci in P. brevicompactum, enabling the evaluation of the mating type of 20 natural isolates. The conventional PCR strategy, RT-PCR, and Sanger sequencing demonstrated that the organization of both MAT loci from P. brevicompactum resembles the structure of those loci from P. chrysogenum (Hoff et al. 2008). Both loci are surrounded by SLA2 and APN2. This is consistent with flanking regions of most Aspergillus (Paoletti et al. 2005; Ramirez-Prado et al. 2008) and Penicillium species (Hoff et al. 2008; Eagle 2009; Ropars et al. 2014), but distinct from the plant pathogen Cochliobolus heterostrophus (Wirsel et al. 1998), the human pathogen Histoplasma capsulatum (Fraser et al. 2007), and P. citrinum (investigated in this study). Similar to other heterothallic Penicillium species, both MAT1-1 and MAT1-2 loci from P. brevicompactum contain just one ORF, namely MAT1-1-1 or MAT1-2-1, respectively. Compared to the MAT1-1-1 ORF from P. chrysogenum, MAT1-1-1 from P. brevicompactum is 61 bp longer in size (1.138 bp) but similarly harbors a gene encoding a protein with conserved alpha-box domain, in which one intron of 49 bp is located. This intron in P. chrysogenum is 48 bp long. Thus, the putative TF encoded by MAT1-1-1 from P. brevicompactum consists of 362 amino acid residues; 20 amino acids more than V. Discussion 38

P. chrysogenum. Similar to P. chrysogenum, MAT-1-2-1 gene from P. brevicompactum comprises three exons and two introns and encodes a TF with a conserved MATa-HMG domain. The second intron of the P. brevicompactum MAT-1-2-1 gene resides in a serine (S) codon of the HMG domain. This splice site is conserved in all of the ascomycete HMG mating type genes known so far (Debuchy and Turgeon 2006; Hoff et al. 2008). Comparing the 3´ and 5´ flanking sequences of about 1.2 kb from the MAT1-1 and MAT1-2 loci of P. brevicompactum revealed a similarity of more than 95% between the intergenic regions of these idiomorphs. This is in accordance with the architecture of MAT loci in many heterothallic filamentous fungi (Hoff et al. 2008).

As shown in chapter IV, 20 P. brevicompactum strains were investigated and the global geographic distribution of both MAT loci in approximately equal numbers indicates a heterothallic breeding system for this species. If P. brevicompactum would be a truly asexual species, it would be expected that in the natural populations nearly all isolates are clonal and of the same mating type (Dyer and Kück 2017). The worldwide distribution of MAT loci was previously used as a powerful indicator of the potential of sexual reproduction in asexual species Besides the representatives from the genus Penicillium (Henk et al. 2011), the global samples of the genera Aspergillus, Cochliobolus, Fusarium, and Trichoderma have shown a ratio of near 1:1 for MAT1-1 and MAT1-2 as well (Dyer and Kück 2017). In contrast, analysis of MAT distribution of 88 Magnaporthe oryzae isolates from East Africa, West Africa, and the Philippines revealed that MAT1-1 was dominant in these populations. Whether M. oryzae is able to reproduce sexually is still unclear, although most likely it reproduces asexually (Onaga et al. 2015). Moreover, in the majority of the dermatophyte species an imbalanced distribution of the MAT loci was evident (Kosanke et al. 2018). So far, no genome sequencing data is available for Penicillium bialowiezense and Penicillium olsonii which are closely related species to P. brevicompactum. Eagle (2009) searched for mating type genes in seven P. bialowiezense and six P. olsonii strains. Interestingly, he could not find any MAT genes for P. bialowiezense and reported a homothallic state for all P. olsonii isolates. However, our sequencing data of the entire MAT loci confirmed a heterothallic life cycle in both species The observed discrepancy between both studies is caused because Eagle (2009) used the sequences of conserved DNA-binding HMG- or α-domains for determination of mating type. Previously, alignment of α-domain and MATA-HMG domain sequences from ascomycetes identified certain similarities and conserved sequences between both domains (Martin et al. 2010). This could explain why in Eagle's study both MAT genes were found in P. olsonii strains. To sum up, the whole ORF of both mating type genes from three sister species P. brevicompactum, V. Discussion 39

P. bialowiezense, and P. olsonii was described for the first time. These data suggest the existence of a sexual cycle for these species in nature. Further, population analyses with polymorphic markers and assessment of footprints of sexual recombination in strains could be used to aid the demonstration of a sexual cycle in these species. Moreover, as genome data is accessible for P. brevicompactum, detection of RIP (repeat-induced point mutation) as well as meiosis- and sex-associated genes can also be used as indirect evidence of the occurrence of sex in this fungus. The signatures of RIP were identified in P. chrysogenum (Braumann et al. 2008; Hoff et al. 2008) and P. roqueforti (Ropars et al. 2012). RIP in fungi is a genome defense mechanism that occurs during the dikaryotic phase between karyogamy and meiosis and causes repeat sequences to be mutated (Gladyshev 2017).

3. Different attempts for induction of a sexual cycle in P. brevicompactum were unsuccessful P. brevicompactum is believed to lack a sexual cycle, as neither in natural nor in laboratory conditions sexual structures have ever been observed. Since P. brevicompactum is currently in an industrial line to produce MPA, the discovery of suitable conditions for the induction of a sexual cycle in the laboratory offers the opportunity to generate recombinant strains for industrial purposes. Furthermore, such an approach could be used for classical genetic studies. In this study, several attempts were carried out to induce a sexual cycle in this fungus (shown in Supplementary Table 1). The experimental conditions were set based on the previously described procedures required for the sexual reproduction of other filamentous ascomycetes (Houbraken and Dyer 2015). To induce a sexual cycle in heterothallic species, the selection of suitable mating partners is not the only prerequisite. The selection of appropriate agar media, inoculation methods, and incubation conditions is also essential (Houbraken and Dyer 2015). Further, sexual reproduction is influenced by multiple environmental factors such as light, oxygen, and nutrient availability (Dyer and O'Gorman 2012). In this study, first parafilm-sealed Petri dishes, containing an oatmeal agar medium supplemented with 64 µg/ml biotin were used and incubated at 20 °C in darkness. These conditions were recently reported as optimal conditions for the mating of P. chrysogenum (Böhm et al. 2013) and P. roqueforti (Ropars et al. 2014). As this strategy was not successful, the influence of various temperatures, media and additives were tested for P. brevicompactum. The V-8 agar medium (vegetable juice medium) is a suitable medium for the induction of a sexual cycle in Fusarium keratoplasticum and Cryptococcus neoformans (Kent et al. 2008; Short et al. 2013). This medium contains copper, which is connected to sexual reproduction by upregulation of mating pheromone genes (Kent et al. 2008). Inositol containing Murashige-Skoog (MS) medium has been described as a V. Discussion 40 suitable medium to trigger the unisexual reproduction of the basidiomycete C. neoformans (Phadke et al. 2013). Czapek yeast agar (CYA) has been used to induce mating in Aspergillus tubingensis (Horn et al. 2013). All media were inappropriate for production of cleistothecia in P. brevicompactum.

Several media additives have been described to positively affect sexual development. The addition of myo-inositol in the growth medium of Podospora anserina increased the number of developing fruiting bodies (Xie et al. 2017). For P. brevicompactum, inositol could not induce the sexual reproduction. Calcium influx and signaling has been reported to be associated with sexual reproduction in fungi as well. For example, the low-affinity calcium uptake system, which is active at high calcium levels, is involved in the sexual development of the yeast S. cerevisiae (Iida et al. 1994) and the plant pathogen Fusarium graminearum (Cavinder and Trail 2012). Wang et al. (2010) indicated that overexpression of A. nidulans striatin (StrA) is associated with an increased number of cleistothecia, probably through binding to calmodulin 2+ in the endoplasmic reticulum in a Ca -dependent manner. Thus, the effect of CaCO3 as a source of Ca2+ on the sexual reproduction of Penicillium was investigated in this study. Surprisingly, supplementation of oatmeal medium with 27 g/L CaCO3 correlated significantly with an increased number of cleistothecia, when P. chrysogenum strains were crossed, as shown in figure 6. However, no cleistothecia were observed for P. brevicompactum under this condition.

Figure 6. Sexual reproduction of P. chrysogenum. (A) Crossing culture of PC088B (MAT1-1) and PC3 (MAT1-2) incubated for 4 weeks at 20 °C in the dark on oatmeal agar supplemented with biotin and CaCO3. (B) Cleistothecia found at the barrage zones of mycelia from both mating types. (C) One cleistothecium in closer view.

There might be various reasons for the unsuccessful mating experiments with P. brevicompactum. One explanation may be that mating partners have incompatible karyotypes, as it was demonstrated by pulsed-field gel electrophoresis analysis for six representative strains. The observed morphological heterogeneity of all isolates even with an identical geographical origin (chapter IV, supplementary materials) could reflect the V. Discussion 41 heterogeneous karyotypes of other strains as well. It has been previously described that sexual incompatibility, reduced sexual fertility, and ensuing decreased meiotic recombination can be elicited by karyotype heterogeneity and chromosomal rearrangements (Dahlmann et al. 2015). The intraspecific infertility phenotypes were associated with karyotype heterogeneity in homothallic S. macrospora strains isolated from diverse geographic regions (Pöggeler et al. 2000). The inefficient breeding of the industrial P. chrysogenum strain P2niaD18 (MAT1-1) with the wild type Pc3 (MAT1-2) was also due to their karyotype heterogeneity and chromosomal rearrangement in P2niaD18 (Böhm et al. 2015). To overcome this barrier for P. brevicompactum, two mating partners with the same background could be genetically engineered and used for breeding. For instance, the MAT1-1-1 deletion strains constructed in this study could be complemented with a MAT1-2-1 gene and subsequently crossed with its recipient strain. A recent study presented a successful mating type swapping by genetic engineering of A. oryzae. The MAT1-2-1 could be expressed efficiently in a ΔMAT1-1-1 strain and was functional in regulating expression of sex-related genes (Wada et al. 2012). However, although this strategy would be useful for confirmation of a sexual cycle and the effect of karyotype heterogeneity on fertility in P. brevicompactum, it is not applicable to obtain progeny with novel characteristics.

Dyer and Paoletti (2005) suggested that evolution of asexuality could have been mediated by a slow decline in sexual fertility within species. It has been reported for divers ascomycota that newly isolated strains show a higher fertility rate compared to the strains sustained for a prolonged period in culture collections (Houbraken and Dyer 2015). For example, while the Paecilomyces variotii strains freshly isolated from heat-treated products could undergo sexual reproduction, the older strains from culture collection were infertile (Houbraken et al. 2008). Similarly, it was shown for the heterothallic species H. capsulatum that isolates that were maintained for eight months had lost their fertility, while isolates used for mating experiment after three months still kept their capability for sexual reproduction (Kwon-Chung et al. 1974). In this context, using fresh isolates of P. brevicompactum may lead to effective sexual reproduction and production of cleistothecia, asci, and ascospores.

It is important to mention that even freshly isolated strains could have a different degree of fertility, depending on variable physiological and genetic reasons, as it has been displayed for Aspergillus fumigatus (O'Gorman et al. 2009; Sugui et al. 2011), Aspergillus lentulus (Swilaiman et al. 2013), P. chrysogenum (Böhm et al. 2013), and P. roqueforti (Ropars et al. 2014). Not only the karyotype divergence is correlated with strain-specific fertility, but also other factors e.g. variable expression of genes required for sexual propagation, variable V. Discussion 42 production of key sexual morphogens or pheromones and pheromone receptors. Furthermore, the presence of secondary sexual incompatibility systems or heterogenic incompatibility, and potential incongruity in cytoplasmic factors can affect the fertility rate (Dyer and Paoletti 2005). It could be concluded for P. brevicompactum that a very special environment or specific nutrition might be necessary for a sexual cycle. Such a condition may occur sporadically in nature; thus, much tedious work is required to detect those conditions. Furthermore, there may be a ‘slow decline’ in fertility rate amongst P. brevicompactum populations; thus, it is crucial to screen many isolates for identification of probably rare compatible mating partners. Those partners that maintained the capability of sexual propagation have a high impact on the evolutionary population structure. Since, they were able to overwhelm the asexuality-correlated evolutionary obstacles, albeit at a low rate (Dyer and Paoletti 2005).

4. Mating type genes are a suitable molecular marker for identification and phylogeny of Penicillium species Back in time, the taxonomy of Penicillium species was based on morphological concepts but, with the advent of DNA sequencing, a large number of morphologically similar species were considered to be misidentified (Visagie et al. 2014). Currently, various genetic markers are accessible for DNA barcoding of fungal species. Reliable identification is linked, however, to a regularly updated reference data set. Internal transcribed spacer rDNA (ITS) was accepted officially as universal molecular barcode for fungi (Schoch et al. 2012). However, ITS from Penicillium and other ascomycetes are not divergent enough for a strong intraspecies resolution (Skouboe et al. 1999; Schoch et al. 2012), and databases contain many unidentified or misidentified sequences (Kõljalg et al. 2013; Visagie et al. 2014). Therefore, additional markers are required for reliable species identification (Frisvad and Samson 2004; Visagie et al. 2014). The sequence of β-tubulin coding gene benA is a widely used marker for the classification of Penicillium species and the results are usually in accordance with the observed phenotypical traits (Samson et al. 2004). In this study, ITS, benA, and MAT regions were amplified to evaluate the taxonomic classification of the Penicillium species obtained from international type culture collections. While the universal primers ITS1/ITS4 and Bt2a /Bt2b were used for ITS and benA, respectively (Glass and Donaldson 1995; Samson et al. 2004), for MAT we designed species- specific primer pairs. As described in chapter IV, sequence comparison revealed that some Penicillium brevicompactum strains, received from culture collections, were misidentified as P. brevicompactum. After strain identification, the partial β-tubulin sequences were used for phylogenetic analysis. Previous phylogenetic studies based on β-tubulin divided the subgenus Penicillium into 6 sections, namely Coronata, Chrysogena, Roqueforti, Penicillium, Digitata, V. Discussion 43 and Viridicata (Samson et al. 2004). That study placed P. brevicompactum, P. bialowiezense, and P. olsonii in the section Coronata. The species of this section displayed a high evolutionary distance to each other and to the other sections of the subgenus Penicillium. Remarkably, the diversity among strains of the species in this section was more prominent compared to species of other sections. This data matches our phylogenetic survey, as the section Coronata created a robust monophyletic clade with a high bootstrap value and the length of the branches supports the distantly relation of the lineages within this section. The branch separating P. olsonii from its two sister species received a high bootstrap value, whereas the phylogenetic relationships of P. brevicompactum and P. bialowiezense was resolved weakly. The corresponding node in the previous study using benA was highly supported (Samson et al. 2004). Another phylogenetic study based on four other molecular markers also strongly supported branches for the separation of P. brevicompactum and P. bialowiezense (Houbraken and Samson 2011). Unexpectedly, the P. egyptiacum strain CBS 244.32, which is the ex-type strain of this species (Visagie et al. 2014) is located outside section Chrysogena next to P. roqueforti. However, in other studies it was grouped with section Chrysogena (Samson et al. 2004; Houbraken and Samson 2011).

In some Penicillium species intraspecies divergence arises in benA. Thus, additional sequences to reference ex-type culture may be required to ensure the accuracy of phylogenetic investigations (Visagie et al. 2014). The ex-type strain of a species is a dead, dried type culture that is deposited often in several culture collections and sustained separately over years under different conditions. Thus, they can be damaged over time (Kuhls et al. 1995). In our phylogenetic analysis, using additional representatives of P. egyptiacum might resolve the contradictory arrangement of this lineage. Note that the isolate CBS 244.32, isolated from desert habitat, showed a secondary metabolite profile different than other strains and Samson et al. (2004) proposed a probable division of desert isolates into a distinct sister group. A comparable situation was observed for Penicillium kongii, which was described as a closely related species to P. brevicompactum (Wang and Wang 2013). Although P. kongii was placed in a distinct clade from ex-type of P. brevicompactum, evaluation of additional P. brevicompactum strains demonstrated that both lineages form a monophyletic group. Our β-tubulin phylogeny tree indicated poorly supported branches within the sections Penicillium, Roqueforti, and Viridicata. It was discussed by Visagie et al. (2014), that although benA is a suitable marker for strain identification, some misleading alignment sites could lead to unprecise phylogenetic analysis of Penicillium species Previous studies located P. chrysogenum and P. rubens in separate linkage groups (Houbraken et al. 2011). Using a representative strain from each group in our V. Discussion 44 study resulted in the placement of some isolates previously described as P. chrysogenum now into group of P. rubens.

In our study we compared the benA phylogenetic tree with phylograms based on the coding sequence of the MAT1-1-1 and MAT1-2-1 genes, using maximum likelihood analysis. Mating type genes seem to evolve more rapidly than other protein-coding genes (Turgeon 1998; Voigt et al. 2005); therefore, they have been subjected to phylogenetic studies of several ascomycetes (Pöggeler 1999; O’Donnell et al. 2004; Geng et al. 2014). Some studies have described a high interspecies divergence and low intraspecific variations for mating type genes, suggesting their applicability for phylogenetic investigations (López-Villavicencio et al. 2010; Pöggeler et al. 2011; Chai et al. 2019). Nevertheless, other studies have shown some limitations regarding their usage for phylogenetics at the species level (Strandberg et al. 2010; Geng et al. 2014). Our phylogenetic analysis of Penicillium species showed a consistent topology between benA and MAT trees. This topological similarity between MAT genes and other loci has been depicted for the genera Cochliobolus (Turgeon 1998), Leptosphaeria (Voigt et al. 2005), and Morchella (Chai et al. 2019). In some other genera e.g. Neurospora (Strandberg et al. 2010) or Ulocladium (Geng et al. 2014), diverse discordant topological patterns were found between the trees generated with MAT genes and other genes. For Ulocladium also contradictory topologies between MAT1-1-1 and MAT1-2-1 phylograms were observed, indicating separate evolutionary events of MAT1-1 and MAT1-2 strains. In our data both MAT genes displayed consistent genealogies. The comparison of our MAT and benA phylogenetic trees revealed a higher resolution and significantly better support for the separation of different Penicillium species when using MAT genes. In particular, MAT sequences robustly resolved the weak separation inside the sect. Coronata and located the P. egyptiacum strain CBS 244.32 correctly within the section Chrysogena. Remarkably, P. chrysogenum and P. rubens clustered together in the phylogenetic trees of both MAT genes. Similarly, for the genus Ulocladium it was demonstrated that the isolates belonging to distinct species, based on other genomic sequences, possess highly similar MAT sequences (Geng et al. 2014). This resemblance could be elicited by evolutionary processes such as horizontal gene transfer, or introgression. It was hypothesized for Neurospora species that reproductive genes are more permeable to gene flow than other parts of the genome (Strandberg et al. 2010). Collectively, our findings suggest that MAT sequences are appropriate markers for phylogenetic analysis of Penicillium species and differentiation between closely related species. However, distinguishing between P. chrysogenum and P. rubens is not possible using MAT genes. Yet, MAT genes seem to be a very promising marker for phylogeny of section V. Discussion 45

Coronata, and thus it would be of significant interest to investigate and characterize those genes in other species of Coronata.

5. MAT1-1-1 controls asexual development of P. brevicompactum It is known that the asexual sporulation in filamentous fungi is controlled by environmental factors, such as light, nutrient supply, oxygen influx or signal peptides (Han et al. 2003; Roncal and Ugalde 2003). Light is shown to be a positive stimulus for the conidiation of some species. While light-dependent conidia formation has been demonstrated for Aspergillus species (Sarikaya Bayram et al. 2010; Krijgsheld et al. 2013), conidiation in Fusarium species is not affected by light (Estrada and Avalos 2008; Bodor et al. 2012). For some Penicillium species e.g. P. chrysogenum and P. cyclopium (Pažout et al. 1982; Hoff et al. 2010a), a light-dependent sporulation effect has been reported. However, light is usually not a prerequisite for conidiation (Roncal and Ugalde 2003). In our study, we did not observe any difference in the conidiogenesis rate of P. brevicompactum strains, in the presence or absence of light, implying the effect of other stimuli for sporulation.

Our analysis of MAT1-1-1 and MAT1-2-1 genes from P. brevicompactum provided no evidence for loss-of-function mutations. Furthermore, RT-PCR revealed the constitutive expression of both MAT genes. Moreover, GFP-tagged MAT1-1-1 translation and its nuclear localization propose MAT genes being fully functional in P. brevicompactum. These findings comply with previous studies. In the sequencing data of nearly all apparently asexual species, no loss-of- function mutation was evident (López-Villavicencio et al. 2010). The entomopathogenic fungus Cordyceps takaomontana is the only species whose MAT loci are considered to harbor pseudogenes (Yokoyama et al. 2003).

In heterothallic species, mating type genes encode for TFs that govern sex determination and sexual reproduction processes by transcriptional regulation of pheromone precursor and receptor genes. In N. crassa, MAT-dependent expression of pheromone receptor genes pre-1 and pre-2 and the pheromone precursor gene ccg-4 (encoding the PRE-2 pheromone precursor), as well as the correlation of those TFs with perithecia and ascospores production, has been described (Kim et al. 2012). Such effects are consistent with reports demonstrating the role of MAT TFs in A. fumigatus (Yu et al. 2018), A. oryzae, (Wada et al. 2012), and P. chrysogenum (Böhm et al. 2013). Several studies indicated the multilateral functions of MAT proteins beyond the sexual processes in euascomycetes. For instance, correlation between MAT genes and pathogenicity in the plant-pathogenic ascomycete F. graminearum (Zheng et al. 2013) and in the human pathogenic basidiomycete C. neoformans (Mead et al. 2015) is evident. Some studies V. Discussion 46 presented that MAT TFs can control the expression of genes or gene clusters involved in secondary metabolism. Prominent examples include the role of MAT genes in fumagillin and pseurotin biosynthesis by A. fumigatus, (Yu et al. 2018), carotenoid production by Fusarium verticillioides (Zheng et al. 2013), and penicillin biosynthesis by P. chrysogenum (Böhm et al. 2013). It was also elucidated that some other phenotypical traits of P. chrysogenum, including hyphal morphology and conidiogenesis, are affected by MAT genes. Moreover, in the scope of target genes of MAT1-1-1 protein detected by ChIP-seq analyses, are not only the genes responsible for sexual reproduction but also genes involved in asexual reproduction, morphogenesis, and amino acid- and iron metabolism (Becker et al. 2015). Ádám et al. (2011) suggested that MAT genes are functionally maintained even along with the asexual part of the life cycle, probably since they contribute to the beneficial selective influence on imperative processes unrelated to sexual development.

In our functional analysis, the P. brevicompactum MAT1-1-1 deletion strain achieved a dramatic increase of 50 % in asexual spore production. This finding is in accordance with the enhanced conidiogenesis in ΔMAT1-1-1 strains of P. chrysogenum. However, the difference between the wild type and mutant strains of P. chrysogenum was less prominent (about 25 %) (Böhm et al. 2013). The exact mode of action of MAT1-1-1 as a regulator of asexual reproduction in P. brevicompactum has yet to be defined by comparative transcriptomic analysis and protein interaction studies. However, one can assume the function of this TF through a review of previous studies. The regulatory pathway of asexual reproduction in

A. nidulans has been described. Activation of brlA, encoding a C2H2 zinc finger TF, is crucial for conidiogenesis (Adams et al. 1988). BrlA together with two other TFs, AbaA and WetA form the core regulatory cascade that elicits conidiophore development and spore maturation by regulation of temporal and spatial expression of conidiation-specific genes (Clutterbuck 1969; Yu 2010). In turn, the expression of brlA is activated by the upstream regulators of asexual development, FluG and Flb proteins (FlbA-E), which had been expressed and activated by exogenous stimuli (Adams et al. 1998). In a microarray survey of P. chrysogenum a 1.5-fold upregulation of flbA expression after 36 h of growth was evident. Further investigations are required to elucidate whether this gene is also differentially expressed in P. brevicomactum ΔMAT1-1-1 strains and if it is responsible for the conidiation phenotype. Böhm et al. (2013) discussed that another differentially expressed gene may convey the increase of sporulation in MAT1-1-1 deletion strains. They observed the 5.8 times downregulation of a gene homologous to ppoA. It has been shown that the deletion of ppoA, encoding a fatty acid dioxygenase in Aspergilli, promotes significantly the conidiospores formation by reduction of oxylipin psiBα V. Discussion 47

(precocious sexual inducer) biosynthesis (Tsitsigiannis et al. 2004). psiBα is an endogenous oxylipin and serves as a primary signal for the induction of sexual reproduction in Aspergilli (Champe and el-Zayat 1989). The transcriptional regulatory impact of the MAT1-1-1 from P. brevicompactum on ppoA needs further analysis.

6. P. brevicompactum MAT1-1-1 transcription factor is associated with germ tube formation and pellet morphology As described in the introduction of this thesis, several Penicillium species are used for the biosynthesis of secondary metabolites at an industrial scale, mostly by the process of submerged fermentation. Such processes are influenced by various parameters. Hyphal morphology is one of the most critical points, and depends on the medium composition, broth viscosity, spore concentration and viability, pH, temperature, oxygen concentration, and agitation (Zhang and Zhang 2016; Veiter et al. 2018). In submerged culture, filamentous fungi grow either in spherical pellets, composed of compact hyphal aggregates or in filamentous form, appearing as free mycelia (Pirt 1966). Depending on producing organisms and desirable products, one of these forms is preferred (Veiter et al. 2018). Previously, investigation of P. brevicompactum spore development in shaking flask culture pointed to the initiation of MPA production by aggregation of the hyphae of germinating spores for pellet formation (Doerfler et al. 1978). Concerning formation mode, fungal pellets are categorized either as coagulative or non- coagulative. In the coagulative form, spores first aggregate then, germinate, and hyphal growth shapes the pellet. In the non-coagulative type, spores first germinate and then form pellets (Zhang and Zhang 2016). In P. brevicompactum pellets formation is accompanied by both types (Doerfler et al. 1978). Most interestingly, our analysis of the recombinant MAT1-1-1 strains showed marked changes in pellet morphology. The deletion of MAT1-1-1 led to smaller pellet sizes of 1-1.5 mm compared to 2mm in the wild type. This effect is associated with a reduced numbers of germ tubes. This result contrasted with P. chrysogenum MAT1-1-1-dependent pellet morphology. P. chrysogenum MAT1-1-1 deletion and overexpression strains displayed significant bigger pellet and increased germ tube formation. The MAT-dependent pellet size and germination rate in P. brevicompactum could be discussed based on findings in A. nidulans and P. chrysogenum. Different features like electrostatics, hydrophobicity and interaction between spore wall constituents are attributed to pellet formation (Jones 1994; Zhang and Zhang 2016). In A. nidulans, small hydrophobic proteins, DewA-E and RodA located in spore walls mediate the hydrophobicity of conidiospores (Stringer and Timberlake 1995; Grunbacher et al. 2014). The absence of either hydrophobin resulted in a reduction of the average size of the pellets in A. nidulans. dewA was identified as a target gene for P. chrysogenum MAT1-1-1 V. Discussion 48 in the ChIP-seq data (Becker et al. 2015). The downregulation of this gene was also confirmed by microarray and qRT-PCR analysis (Böhm et al. 2013; Becker et al. 2015). sidD and artA are two other MAT1-1-1 target genes revealed in ChIP-seq analysis in P. chrysogenum. artA encodes a protein that was shown to contribute to conidial germination in A. nidulans (Kraus et al. 2002) and deletion of this gene in P. chyrsogenum led to a drastic increase of conidiospore germination (Becker et al. 2015). sidD encodes a non-ribosomal siderophore-peptide synthetase. In Aspergillus it was demonstrated that deletion of components of the siderophore metabolism causes delayed germination of conidia (Eisendle et al. 2006). It is most likely that these genes are also directly or indirectly regulated by MAT in P. brevicompactum. Remarkably, no defining phenotype was observed following MAT1-2-1 gene deletion in P. brevicompactum, while P. chrysogenum ΔMAT1-2-1 strains showed elevated sporulation in darkness. Those strains also exhibited increased germ tube formation. Taken together, our phenotypic observations point to similar, but also distinct functions of homologous mating type TFs in Penicillium species. A reason for some contradictory effects of MAT TF could be the sequence divergence. Our amino acid sequence alignment of MAT TFs from P. brevicompactum and P. chrysogenum showed that the DNA-binding domains of those TFs are highly conserved and almost 70 % identical; however, adjacent N- and C-terminal regions were dissimilar. This divergence was even more profound in the C-terminal part. The high dissimilarity of C-terminal regions has been also reported for the Morchella (Chai et al. 2019) and Leptosphaeria species For Leptosphaeria species, also a highly variable N-terminal region was indicated (Voigt et al. 2005). TFs regulate the gene expression by sequence-specific DNA recognition and binding to upstream regulatory regions. The DNA binding sites of TF can be defined by a DNA-binding domain and/ or by direct and indirect interactions with other TFs and cofactors (Georges et al. 2009; Slattery et al. 2014). It is known that homo- and heteromerization of TF can modulate their DNA-binding activity and/or specificity (Georges et al. 2009). For the interaction of TFs with other proteins, C-terminal domains comprising protein-interacting sites also play a role aside from DNA binding domains. The C-terminal regions guarantee the accuracy of transcriptional regulation. For example, transcriptional activation by human HMG box containing SOX9 TF is controlled by its interaction with other protein partners at the two transactivation sites located in the C-terminal region of SOX9. In concert with other TFs, including WT1, SOX9 controls the transcription of the AMH gene (encoding anti-Mullerian hormone). Heat shock protein 70 (HSP70) links the TFs WT1 and SOX9. For the interaction of SOX9 with HSP70, the C-terminus of HSP70 interacts with regions of 100 amino acids at the C-terminal domain of SOX9 (with no structural domain) V. Discussion 49

(Maheswaran et al. 1998; Harley et al. 2003). Thus, the C-terminal amino acid diversity in MAT TFs from P. brevicompactum and P. chrysogenum may be associated with their distinct functions. To clarify the mode of action of MAT1-1 and MAT1-2 TFs from P. brevicompactum, further RNA-sequencing strategies could be used to compare the transcriptomes of wild type and deletion strains and search for differentially expressed genes in wild type and mutant strains. The MAT-dependent expression of identified genes could be then verified by qRT-PCR analysis. Generation of genome-wide MAT DNA-binding profile, by the mean of ChIP-seq technique, is also a useful tool for identification of MAT target genes. To confirm the predicted MAT target genes, different approaches such as electrophoretic mobility shift essays (EMSAs), yeast one-hybrid (Y1H), and using promoter regions for reporter gene assays can be applied for DNA-binding studies. Finally, functional characterization of identified genes would provide confident insights about the mechanism of action of MAT TFs. Moreover, different protein- protein interaction methods e.g. pull-down assays, co-immunoprecipitation, yeast two-hybrid (Y2H), and bimolecular fluorescence complementation (BiFC) approach can be used for identification of MAT interaction partners. VI. Summary 50

VI. Summary The filamentous ascomycete Penicillium brevicompactum is the industrial producer of the immunosuppressant agent mycophenolic acid (MPA), whose derivatives are used for the treatment of transplantation patients to inhibit organ rejection. Accordingly, targeted strain optimization to upscale the production yield of MPA and improve morphological traits for fermentation procedures is of major importance. The aim of this thesis was to develop a directed DNA-mediated transformation system for P. brevicompactum and to investigate the cryptic sexuality in this fungus, as an additional tool for strain improvement.

In this work, we introduced a codon-optimized nat1 gene along with the commercially available ble as a dominant selection marker for site specific genetic manipulation of P. brevicompactum. The application of both antibiotic resistance genes was confirmed by successful disruption of two nuclear genes, flbA and MAT1-2-1, followed by complementation studies.

Using conventional PCR approaches, we resolved the genomic organization of the MAT1-1 and MAT1-2 loci in P. brevicompactum and the transcriptional expression of both loci was approved by RT-PCR analysis. Investigation of MAT loci from 22 P. brevicompactum strains and their worldwide distribution revealed a ratio of near 1:1 for both loci, suggesting a heterothallic reproduction system for this fungus. To induce targeted crosses, we used ITS, BenA and MAT genes for strain identification of P. brevicompactum isolates. Our experiments revealed the misidentification of some strains. Moreover, our phylogenetic analysis of 10 different Penicillium species demonstrated that MAT genes are suitable molecular markers to clarify phylogenetic relationship among species closely related to P. brevicompactum. Despite performing several directed crosses, induction of a sexual cycle in this study was not successful, more likely due to the incompatible karyotypes of the mating partners, as it was determined by pulsed-field gel electrophoresis analysis for six representative strains. Our functional analysis provided important insights into the key roles of the MAT1-1-1 TF on developmental processes of P. brevicompactum. MAT1-1-1 act as a negative regulator of asexual sporulation, whereas it is associated with the formation of larger pellets in shaking culture and increased germ tube formation. MAT1-2-1 in contrast, is not involved in asexual reproduction and hyphal morphology.

In this study we provided extended opportunities for genetic manipulation of P. brevicompactum, which can be exploited for metabolic engineering purposes, to enhance the production of MPA. Moreover, our data suggest a putative sexual cycle in P. brevicompactum, which can be a valuable tool to generate recombinant strains for biotechnological application. VII. Zusammenfassung 51

VII. Zusammenfassung Der filamentöse Ascomycet Penicillium brevicompactum ist der industrielle Produzent von Mycophenolsäure (MPA) und besitz insofern eine hohe Relevanz. Die immunsupprimierende Wirkung von MPA wird in der Behandlung von Transplantationspatienten zur Verhinderung von Organabstoßungen eingesetzt. Entsprechend ist eine gezielte Stammoptimierung zwecks Steigerung der Produktionsausbeute und zur Verbesserung der morphologischen Merkmale während der Fermentation von großer Bedeutung. Im Rahmen dieser Arbeit sollte ein gerichtetes DNA-vermitteltes Transformationssystem für P. brevicompactum entwickelt werden. Außerdem wurde der kryptische sexuelle Lebenszyklus von P. brevicompactum untersucht, um zu klären, ob dieser sich als ein zusätzliches Werkzeug für die Stammoptimierung eignet.

In dieser Arbeit wurde ein Codon-optimiertes nat1-Gen zusammen mit dem im bereits verfügbaren ble-Gen als dominante Selektionsmarker für die gezielte genetische Manipulation von P. brevicompactum eingeführt. Die Anwendung dieser Selektionsmarker wurde durch die erfolgreiche Deletion zweier Gene, flbA und MAT1-2-1, sowie die darauffolgenden Komplementationsstudien bestätigt. Auf Basis von Sequenzauswertungen der genomischen DNA verschiedener Stämme, konnte die Organisation der MAT1-1 und MAT1-2-Loci in P. brevicompactum gezeigt werden. Die transkriptionelle Expression beider Loci wurde durch RT-PCR-Analyse nachgewiesen. Die Untersuchung von MAT-Loci aus 22 P. brevicompactum-Stämmen ergab ein Verhältnis von nahezu 1:1 für beide Loci, welches darauf hindeutet, dass dieser Pilz einen heterothallischen Lebenszyklus besitzt. Um gezielte Kreuzungen zu induzieren, wurden ITS, BenA- und MAT-Gene zur Stammidentifizierung der P. brevicompactum-Stämme verwendet. Unsere Experimente zeigten, dass einige Stämme falsch identifiziert wurden. Unsere phylogenetische Untersuchung von 10 verschiedenen Penicillium Arten zeigte, dass die MAT-Gene geeignete molekulare Marker sind, um die phylogenetische Beziehung zwischen den mit P. brevicompactum verwandten Arten zu ermitteln. Trotz mehrerer Versuche war die Induktion eines sexuellen Zyklus in dieser Studie bislang nicht erfolgreich, was wahrscheinlich auf die inkompatiblen Karyotypen der Paarungspartner, die für sechs repräsentative Stämme bestimmt wurde, zurückzuführen ist. Die funktionelle Analyse des Transkriptionsfaktors (TF) MAT1-1-1 lieferte wichtige Erkenntnisse über seine Schlüsselrollen für Entwicklungsprozesse mit fermentationstechnischer Relevanz. MAT1-1-1 wirkt als negativer Regulator der asexuellen Sporulation, während er mit der Bildung größerer Pellets in Schüttelkultur und einer erhöhten Keimschlauchbildung verbunden VII. Zusammenfassung 52 ist. Im Gegensatz dazu, ist MAT1-2-1 nicht an der asexuellen Vermehrung und Hyphenmorphologie beteiligt.

Im Rahmen dieser Arbeit konnte ein zusätzliches Transformationverfahren zur genetischen Manipulation von P. brevicompactum etabliert werden, welches nachfolgend über gezielte genetische Manipulation zur Steigerung der MPA-Produktion verwendet werden kann. Darüber hinaus weisen die im Rahmen der Studie gewonnenen Daten auf einen heterothallischen Sexualzyklus für diesen Pilz hin. Letzterer könnte zwecks gezielter Stammoptimierung eingesetzt werden und sich insofern für die biotechnologische Verwendung von P. brevicompactum als außerordentlich nützlich erweisen. VIII. References 53

VIII. References Ádám AL, García-Martínez J, Szűcs EP, Avalos J, Hornok L (2011) The MAT1-2-1 mating- type gene upregulates photo-inducible carotenoid biosynthesis in Fusarium verticillioides. FEMS microbiology letters 318 (1):76-83. Adams TH, Boylan MT, Timberlake WE (1988) brlA is necessary and sufficient to direct conidiophore development in Aspergillus nidulans. Cell 54 (3):353-362. Adams TH, Wieser JK, Yu JH (1998) Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 62 (1):35-54. Adrio JL, Demain AL (2006) Genetic improvement of processes yielding microbial products. FEMS microbiology reviews 30 (2):187-214. Aerts D, Hauer EE, Ohm RA, Arentshorst M, Teertstra WR, Phippen C, Ram AFJ, Frisvad JC, Wosten HAB (2018) The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production. Antonie van Leeuwenhoek 111 (3):311-322. Afiyatullov SS, Leshchenko EV, Berdyshev DV, Sobolevskaya MP, Antonov AS, Denisenko VA, Popov RS, Pivkin MV, Udovenko AA, Pislyagin EA, von Amsberg G, Dyshlovoy SA (2017) Zosteropenillines: Polyketides from the marine- derived fungus Penicillium thomii. Marine drugs 15 (2):46. Agrawal R, Deepika NU, Joseph R (1999) Strain improvement of Aspergillus sp. and Penicillium sp. by induced mutation for biotransformation of alpha-pinene to verbenol. Biotechnology and bioengineering 63 (2):249-252. Aleksenko AY, Makarova NA, Nikolaev IV, Clutterbuck AJ (1995) Integrative and replicative transformation of Penicillium canescens with a heterologous nitrate- reductase gene. Current genetics 28 (5):474-477. Aleksieva P, Tchorbanov B, Nacheva L (2010) High-yield production of alpha-galactosidase excreted from Penicillium Chrysogenum and Aspergillus Niger. Biotechnology & Biotechnological Equipment 24 (1):1620-1623. Anderson JB, Kohn LM (1998) Genotyping, gene genealogies and genomics bring fungal population genetics above ground. Trends in ecology & evolution 13 (11):444-449. Anné J (1982) Comparison of produced by inter-species hybrids from Penicillium chrysogenum. European J Appl Microbiol Biotechnol 15 (1):41-46. Anné J, Eyssen H, De Somer P (1976) Somatic hybridisation of Penicillium roqueforti with P. chrysogenum after protoplast fusion. Nature 262 (5570):719-721. Ashton GD, Dyer PS (2016) Sexual development in fungi and its uses in gene expression systems. In: Schmoll M DC (ed) Gene Expression Systems of Fungi: Applications and Advancements. Fungal Biology. Springer, Cham, Switzerland, pp 335–350. Austin B, Hall RM, Tyler BM (1990) Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance. Gene 93 (1):157-162. VIII. References 54

Balkan B, Ertan F (2005) Production and properties of α‐Amylase from Penicillium chrysogenum and its application in starch hydrolysis. Preparative biochemistry & biotechnology 35 (2):169-178. Barreiro C, Martin JF, Garcia-Estrada C (2012) Proteomics shows new faces for the old penicillin producer Penicillium chrysogenum. Journal of biomedicine & biotechnology 2012:105109. Barron G (2011) The parasexual cycle and linkage relationships in the storage rot fungus Penicillium expansum. Canadian Journal of Botany 40:1603-1613. Barton NH (2009) Why sex and recombination? Cold Spring Harbor symposia on quantitative biology 74:187-195. Becker K, Beer C, Freitag M, Kück U (2015) Genome-wide identification of target genes of a mating-type alpha-domain transcription factor reveals functions beyond sexual development. Molecular microbiology 96 (5):1002-1022. Benjamin CR (1955) Ascocarps of Aspergillus and Penicillium. Mycologia 47 (5):669-687. Bentley R (2000) Mycophenolic acid: a one hundred year odyssey from antibiotic to immunosuppressant. Chemical reviews 100 (10):3801-3826. Berbee ML, Yoshimura A, Sugiyama J, Taylor JW (1995) Is Penicillium monophyletic? An evaluation of phylogeny in the family Trichocomaceae from 18S, 5.8S and ITS Ribosomal DNA sequence data. Mycologia 87 (2):210-222. Beri RK, Turner G (1987) Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker. Current genetics 11 (8):639-641. Bird BA, Campbell IM (1982) Disposition of mycophenolic acid, brevianamide A, asperphenamate, and ergosterol in solid cultures of Penicillium brevicompactum. Applied and environmental microbiology 43 (2):345-348. Bird BA, Remaley AT, Campbell IM (1981) Brevianamides A and B are formed only after conidiation has begun in solid cultures of Penicillium brevicompactum. Applied and environmental microbiology 42 (3):521-525. Bird D, Bradshaw R (1997) Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Molecular & general genetics : MGG 255 (2):219-225. Bloemendal S, Loper D, Terfehr D, Kopke K, Kluge J, Teichert I, Kück U (2014) Tools for advanced and targeted genetic manipulation of the beta-lactam antibiotic producer Acremonium chrysogenum. Journal of biotechnology 169:51-62. Bodor Á, Nagygyörgy E, Hornok L (2012) Inactivation of the mat1-2-1 mating type gene results in reduced asexual sporulation and con10 expression in Fusarium verticillioides. Acta Phytopathologica et Entomologica Hungarica 47:7-15. Böhm J, Dahlmann TA, Gümüser H, Kück U (2015) A MAT1-2 wild-type strain from Penicillium chrysogenum: functional mating-type locus characterization, genome sequencing and mating with an industrial penicillin-producing strain. Molecular microbiology 95 (5):859-874. VIII. References 55

Böhm J, Hoff B, O'Gorman CM, Wolfers S, Klix V, Binger D, Zadra I, Kürnsteiner H, Pöggeler S, Dyer PS, Kück U (2013) Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. Proceedings of the National Academy of Sciences of the United States of America 110 (4):1476-1481. Braumann I, van den Berg M, Kempken F (2008) Repeat induced point mutation in two asexual fungi, Aspergillus niger and Penicillium chrysogenum. Current genetics 53 (5):287-297. Bugeja HE, Boyce KJ, Weerasinghe H, Beard S, Jeziorowski A, Pasricha S, Payne M, Schreider L, Andrianopoulos A (2012) Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei. Fungal genetics and biology : FG & B 49 (10):772-778. Buron-Moles G, López-Pérez M, González-Candelas L, Viñas I, Teixidó N, Usall J, Torres R (2012) Use of GFP-tagged strains of Penicillium digitatum and Penicillium expansum to study host-pathogen interactions in oranges and apples. International journal of food microbiology 160 (2):162-170. Carthew RW, Sontheimer EJ (2009) Origins and Mechanisms of miRNAs and siRNAs. Cell 136 (4):642-655. Casqueiro J, Gutierrez S, Banuelos O, Hijarrubia MJ, Martin JF (1999) Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene leads to penicillin overproduction. Journal of bacteriology 181 (4):1181-1188. Cavinder B, Trail F (2012) Role of Fig1, a component of the low-affinity calcium uptake system, in growth and sexual development of filamentous fungi. Eukaryotic cell 11 (8):978-988. Chai H, Chen W, Zhang X, Su K, Zhao Y (2019) Structural variation and phylogenetic analysis of the mating-type locus in the genus Morchella. Mycologia 111 (4):551-562. Chakraborty BN, Patterson NA, Kapoor M (1991) An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi. Canadian journal of microbiology 37 (11):858-863. Champe SP, el-Zayat AA (1989) Isolation of a sexual sporulation hormone from Aspergillus nidulans. Journal of bacteriology 171 (7):3982-3988. Chávez R, Fierro F, García-Rico RO, Laich L (2011) Chapter 5-Mold-fermented foods: Penicillium spp. as ripening agents in the elaboration of cheese and meat products. In: Monteiro Al, Leitao D (eds) Mycofactories. Bentham Science, Chile, pp 73-98. Chen Y, Rice PA (2003) New insight into site-specific recombination from Flp recombinase- DNA structures. Annual Review of Biophysics and Biomolecular Structure 32 (1):135- 159. Chinaglia S, Chiarelli LR, Maggi M, Rodolfi M, Valentini G, Picco AM (2014) Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides. Bioscience, biotechnology, and biochemistry 78 (2):245-254. VIII. References 56

Chuprina VP, Lipanov AA, Fedoroff O, Kim SG, Kintanar A, Reid BR (1991) Sequence effects on local DNA topology. Proceedings of the National Academy of Sciences of the United States of America 88 (20):9087-9091. Clutterbuck AJ (1969) A mutational analysis of conidial development in Aspergillus nidulans. Genetics 63 (2):317-327. Coppin E, Debuchy R, Arnaise S, Picard M (1997) Mating types and sexual development in filamentous ascomycetes. Microbiol Mol Biol Rev 61 (4):411-428. Cruz R, D'Arcadia Cruz Vc, Belote JG, de Oliveira Khenayfes M, Dorta C, dos Santos Oliveira LzH, Ardiles E, Galli A (1999) Production of transgalactosylated oligosaccharides (TOS) by galactosyltransferase activity from Penicillium simplicissimum. Bioresource technology 70 (2):165-171. Dahlmann TA, Böhm J, Becker K, Kück U (2015) Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains. Current genetics 61 (4):679- 683. Dang Y, Yang Q, Xue Z, Liu Y (2011) RNA interference in fungi: pathways, functions, and applications. Eukaryotic cell 10 (9):1148. Danial EN, Ayaz NOA, Alnahdi HSO (2015) Production of inulinase by free and immobilized cells of Penicillium funiculosum p.36. Braz Arch Biol Technol 58 (4):636-642. de Boer P, Bastiaans J, Touw H, Kerkman R, Bronkhof J, van den Berg M, Offringa R (2010) Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in Δlig4 or Δku70 mutants. Fungal Genetics and Biology 47 (10):839-846. de Boer P, Bronkhof J, Dukismall kje Macedonian K, Kerkman R, Touw H, van den Berg M, Offringa R (2013) Efficient gene targeting in Penicillium chrysogenum using novel Agrobacterium-mediated transformation approaches. Fungal genetics and biology : FG & B 61:9-14. de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16 (9):839-842. Debuchy R, Turgeon BG (2006) Chapter 15- Mating-type structure, evolution, and function in Euascomycetes. In: Kües U, Fischer R (eds) Growth, Differentiation and Sexuality. The Mycota (A comprehensive treatise on fungi as experimental systems for basic and applied research), vol 1. Springer, Berlin, Heidelberg, pp 293-324. Del-Cid A, Gil-Durán C, Vaca I, Rojas-Aedo JF, García-Rico RO, Levicán G, Chávez R (2016) Identification and functional analysis of the mycophenolic acid gene cluster of Penicillium roqueforti. PloS one 11 (1):e0147047. Derntl C, Kluger B, Bueschl C, Schuhmacher R, Mach RL, Mach-Aigner AR (2017) Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism. Proceedings of the National Academy of Sciences of the United States of America 114 (4):E560-E569. VIII. References 57

Doerfler DL, Nulton CP, Bartman CD, Gottlieb FJ, Campbell IM (1978) Spore germination, colony development, and secondary metabolism in Penicillium brevicompactum: a radiogas chromatographic and morphological study. Canadian journal of microbiology 24 (12):1490-1501. Dong Y, Zhang J, Xu R, Lv X, Wang L, Sun A, Wei D (2016) Insertion mutation in HMG- CoA lyase increases the production yield of MPA through Agrobacterium tumefaciens- mediated transformation. Journal of microbiology and biotechnology 26 (11):1924- 1932. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR- Cas9. Science 346 (6213):1258096. Doyle EM, Kelly CT, Fogarty WM (1989) The high maltose-producing α-amylase of Penicillium expansum. Applied microbiology and biotechnology 30 (5):492-496. Du L, Yang X, Zhu T, Wang F, Xiao X, Park H, Gu Q (2009) Diketopiperazine alkaloids from a deep ocean sediment derived fungus Penicillium sp. Chemical & pharmaceutical bulletin 57 (8):873-876. Durand N, Reymond P, Fèvre M (1991) Transformation of Penicillium roqueforti to phleomycin- and to hygromycin B-resistance. Current genetics 19 (2):149-153. Dyer PS, Kück U (2017) Sex and the imperfect fungi. Microbiology spectrum 5 (3). Dyer PS, O'Gorman CM (2011) A fungal sexual revolution: Aspergillus and Penicillium show the way. Current opinion in microbiology 14 (6):649-654. Dyer PS, O'Gorman CM (2012) Sexual development and cryptic sexuality in fungi: insights from Aspergillus species. FEMS microbiology reviews 36 (1):165-192. Dyer PS, Paoletti M (2005) Reproduction in Aspergillus fumigatus: sexuality in a supposedly asexual species? Medical mycology 43 Suppl 1:S7-14. Eagle C (2009) Mating-type genes and sexual potential in the ascomycete genera Aspergillus and Penicillium. PhD thesis, University of Nottingham, England, UK. Echeverría V, Eyzaguirre J (2019) Penicillium purpurogenum produces a set of endoxylanases: Identification, heterologous expression, and characterization of a fourth xylanase, XynD, a novel enzyme belonging to glycoside hydrolase family 10. Applied biochemistry and biotechnology 187 (1):298-309. Eisendle M, Schrettl M, Kragl C, Müller D, Illmer P, Haas H (2006) The intracellular siderophore ferricrocin is involved in iron storage, oxidative-stress resistance, germination, and sexual development in Aspergillus nidulans. Eukaryotic cell 5 (10):1596. Emmons CW (1935) The ascocraps in species of Penicillium. Mycologia 27:128-150. Eryomin AN, Makarenko MV, Zhukovskaya LA, Mikhailova RV (2006) Isolation and characterization of extracellular glucose oxidase from Penicillium adametzii LF F- 2044.1. Applied Biochemistry and Microbiology 42 (3):304-311. VIII. References 58

Estrada AF, Avalos J (2008) The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation. Fungal Genetics and Biology 45 (5):705-718. Fang X, Shen Y, Zhao J, Bao X, Qu Y (2010) Status and prospect of lignocellulosic bioethanol production in China. Bioresource technology 101 (13):4814-4819. Fierro F, Kosalkova K, Gutierrez S, Martin JF (1996) Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum. Current genetics 29 (5):482- 489. Fierro F, Laich F, Garcia-Rico RO, Martin JF (2004) High efficiency transformation of Penicillium nalgiovense with integrative and autonomously replicating plasmids. International journal of food microbiology 90 (2):237-248. Fincham JR (1989) Transformation in fungi. Microbiological reviews 53 (1):148-170. Forment JV, Ramon D, MacCabe AP (2006) Consecutive gene deletions in Aspergillus nidulans: application of the Cre/loxP system. Current genetics 50 (3):217-224. Fox EM, Howlett BJ (2008) Secondary metabolism: regulation and role in fungal biology. Current opinion in microbiology 11 (6):481-487. Fraser JA, Stajich JE, Tarcha EJ, Cole GT, Inglis DO, Sil A, Heitman J (2007) Evolution of the mating type locus: insights gained from the dimorphic primary fungal pathogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii. Eukaryotic cell 6 (4):622-629. Frisvad J, Smedsgaard J, Larsen TO, Samson R (2004) Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Studies in mycology 2004:201-241. Frisvad JC (1981) Physiological criteria and mycotoxin production as AIDS in identification of common asymmetric penicillia. Applied and environmental microbiology 41 (3):568-579. Frisvad JC, Lund F, Elmholt S (2005) Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability. Journal of applied microbiology 98 (3):684-692. Frisvad JC, Samson RA (2004) Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Studies in mycology 49:1-174. Frisvad JCT, U.; Filtenborg, O. (1998) Role and use of secondary metabolites in fungal taxonomy. In: Frisva, J C; Bridge, PD; Arora, P D (eds) Chemical Fungal Taxonomy. Marcel Dekker, New York 289-319. Gandia M, Xu S, Font C, Marcos JF (2016) Disruption of ku70 involved in non-homologous end-joining facilitates homologous recombination but increases temperature sensitivity in the phytopathogenic fungus Penicillium digitatum. Fungal biology 120 (3):317-323. VIII. References 59

Garber ED, Beraha L (1965) Genetics of phytopathogenic fungi. XIV. The parasexual cycle in Penicillium expansum. Genetics 52 (3):487-492. Gautschi JT, Amagata T, Amagata A, Valeriote FA, Mooberry SL, Crews P (2004) Expanding the strategies in natural product studies of marine-derived fungi: a chemical investigation of Penicillium obtained from deep water sediment. Journal of natural products 67 (3):362-367. Geisen R, Leistner L (1989) Transformation of Penicillium nalgiovense with the amdS gene of Aspergillus nidulans. Current genetics 15 (4):307-309. Gems D, Johnstone IL, Clutterbuck AJ (1991) An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency. Gene 98 (1):61-67. Geng Y, Li Z, Xia LY, Wang Q, Hu XM, Zhang XG (2014) Characterization and phylogenetic analysis of the mating-type loci in the asexual ascomycete genus Ulocladium. Mycologia 106 (4):649-665. Georges AB, Benayoun BA, Caburet S, Veitia RA (2009) Generic binding sites, generic DNA-binding domains: where does specific promoter recognition come from? The FASEB Journal 24 (2):346-356. Ghosh D, Venkataramani P, Nandi S, Bhattacharjee S (2019) CRISPR–Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics. Cancer Cell International 19 (1):12. Gladyshev E (2017) Repeat-induced point mutation and other genome defense mechanisms in fungi. Microbiology spectrum 5 (4). Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and environmental microbiology 61 (4):1323-1330. Glass NL, Jacobson DJ, Shiu PKT (2000) The genetics of hyphal fusion and vegetative incompatibility in filamentous ascomycete fungi Annual review of genetics 34 (1):165- 186. Gooch VD, Mehra A, Larrondo LF, Fox J, Touroutoutoudis M, Loros JJ, Dunlap JC (2008) Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock. Eukaryotic cell 7 (1):28-37. Gouka RJ, van Hartingsveldt W, Bovenberg RA, van den Hondel CA, van Gorcom RF (1991) Cloning of the nitrate-nitrite reductase gene cluster of Penicillium chrysogenum and use of the niaD gene as a homologous selection marker. Journal of biotechnology 20 (2):189-199. Grijseels S, Nielsen JC, Nielsen J, Larsen TO, Frisvad JC, Nielsen KF, Frandsen RJN, Workman M (2017) Physiological characterization of secondary metabolite producing Penicillium cell factories. Fungal biology and biotechnology 4:8. Griswold KE, Mahmood NA, Iverson BL, Georgiou G (2003) Effects of codon usage versus putative 5′-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm. Protein Expression and Purification 27 (1):134-142. VIII. References 60

Grunbacher A, Throm T, Seidel C, Gutt B, Rohrig J, Strunk T, Vincze P, Walheim S, Schimmel T, Wenzel W, Fischer R (2014) Six hydrophobins are involved in hydrophobin rodlet formation in Aspergillus nidulans and contribute to hydrophobicity of the spore surface. PloS one 9 (4):e94546. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends in biotechnology 22 (7):346-353. Guzman-Chavez F, Zwahlen RD, Bovenberg RAL, Driessen AJM (2018) Engineering of the filamentous fungus Penicillium chrysogenum as cell factory for natural products. Frontiers in microbiology 9:2768. Hamlyn PF, Bradshaw RE, Mellon FM, Santiago CM, Wilson JM, Peberdy JF (1981) Efficient protoplast isolation from fungi using commercial enzymes. Enzyme and microbial technology 3 (4):321-325. Han KH, Le eDB, Kim JH, Kim MS, Han KY, Kim WS, Park YS, Kim HB, Han DM (2003) Environmental factors affecting development of Aspergillus nidulans. J Microbiol 41:34-40. Harley VR, Clarkson MJ, Argentaro A (2003) The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocrine reviews 24 (4):466- 487. Hashimoto H, Iwaasa T, Yokotsuka T (1972) Thermostable acid protease produced by Penicillium duponti K1014, a true thermophilic fungus newly isolated from compost. Applied microbiology 24 (6):986-992. Hawksworth DL, Crous PW, Redhead SA, Reynolds DR, Samson RA, Seifert KA, Taylor JW, Wingfield MJ, Abaci O, Aime C, Asan A, Bai FY, de Beer ZW, Begerow D, Berikten D, Boekhout T, Buchanan PK, Burgess T, Buzina W, Cai L, Cannon PF, Crane JL, Damm U, Daniel HM, van Diepeningen AD, Druzhinina I, Dyer PS, Eberhardt U, Fell JW, Frisvad JC, Geiser DM, Geml J, Glienke C, Grafenhan T, Groenewald JZ, Groenewald M, de Gruyter J, Gueho-Kellermann E, Guo LD, Hibbett DS, Hong SB, de Hoog GS, Houbraken J, Huhndorf SM, Hyde KD, Ismail A, Johnston PR, Kadaifciler DG, Kirk PM, Koljalg U, Kurtzman CP, Lagneau PE, Levesque CA, Liu X, Lombard L, Meyer W, Miller A, Minter DW, Najafzadeh MJ, Norvell L, Ozerskaya SM, Ozic R, Pennycook SR, Peterson SW, Pettersson OV, Quaedvlieg W, Robert VA, Ruibal C, Schnurer J, Schroers HJ, Shivas R, Slippers B, Spierenburg H, Takashima M, Taskin E, Thines M, Thrane U, Uztan AH, van Raak M, Varga J, Vasco A, Verkley G, Videira SI, de Vries RP, Weir BS, Yilmaz N, Yurkov A, Zhang N (2011) The amsterdam declaration on fungal nomenclature. IMA fungus 2 (1):105-112. He ZM, Price MS, Obrian GR, Georgianna DR, Payne GA (2007) Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus. BMC microbiology 7:104. VIII. References 61

Henk DA, Eagle CE, Brown K, Van Den Berg MA, Dyer PS, Peterson SW, Fisher MC (2011) Speciation despite globally overlapping distributions in Penicillium chrysogenum: the population genetics of Alexander Fleming's lucky fungus. Molecular ecology 20 (20):4288-4301. Henk DA, Fisher MC (2011) Genetic diversity, recombination, and divergence in animal associated Penicillium dipodomyis. PloS one 6 (8):e22883. Hoff B, Kamerewerd J, Sigl C, Mitterbauer R, Zadra I, Kürnsteiner H, Kück U (2010a) Two components of a velvet-like complex control hyphal morphogenesis, conidiophore development, and penicillin biosynthesis in Penicillium chrysogenum. Eukaryotic cell 9 (8):1236-1250. Hoff B, Kamerewerd J, Sigl C, Zadra I, Kück U (2010b) Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway. Applied microbiology and biotechnology 85 (4):1081- 1094. Hoff B, Pöggeler S, Kück U (2008) Eighty years after its discovery, Fleming's Penicillium strain discloses the secret of its sex. Eukaryotic cell 7 (3):465-470. Honary S, Gharaei-Fathabad E, Barabadi H, Naghibi F (2013) Fungus-mediated synthesis of gold nanoparticles: a novel biological approach to nanoparticle synthesis. Journal of Nanoscience and Nanotechnology 13 (2):1427-1430. Horn BW, Olarte RA, Peterson SW, Carbone I (2013) Sexual reproduction in Aspergillus tubingensis from section Nigri. Mycologia 105 (5):1153-1163. Houbraken J, de Vries RP, Samson RA (2014a) Modern taxonomy of biotechnologically important Aspergillus and Penicillium species. Advances in applied microbiology 86:199-249. Houbraken J, Dyer PS (2015) Induction of the sexual cycle in filamentous ascomycetes. In: van den Berg M, Maruthachalam K (eds) Genetic Transformation Systems in Fungi. Fungal Biology, vol 2. Springer, Cham. Houbraken J, Frisvad JC, Samson RA (2011) Fleming's penicillin producing strain is not Penicillium chrysogenum but P. rubens. IMA fungus 2 (1):87-95. Houbraken J, Frisvad JC, Seifert KA, Overy DP, Tuthill DM, Valdez JG, Samson RA (2012) New penicillin-producing Penicillium species and an overview of section Chrysogena. Persoonia 29:78-100. Houbraken J, Samson RA (2011) Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Studies in mycology 70 (1):1-51. Houbraken J, Varga J, Rico-Munoz E, Johnson S, Samson RA (2008) Sexual reproduction as the cause of heat resistance in the food spoilage fungus Byssochlamys spectabilis (anamorph Paecilomyces variotii). Applied and environmental microbiology 74 (5):1613. Houbraken J, Visagie CM, Meijer M, Frisvad JC, Busby PE, Pitt JI, Seifert KA, Louis- Seize G, Demirel R, Yilmaz N, Jacobs K, Christensen M, Samson RA (2014b) A VIII. References 62

taxonomic and phylogenetic revision of Penicillium section Aspergilloides. Studies in mycology 78:373-451. Iida H, Nakamura H, Ono T, Okumura MS, Anraku Y (1994) MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating. Molecular and cellular biology 14 (12):8259-8271. Janus D, Hoff B, Kück U (2009) Evidence for Dicer-dependent RNA interference in the industrial penicillin producer Penicillium chrysogenum. Microbiology 155 (Pt 12):3946-3956. Jiang B, Zhang R, Feng D, Wang F, Liu K, Jiang Y, Niu K, Yuan Q, Wang M, Wang H, Zhang Y, Fang X (2016) A Tet-on and Cre-loxP based genetic engineering system for convenient recycling of selection markers in Penicillium oxalicum. Frontiers in microbiology 7:485. Jinks JL (1952a) Heterokaryosis in wild Penicillium. Heredity 6:77-87. Jinks JL (1952b) Heterokaryosis; a system of adaption in wild fungi. Proceedings of the Royal Society of London Series B, Biological sciences 140 (898):83-99. Jones EBG (1994) Fungal adhesion. Mycological research 98 (9):961-981. Jørgensen H, Olsson L (2006) Production of cellulases by Penicillium brasilianum IBT 20888—Effect of substrate on hydrolytic performance. Enzyme and microbial technology 38 (3):381-390. Julca I, Droby S, Sela N, Marcet-Houben M, Gabaldon T (2015) Contrasting genomic diversity in two closely related postharvest pathogens: Penicillium digitatum and Penicillium expansum. Genome biology and evolution 8 (1):218-227. Keller NP (2019) Fungal secondary metabolism: regulation, function and drug discovery. Nature reviews Microbiology 17 (3):167-180. Keller NP, Turner G, Bennett JW (2005) Fungal secondary metabolism - from biochemistry to genomics. Nature reviews Microbiology 3 (12):937-947. Kent CR, Ortiz-Bermúdez P, Giles SS, Hull CM (2008) Formulation of a defined V8 medium for induction of sexual development of Cryptococcus neoformans. Applied and environmental microbiology 74 (20):6248-6253. Kim H, Wright SJ, Park G, Ouyang S, Krystofova S, Borkovich KA (2012) Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa. Genetics 190 (4):1389-1404. Klasen M, Wabl M (2004) Silent point mutation in DsRed resulting in enhanced relative fluorescence intensity. BioTechniques 36 (2):236-238. Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327 (6117):70-73. Kluge J, Terfehr D, Kück U (2018) Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi. Applied microbiology and biotechnology 102 (15):6357-6372. VIII. References 63

Kojima R, Arai T, Kasumi T, Ogihara J (2015) Construction of transformation system in Penicillium purpurogenum. Journal of bioscience and bioengineering 119 (3):314-316. Kolar M, Punt PJ, van den Hondel CAMJJ, Schwab H (1988) Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene 62 (1):127-134. Kõljalg U, Nilsson RH, Abarenkov K, Tedersoo L, Taylor AFS, Bahram M, Bates ST, Bruns TD, Bengtsson-Palme J, Callaghan TM, Douglas B, Drenkhan T, Eberhardt U, Dueñas M, Grebenc T, Griffith GW, Hartmann M, Kirk PM, Kohout P, Larsson E, Lindahl BD, Lücking R, Martín MP, Matheny PB, Nguyen NH, Niskanen T, Oja J, Peay KG, Peintner U, Peterson M, Põldmaa K, Saag L, Saar I, Schüßler A, Scott JA, Senés C, Smith ME, Suija A, Taylor DL, Telleria MT, Weiss M, Larsson K-H (2013) Towards a unified paradigm for sequence-based identification of fungi. Molecular ecology 22 (21):5271-5277. Kopke K, Hoff B, Kück U (2010) Application of the Saccharomyces cerevisiae FLP/FRT recombination system in filamentous fungi for marker recycling and construction of knockout strains devoid of heterologous genes. Applied and environmental microbiology 76 (14):4664-4674. Kosanke S, Hamann L, Kupsch C, Moreno Garcia S, Chopra A, Graser Y (2018) Unequal distribution of the mating type (MAT) locus idiomorphs in dermatophyte species. Fungal genetics and biology : FG & B 118:45-53. Krappmann S (2007) Gene targeting in filamentous fungi: the benefits of impaired repair. Fungal Biology Reviews 21 (1):25-29. Krappmann S, Bayram O, Braus GH (2005) Deletion and allelic exchange of the Aspergillus fumigatus veA locus via a novel recyclable marker module. Eukaryotic cell 4 (7):1298- 1307. Kraus PR, Hofmann AF, Harris SD (2002) Characterization of the Aspergillus nidulans 14- 3-3 homologue, ArtA. FEMS microbiology letters 210 (1):61-66. Krijgsheld P, Bleichrodt R, van Veluw GJ, Wang F, Müller WH, Dijksterhuis J, Wösten HA (2013) Development in Aspergillus. Studies in mycology 74 (1):1-29. Kroon PA, Williamson G, Fish NM, Archer DB, Belshaw NJ (2000) A modular esterase from Penicillium funiculosum which releases ferulic acid from plant cell walls and binds crystalline cellulose contains a carbohydrate binding module. European journal of biochemistry 267 (23):6740-6752. Kück U, Bloemendal S, Teichert I (2014) Putting fungi to work: harvesting a cornucopia of drugs, toxins, and antibiotics. PLoS pathogens 10 (3):e1003950. Kück U, Hoff B (2006) Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi. Fungal Genetics Reports 53:9-11. Kück U, Hoff B (2010) New tools for the genetic manipulation of filamentous fungi. Applied microbiology and biotechnology 86 (1):51-62. VIII. References 64

Kuhls K, Lieckfeldt E, Börner T (1995) PCR-fingerprinting used for comparison of ex-type strains of Trichoderma species deposited in different culture collections. Microbiological research 150 (4):363-371. Kühn R, Torres RM (2002) Cre/loxP recombination system and gene targeting. Methods in molecular biology 180:175-204. Kwon-Chung KJ, Weeks RJ, Larsh HW (1974) Studies on Emmonsiella capsulata (Histoplasma capsulatum). II. Distribution of the two mating types in 13 endemic states of the United States. American journal of epidemiology 99 (1):44-49. Lee BN, Adams TH (1994) Overexpression of FlbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development. Molecular microbiology 14 (2):323-334. Leslie JF (1993) Fungal vegetative compatibility. Annual review of phytopathology 31:127- 150. Li D, Tang Y, Lin J, Cai W (2017a) Methods for genetic transformation of filamentous fungi. Microbial cell factories 16 (1):168. Li L, Jiang W, Lu Y (2017b) New strategies and approaches for engineering biosynthetic gene clusters of microbial natural products. Biotechnol Adv 35 (8):936-949. Li W, Fan A, Wang L, Zhang P, Liu Z, An Z, Yin WB (2018) Asperphenamate biosynthesis reveals a novel two-module NRPS system to synthesize amino acid esters in fungi. Chemical science 9 (9):2589-2594. Li ZH, Du CM, Zhong YH, Wang TH (2010) Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens. Applied microbiology and biotechnology 87 (3):1065-1076. Lima JO, Pereira JF, Araujo EF, Queiroz MV (2017) Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 48 (3):602-606. LoBuglio KF, Pitt JI, Taylor JW (1993) Phylogenetic analysis of two ribosomal DNA regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia 85 (4):592-604. López-Villavicencio M, Aguileta G, Giraud T, de Vienne DM, Lacoste S, Couloux A, Dupont J (2010) Sex in Penicillium: Combined phylogenetic and experimental approaches. Fungal Genetics and Biology 47 (8):693-706. Lund F, Nielsen AB, Skouboe P (2003) Distribution of Penicillium commune isolates in cheese dairies mapped using secondary metabolite profiles, morphotypes, RAPD and AFLP fingerprinting. Food microbiology 20 (6):725-734. Maeda RN, Barcelos CA, Anna LMMS, Pereira N (2013) Cellulase production by Penicillium funiculosum and its application in the hydrolysis of sugar cane bagasse for second generation ethanol production by fed batch operation. Journal of biotechnology 163 (1):38-44. VIII. References 65

Mahapatra S, Banerjee D (2013) Fungal exopolysaccharide: production, composition and applications. Microbiology insights 6:1-16. Maheswaran S, Englert C, Zheng G, Lee SB, Wong J, Harkin DP, Bean J, Ezzell R, Garvin AJ, McCluskey RT, DeCaprio JA, Haber DA (1998) Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70. Genes & development 12 (8):1108-1120. Mahmoudjanlou Y, Hoff B, Kück U (2019) Construction of a codon-adapted nourseotricin- resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum. Journal of fungi 5 (4). Mandel MA, Barker BM, Kroken S, Rounsley SD, Orbach MJ (2007) Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition. Eukaryotic cell 6 (7):1189-1199. Manzoni M, Rollini M (2002) Biosynthesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowering drugs. Applied microbiology and biotechnology 58 (5):555-564. Martin T, Lu SW, van Tilbeurgh H, Ripoll DR, Dixelius C, Turgeon BG, Debuchy R (2010) Tracing the origin of the fungal alpha1 domain places its ancestor in the HMG- box superfamily: implication for fungal mating-type evolution. PloS one 5 (12):e15199. McDonald BA, Linde C (2002) Pathogen, population genetics, evolutionary potential, and durable resistance. Annual review of phytopathology 40 (1):349-379. McRae C, Hocking A, Seppelt R (1999) Penicillium species from terrestrial habitats in the Windmill Islands, East Antarctica, including a new species, Penicillium antarcticum. Polar Biol 21 (2):97–111. Mead ME, Stanton BC, Kruzel EK, Hull CM (2015) Targets of the sex inducer homeodomain proteins are required for fungal development and virulence in Cryptococcus neoformans. Molecular microbiology 95 (5):804-818. Metzenberg RL, Glass NL (1990) Mating type and mating strategies in Neurospora. BioEssays : news and reviews in molecular, cellular and developmental biology 12 (2):53-59. Meyer V (2008) Genetic engineering of filamentous fungi-progress, obstacles and future trends. Biotechnol Adv 26 (2):177-185. Michielse CB, Hooykaas PJ, van den Hondel CA, Ram AF (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current genetics 48 (1):1-17. Mishra A, Tripathy SK, Wahab R, Jeong S-H, Hwang I, Yang Y-B, Kim Y-S, Shin H-S, Yun S-I (2011) Microbial synthesis of gold nanoparticles using the fungus Penicillium brevicompactum and their cytotoxic effects against mouse mayo blast cancer C2C12 cells. Applied microbiology and biotechnology 92 (3):617-630. Mizutani O, Masaki K, Gomi K, Iefuji H (2012) Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue. Applied and environmental microbiology 78 (12):4126-4133. VIII. References 66

Muralidhar R, Panda T (2000) Fungal protoplast fusion – a revisit. Bioprocess Engineering 22 (5):429/431. Nara F, Watanabe I, Serizawa N (1993) Development of a transformation system for the filamentous, ML-236B (compactin) - producing fungus Penicillium citrinum. Current genetics 23 (1):28-32. Nassiri-Koopaei N, Faramarzi MA (2015) Recent developments in the fungal transformation of steroids. Biocatalysis and Biotransformation 33 (1):1-28. Navarrete K, Roa A, Vaca I, Espinosa Y, Navarro C, Chavez R (2009) Molecular characterization of the niaD and pyrG genes from Penicillium camemberti, and their use as transformation markers. Cell Mol Biol Lett 14 (4):692-702. Nielsen JC, Grijseels S, Prigent S, Ji B, Dainat J, Nielsen KF, Frisvad JC, Workman M, Nielsen J (2017) Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species. Nature Microbiology 2:17044. Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proceedings of the National Academy of Sciences of the United States of America 101 (33):12248-12253. Normark BB, Judson OP, Moran NA (2003) Genomic signatures of ancient asexual lineages. Biological Journal of the Linnean Society 79 (1):69-84. Norvell LL (2011) Fungal nomenclature. Melbourne approves a new code. Mycotaxon 166: 481-490. O'Gorman CM, Fuller H, Dyer PS (2009) Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature 457 (7228):471-474. O’Donnell K, Ward TJ, Geiser DM, Corby Kistler H, Aoki T (2004) Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genetics and Biology 41 (6):600-623. Onaga G, Wydra K, Koopmann B, Sere Y, von Tiedemann A (2015) Population structure, pathogenicity, and mating type distribution of Magnaporthe oryzae Isolates from East Africa. Phytopathology 105 (8):1137-1145. Osinska-Jaroszuk M, Jarosz-Wilkolazka A, Jaroszuk-Scisel J, Szalapata K, Nowak A, Jaszek M, Ozimek E, Majewska M (2015) Extracellular polysaccharides from Ascomycota and Basidiomycota: production conditions, biochemical characteristics, and biological properties. World journal of microbiology & biotechnology 31 (12):1823-1844. Oxford AE, Raistrick H, Simonart P (1939) Studies in the biochemistry of microorganisms: Griseofulvin, C17H17O6Cl, a metabolic product of Penicillium griseofulvum Dierckx. Biochemical Journal 33 (2):240-248. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R (2000) Advances in microbial amylases. Biotechnology and applied biochemistry 31 ( Pt 2):135-152. VIII. References 67

Paoletti M, Rydholm C, Schwier EU, Anderson MJ, Szakacs G, Lutzoni F, Debeaupuis JP, Latge JP, Denning DW, Dyer PS (2005) Evidence for sexuality in the opportunistic fungal pathogen Aspergillus fumigatus. Current biology : CB 15 (13):1242-1248. Papagianni M, Sergelidis D (2014) Purification and biochemical characterization of a novel alkaline protease produced by Penicillium nalgiovense. Applied biochemistry and biotechnology 172 (8):3926-3938. Parekh S, Vinci VA, Strobel RJ (2000) Improvement of microbial strains and fermentation processes. Applied microbiology and biotechnology 54 (3):287-301. Patel G, Patil MD, Soni S, Khobragade TP, Chisti Y, Banerjee UC (2016) Production of mycophenolic acid by Penicillium brevicompactum-A comparison of two methods of optimization. Biotechnology reports 11:77-85. Paterson RRM, Simmonds MJS, Kemmelmeier C, Blaney WM (1990) Effects of brevianamide A, its photolysis product brevianamide D, and ochratoxin A from two Penicillium strains on the insect pests Spodoptera frugiperda and Heliothis virescens. Mycological research 94 (4):538-542. Pažout J, Pažoutová S, Vanĉura V (1982) Effects of light, phosphate, and oxygen on ethylene formation and conidiation in surface cultures of penicillium cyclopium westling. Current microbiology 7 (3):133-136. Peng X, Wang Y, Sun K, Liu P, Yin X, Zhu W (2011) Cerebrosides and 2-pyridone alkaloids from the halotolerant fungus Penicillium chrysogenum grown in a hypersaline medium. Journal of natural products 74 (5):1298-1302. Pereira JF, de Queiroz MV, Lopes FJ, Rocha RB, Daboussi MJ, de Araújo EF (2004) Characterization, regulation, and phylogenetic analyses of the Penicillium griseoroseum nitrate reductase gene and its use as selection marker for homologous transformation. Canadian journal of microbiology 50 (11):891-900. Pereira JF, Vieira de Queiroz M, Aparecida Gomes E, Muro-Abad JI, Fernandes de Araújo E (2002) Molecular characterization and evaluation of pectinase and cellulase production of Penicillium spp. Biotechnology letters 24 (10):831-838. Peterson R (1998) Chemotaxonomy of fungi by unsaponifiable lipids. In: Frisvad, JC ; Bridge, P D; Arora, D K (eds). Chemical fungal taxonomy. Marcel Dekker, New York:183-218. Peterson SW (2000) Phylogenetic analysis of Penicillium species based on ITS and LSUrDNA nucleotide sequences. In: Samson RA, Pitt JI (eds) Integration of modern taxonomic methods for Penicillium and Aspergillus classification Plenum Press, New York, pp 163-178. Phadke SS, Feretzaki M, Heitman J (2013) Unisexual reproduction enhances fungal competitiveness by promoting habitat exploration via hyphal growth and sporulation. Eukaryotic cell 12 (8):1155-1159. VIII. References 68

Pirt SJ (1966) A theory of the mode of growth of fungi in the form of pellets in submerged culture. Proceedings of the Royal Society of London Series B, Biological sciences 166 (1004):369-373. Pitt JI (1979) The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London ; New York. Pitt JI, Cruickshank RH (1990) Speciation and synonymy in Penicillium subgenus Penicillium - towards a definitive taxonomy. In: Samson RA, Pitt JI (eds) Modern concepts in Penicillium and Aspergillus Classification, vol 185. NATO ASI (Series A: Life Sciences). Springer, Boston, MA. Pöggeler, Masloff, Jacobsen, Kück (2000) Karyotype polymorphism correlates with intraspecific infertility in the homothallic ascomycete Sordaria macrospora. Journal of Evolutionary Biology 13 (2):281-289. Pöggeler S (1999) Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes. Curr Genet 36 (4):222-231. Pöggeler S (2001) Mating-type genes for classical strain improvements of ascomycetes. Applied microbiology and biotechnology 56 (5-6):589-601. Pöggeler S, O'Gorman CM, Hoff B, Kück U (2011) Molecular organization of the mating- type loci in the homothallic Ascomycete Eupenicillium crustaceum. Fungal biology 115 (7):615-624. Pohl C, Kiel JAKW, Driessen AJM, Bovenberg RAL, Nygård Y (2016) CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synthetic Biology 5 (7):754- 764. Pontecorvo G (1956) The parasexual cycle in fungi. Annual review of microbiology 10:393- 400. Pontecorvo G, Sermonti G (1954) Para-sexual recombination in Penicillium chrysogenum. Journal of general microbiology 11 (1):94-104. Presnyak V, Alhusaini N, Chen Y-H, Martin S, Morris N, Kline N, Olson S, Weinberg D, Baker KE, Graveley BR, Coller J (2015) Codon optimality is a major determinant of mRNA stability. Cell 160 (6):1111-1124. Punt PJ, van den Hondel CAMJJ (1992) Transformation of filamentous fungi based on hygromycin b and phleomycin resistance markers. In: Methods in Enzymology, vol 216. Academic Press, pp 447-457. Qiao Y-M, Yu R-L, Zhu P (2019) Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites. RSC Advances 9 (60):35124- 35134. Qin X, Li R, Luo X, Lin Y, Feng JX (2017) Deletion of ligD significantly improves gene targeting frequency in the lignocellulolytic filamentous fungus Penicillium oxalicum. Fungal biology 121 (6-7):615-623. VIII. References 69

Quax TEF, Claassens NJ, Söll D, van der Oost J (2015) Codon bias as a means to fine-tune gene expression. Molecular cell 59 (2):149-161. Rabha J, Jha DK (2018) Chapter 12 - Metabolic diversity of Penicillium. In: Gupta VK, Rodriguez-Couto S (eds) New and future developments in microbial biotechnology and bioengineering. Elsevier, Amsterdam, pp 217-234. Rajal VB, Cid AG, Ellenrieder G, Cuevas CM (2009) Production, partial purification and characterization of α-l-rhamnosidase from Penicillium ulaiense. World Journal of Microbiology and Biotechnology 25 (6):1025-1033. Ramirez-Prado JH, Moore GG, Horn BW, Carbone I (2008) Characterization and population analysis of the mating-type genes in Aspergillus flavus and Aspergillus parasiticus. Fungal genetics and biology : FG & B 45 (9):1292-1299. Raper KB, Thom C (1949) A manual of the Penicillia. Williams & Wilkins Baltimore. Raveendran S, Parameswaran B, Ummalyma SB, Abraham A, Mathew AK, Madhavan A, Rebello S, Pandey A (2018) Applications of microbial enzymes in food industry. Food technology and biotechnology 56 (1):16-30. Regueira TB, Kildegaard KR, Hansen BG, Mortensen UH, Hertweck C, Nielsen J (2011) Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum. Applied and environmental microbiology 77 (9):3035-3043. Roncal T, Ugalde U (2003) Conidiation induction in Penicillium. Research in microbiology 154 (8):539-546. Ropars J, Dupont J, Fontanillas E, Rodriguez de la Vega RC, Malagnac F, Coton M, Giraud T, Lopez-Villavicencio M (2012) Sex in cheese: evidence for sexuality in the fungus Penicillium roqueforti. PloS one 7 (11):e49665. Ropars J, Lopez-Villavicencio M, Dupont J, Snirc A, Gillot G, Coton M, Jany JL, Coton E, Giraud T (2014) Induction of sexual reproduction and genetic diversity in the cheese fungus Penicillium roqueforti. Evolutionary applications 7 (4):433-441. Ruiz-Díez B (2002) Strategies for the transformation of filamentous fungi. Journal of applied microbiology 92 (2):189-195. Salazar-Cerezo S, Kun RS, de Vries RP, Garrigues S (2020) CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer. Enzyme and microbial technology 133:109463. Samson RA, Pitt JI (2000) Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Harwood Academicn Publishers, Australia. Samson RA, Seifert KA, Kuijpers AFA, Houbraken JAMP, Frisvad JC (2004) Phylogenetic analysis of Penicillium subgenus Penicillium using partial ß-tubulin sequences. Studies in mycology 49:175-200. Sánchez F, Lozano M, Rubio V, Peñalva MA (1987) Transformation in Penicillium chrysogenum. Gene 51 (1):97-102. VIII. References 70

Sarikaya Bayram O, Bayram O, Valerius O, Park HS, Irniger S, Gerke J, Ni M, Han KH, Yu JH, Braus GH (2010) LaeA control of velvet family regulatory proteins for light- dependent development and fungal cell-type specificity. PLoS genetics 6 (12):e1001226. Saunders VA, Saunders JR (1987) Chapter 6- Microbial strain improvement and novel products. In: Microbial Genetics Applied to Biotechnology. Springer, Boston, pp 265- 305. Schneider WDH, Goncalves TA, Uchima CA, Couger MB, Prade R, Squina FM, Dillon AJP, Camassola M (2016) Penicillium echinulatum secretome analysis reveals the fungi potential for degradation of lignocellulosic biomass. Biotechnology for biofuels 9:66. Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Fungal Barcoding C, Fungal Barcoding Consortium Author L (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109 (16):6241-6246. Sharp PM, Li WH (1987) The codon Adaptation Index-a measure of directional synonymous codon usage bias, and its potential applications. Nucleic acids research 15 (3):1281- 1295. Shin KS, Park HS, Kim YH, Yu JH (2013) Comparative proteomic analyses reveal that FlbA down-regulates gliT expression and SOD activity in Aspergillus fumigatus. Journal of proteomics 87:40-52. Short DP, O'Donnell K, Thrane U, Nielsen KF, Zhang N, Juba JH, Geiser DM (2013) Phylogenetic relationships among members of the Fusarium solani species complex in human infections and the descriptions of F. keratoplasticum sp. nov. and F. petroliphilum stat. nov. Fungal genetics and biology : FG & B 53:59-70. Siebert A, Prejs M, Cholewinski G, Dzierzbicka K (2017) New analogues of mycophenolic acid. Mini reviews in medicinal chemistry 17 (9):734-745. Sigl C, Handler M, Sprenger G, Kürnsteiner H, Zadra I (2010) A novel homologous dominant selection marker for genetic transformation of Penicillium chrysogenum: overexpression of squalene epoxidase-encoding ergA. Journal of biotechnology 150 (3):307-311. Skouboe P, Frisvad JC, Taylor JW, Lauritsen D, Boysen M, Rossen L (1999) Phylogenetic analysis of nucleotide sequences from the ITS region of terverticillate Penicillium species. Mycological research 103 (7):873-881. Slattery M, Zhou T, Yang L, Dantas Machado AC, Gordan R, Rohs R (2014) Absence of a simple code: how transcription factors read the genome. Trends in biochemical sciences 39 (9):381-399. VIII. References 71

Smedsgaard J, Frisvad JC (1996) Using direct electrospray mass spectrometry in taxonomy and secondary metabolite profiling of crude fungal extracts. Journal of microbiological methods 25 (1):5-17. Snoek IS, van der Krogt ZA, Touw H, Kerkman R, Pronk JT, Bovenberg RA, van den Berg MA, Daran JM (2009) Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies. Fungal genetics and biology : FG & B 46 (5):418-426. Sonjak S, Frisvad JC, Gunde-Cimerman N (2006) Penicillium mycobiota in arctic subglacial ice. Microbial ecology 52 (2):207-216. Specht T, Dahlmann TA, Zadra I, Kürnsteiner H, Kück U (2014) Complete sequencing and chromosome-scale genome assembly of the industrial progenitor strain P2niaD18 from the Penicillin producer Penicillium chrysogenum. Genome Announc 2 (4). Stewart MD, Li J, Wong J (2005) Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment. Molecular and cellular biology 25 (7):2525. Strandberg R, Nygren K, Menkis A, James TY, Wik L, Stajich JE, Johannesson H (2010) Conflict between reproductive gene trees and species phylogeny among heterothallic and pseudohomothallic members of the filamentous ascomycete genus Neurospora. Fungal Genetics and Biology 47 (10):869-878. Stringer MA, Timberlake WE (1995) dewA encodes a fungal hydrophobin component of the Aspergillus spore wall. Molecular microbiology 16 (1):33-44. Sugui JA, Losada L, Wang W, Varga J, Ngamskulrungroj P, Abu-Asab M, Chang YC, O'Gorman CM, Wickes BL, Nierman WC, Dyer PS, Kwon-Chung KJ (2011) Identification and characterization of an Aspergillus fumigatus "supermater" pair. mBio 2(6):e00234-11. Sun J, Li X, Feng P, Zhang J, Xie Z, Song E, Xi L (2014) RNAi-mediated silencing of fungal acuD gene attenuates the virulence of Penicillium marneffei. Medical mycology 52 (2):167-178. Swilaiman SS, O'Gorman CM, Balajee SA, Dyer PS (2013) Discovery of a sexual cycle in Aspergillus lentulus, a close relative of A. fumigatus. Eukaryotic cell 12 (7):962-969. Takada M, Udagawa S (1988) A new species of heterothallic Talaromyces. Mycotaxon 31:417-425. Takahashi M, Kagasaki T, Hosoya T, Takahashi S (1993) Adenophostins A and B: potent agonists of inositol-1,4,5-trisphosphate receptor produced by Penicillium brevicompactum. Taxonomy, fermentation, isolation, physico-chemical and biological properties. The Journal of antibiotics 46 (11):1643-1647. Tanaka M, Tokuoka M, Gomi K (2014) Effects of codon optimization on the mRNA levels of heterologous genes in filamentous fungi. Applied microbiology and biotechnology 98 (9):3859-3867. VIII. References 72

Te'o VSJ, Cziferszky AE, Bergquist PL, Nevalainen KMH (2000) Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei. FEMS microbiology letters 190 (1):13-19. Teichert I, Lutomski M, Märker R, Nowrousian M, Kück U (2017) New insights from an old mutant: SPADIX4 governs fruiting body development but not hyphal fusion in Sordaria macrospora. Molecular Genetics and Genomics 292 (1):93-104. Terfehr D, Dahlmann TA, Kück U (2017) Transcriptome analysis of the two unrelated fungal beta-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs. BMC genomics 18 (1):272. Thom C (1910) Cultural studies of species of Penicillium. Bulletin (United States. Bureau of Animal Industry), vol 118 Govt. Print. Off., Washington. Tokuoka M, Tanaka M, Ono K, Takagi S, Shintani T, Gomi K (2008) Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression. Applied and environmental microbiology 74 (21):6538-6546. Torres-Martinez S, Ruiz-Vazquez RM (2017) The RNAi universe in fungi: A varied landscape of small RNAs and biological functions. Annual review of microbiology 71:371-391. Trigui-Lahiani H, Ayadi M, Hadj-Taieb N, Ali MB, Gargouri A (2008) Genomic organization of a polygalacturonase gene from a hyperpectinolytic mutant strain of Penicillium occitanis. FEMS microbiology letters 281 (1):23-29. Tsitsigiannis DI, Zarnowski R, Keller NP (2004) The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans. The Journal of biological chemistry 279 (12):11344-11353. Tsui CK, DiGuistini S, Wang Y, Feau N, Dhillon B, Bohlmann J, Hamelin RC (2013) Unequal recombination and evolution of the mating-type (MAT) loci in the pathogenic fungus Grosmannia clavigera and relatives. G3 3 (3):465-480. Turgeon BG (1998) Application of mating type gene technology to problems in fungal biology. Annual review of phytopathology 36:115-137. Tuthill DE (2004) Genetic variation and recombination in Penicillium miczynskii and Eupenicillium species. Mycological Progress 3 (1):3-12. van Gelder T, Hesselink DA (2015) Mycophenolate revisited. Transplant International 28 (5):508-515. Varavallo M, Queiroz M, Lana T, Brito A, Bonoto Gonçalves D, Araújo E (2007) Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum. Brazilian Journal of Microbiology - BRAZ J MICROBIOL 38:52-57. Varavallo MA, Queiroz MVd, Pereira JF, Ribeiro RA, Soares MA, Ribeiro JB, Araújo EFd (2005) Development of a transformation system for Penicillium brevicompactum VIII. References 73

based on the Fusarium oxysporum nitrate reductase gene. Brazilian Journal of Microbiology 36:184-189. Veiter L, Rajamanickam V, Herwig C (2018) The filamentous fungal pellet-relationship between morphology and productivity. Applied microbiology and biotechnology 102 (7):2997-3006. Visagie CM, Houbraken J, Frisvad JC, Hong SB, Klaassen CH, Perrone G, Seifert KA, Varga J, Yaguchi T, Samson RA (2014) Identification and nomenclature of the genus Penicillium. Studies in mycology 78:343-371. Voigt K, Cozijnsen AJ, Kroymann J, Pöggeler S, Howlett BJ (2005) Phylogenetic relationships between members of the crucifer pathogenic Leptosphaeria maculans species complex as shown by mating type (MAT1-2), actin, and β-tubulin sequences. Molecular phylogenetics and evolution 37 (2):541-557. Wada R, Maruyama J, Yamaguchi H, Yamamoto N, Wagu Y, Paoletti M, Archer DB, Dyer PS, Kitamoto K (2012) Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae. Applied and environmental microbiology 78 (8):2819-2829. Wakelin SA, Warren RA, Harvey PR, Ryder MH (2004) Phosphate solubilization by Penicillium spp. closely associated with roots. Biol Fertil Soils 40 (1):36-43. Walker JR, Corpina RA, Goldberg J (2001) Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412 (6847):607-614. Wang B, Wang L (2013) Penicillium kongii, a new terverticillate species isolated from plant leaves in China. Mycologia 105 (6):1547-1554. Wang CL, Shim WB, Shaw BD (2010) Aspergillus nidulans striatin (StrA) mediates sexual development and localizes to the endoplasmic reticulum. Fungal genetics and biology : FG & B 47 (10):789-799. Wang S, Chen H, Tang X, Zhang H, Chen W, Chen YQ (2017) Molecular tools for gene manipulation in filamentous fungi. Applied microbiology and biotechnology 101 (22):8063-8075. Wirsel S, Horwitz B, Yamaguchi K, Yoder OC, Turgeon BG (1998) Single mating type- specific genes and their 3' UTRs control mating and fertility in Cochliobolus heterostrophus. Molecular & general genetics : MGG 259 (3):272-281. Xie N, Ruprich-Robert G, Chapeland-Leclerc F, Coppin E, Lalucque H, Brun S, Debuchy R, Silar P (2017) Inositol-phosphate signaling as mediator for growth and sexual reproduction in Podospora anserina. Developmental biology 429 (1):285-305. Xu JZ, Zhang JL, Zhang WG (2018) Antisense RNA: the new favorite in genetic research. Journal of Zhejiang University Science B 19 (10):739-749. Xu Q, Zhu CY, Wang MS, Sun XP, Li HY (2014a) Improvement of a gene targeting system for genetic manipulation in Penicillium digitatum. Journal of Zhejiang University Science B 15 (2):116-124. VIII. References 74

Xu X, Chen J, Xu H, Li D (2014b) Role of a major facilitator superfamily transporter in adaptation capacity of Penicillium funiculosum under extreme acidic stress. Fungal genetics and biology : FG & B 69:75-83. Yang Y, Zhao H, Barrero RA, Zhang B, Sun G, Wilson IW, Xie F, Walker KD, Parks JW, Bruce R, Guo G, Chen L, Zhang Y, Huang X, Tang Q, Liu H, Bellgard MI, Qiu D, Lai J, Hoffman A (2014) Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum NRRL 62431. BMC genomics 15 (1):69. Yao G, Li Z, Gao L, Wu R, Kan Q, Liu G, Qu Y (2015) Redesigning the regulatory pathway to enhance cellulase production in Penicillium oxalicum. Biotechnology for biofuels 8:71. Yokoyama E, Yamagishi K, Hara A (2003) Structures of the mating-type loci of Cordyceps takaomontana. Applied and environmental microbiology 69 (8):5019-5022. Yu JH (2010) Regulation of development in Aspergillus nidulans and Aspergillus fumigatus. Mycobiology 38 (4):229-237. Yu JH, Wieser J, Adams TH (1996) The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. The EMBO journal 15 (19):5184-5190. Yu Y, Blachowicz A, Will C, Szewczyk E, Glenn S, Gensberger-Reigl S, Nowrousian M, Wang CCC, Krappmann S (2018) Mating-type factor-specific regulation of the fumagillin/pseurotin secondary metabolite supercluster in Aspergillus fumigatus. Molecular microbiology 110 (6):1045-1065. Yuan L, Li Y, Zou C, Wang C, Gao J, Miao C, Ma E, Sun T (2012) Synthesis and in vitro antitumor activity of asperphenamate derivatives as autophagy inducer. Bioorganic & medicinal chemistry letters 22 (6):2216-2220. Zhang J, Zhang J (2016) The filamentous fungal pellet and forces driving its formation. Critical reviews in biotechnology 36 (6):1066-1077. Zheng Q, Hou R, Juanyu, Zhang, Ma J, Wu Z, Wang G, Wang C, Xu JR (2013) The MAT locus genes play different roles in sexual reproduction and pathogenesis in Fusarium graminearum. PloS one 8 (6):e66980. Zhou Z, Dang Y, Zhou M, Li L, Yu C-h, Fu J, Chen S, Liu Y (2016) Codon usage is an important determinant of gene expression levels largely through its effects on transcription. Proceedings of the National Academy of Sciences 113 (41):E6117. Ziemons S, Koutsantas K, Becker K, Dahlmann T, Kück U (2017) Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes. BMC biotechnology 17 (1):16.

IX. Supplementary Data 75

IX. Supplementary Data Supplementary Table 1. P. brevicompactum strains crossed in various combinations. Numbers represent the conditions used for crossing experiments, that are listed in two next pages.

MAT1-1 CBS 256.31 CBS 257.29 CBS 110069 CBS 110070 CBS 110071 CBS 110072 CBS 118854 DMS 62871 CBS 1, 2, 3, 5 1, 2, 3, 5, 6, 7, 1, 2, 3, 6, 7, 1, 2, 3, 5, 6, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 6, 7, 119.37 6, 7, 8, 10, 11, 8, 10, 11, 12, 17, 18, 22, 7, 10, 22, 23, 22, 23, 24, 5 12, 17, 21, 22, 17, 18, 19, 21, 23, 24, 25, 24, 25, 26, 25, 26, 27 23, 24, 25, 26, 22, 23, 24, 25 26 27 27, 28, 29, 30, 26, 27, 28, 29, 31, 32, 33 30, 31, 32, 33 CBS 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 1, 2, 3, 6, 7, 1, 2, 3, 5, 6, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 6, 7, 287.53 22, 23, 24, 25, 7, 22, 23, 24, 22, 23, 24, 25, 7, 10, 22, 23, 22, 23, 24, 26, 27 25, 26, 27 26, 27 24, 25, 26, 25, 26, 27 27 CBS 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 6, 7, 317.59 8, 10, 17, 22, 7, 8, 9, 10, 10, 22, 23, 24, 7, 10, 22, 23, 22, 23, 24, 23, 24, 25, 26, 12, 13, 14, 25, 26, 27 24, 25, 26, 25, 26, 27 27, 28, 29, 30, 17, 22, 23, 27 31, 32, 33, 34, 24, 25, 26, 35 27, 28, 29, 30, 31, 32, 33, 34, 35 CBS 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 4, 6, 480.84 22, 23, 24, 25, 7, 22, 23, 24, 10, 22, 23, 24, 7, 10, 11, 16, 7, 11, 16, 22, 26, 27 25, 26, 27 25, 26, 27 22, 23, 24, 23, 24, 25, 25, 26, 27 26, 27 CBS 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 1, 2, 3, 6, 7, 1, 2, 3, 5, 6, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 6, 7, 629.66 22, 23, 24, 25, 7, 22, 23, 24, 22, 23, 24, 25, 7,10, 22, 23, 22, 23, 24, 26, 27, 34, 35 25, 26, 27, 26, 27 24, 25, 26, 25, 26, 27

MAT1-2 34, 35 27 CBS 1, 2, 3, 5, 6, 7, 1, 2, 3, 5, 6, 1, 2, 3, 6, 7, 1, 2, 3, 6, 7, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 6, 7, 110067 10, 22, 23, 24, 7, 10, 22, 23, 22, 23, 24, 25, 10, 22, 23, 22, 23, 24, 25, 26, 27 24, 25, 26, 26, 27 24, 25, 26, 25, 26, 27 27 27 CBS 1, 2, 5, 6, 7, 8, 1, 2, 5, 6, 7, 1, 2, 5, 7, 22, 1, 2, 5, 7, 10, 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 7 110068 22, 23, 24, 25, 8, 10,12, 13, 23, 24, 25, 26, 22, 23, 24, 22, 23, 24, 26, 27, 28, 29, 14, 15, 22, 27 25, 26, 27 25, 26, 27 30, 31, 32, 33 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 CBS 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9 112476 DMS 1, 2, 3, 6, 7, 1, 2, 3, 6, 7, 1, 2, 3, 6, 7, 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 1, 2, 3, 6, 7, 62828 10, 22, 23, 24, 10, 22, 23, 10, 22, 23, 24, 10, 22, 23, 25, 26 24, 25, 27 25, 26, 27 24, 25, 26, 27 IMI 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 380329 IMI 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 380346 IMI 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 3, 9, 11 380348

IX. Supplementary Data 76

List of conditions used for crossing experiments of P. brevicompactum

1: Oatmeal (Quicker)+ biotin, 10 ° C, anaerobe, dark, barrage zone

2: Oatmeal (Quicker)+ biotin, 15 ° C, anaerobe, dark, barrage zone

3: Oatmeal (Quicker)+ biotin, 20 ° C, anaerobe, dark, barrage zone

4: Oatmeal (Quicker) agar +TMS+ biotin, 20 ° C, anaerobe, dark, barrage zone

5: Oatmeal (Quicker)+ biotin, 23 ° C, anaerobe, dark, barrage zone

6: Oatmeal (Quicker)+ biotin, room temperature, anaerobe, dark, barrage zone

7: Oatmeal (Quicker)+ biotin, 27 ° C, anaerobe, dark, barrage zone

8: Oatmeal (Quicker)+ biotin, 10 ° C, aerobe, dark, barrage zone

9: Oatmeal (Quicker)+ biotin, 20 ° C, aerobe, dark, barrage zone

10: Oatmeal (Quicker)+ biotin, 23 ° C, aerobe, dark, barrage zone

11: Oatmeal (Quicker)+ CaCO3 (17 g/l) + biotin, 20 ° C, anaerobe, dark, barrage zone

12: Oatmeal (Quicker)+ CaCO3 (3 g/l) + biotin, 20 ° C, anaerobe, dark, barrage zone

13: Oatmeal (Quicker)+ CaCO3 (17 g/l) + biotin, 20 ° C, anaerobe, dark, mixture of protoplasts

14: Oatmeal (Quicker)+ biotin, 20 ° C, anaerobe, dark, mixture of protoplasts

15: CYA, 20 ° C, anaerobe, dark, barrage zone, mixture of protoplasts

16: Oatmeal (Kölln flocken & Frucht) + biotin, 20 ° C, anaerobe, dark, barrage zone

17: Bio-Oatmeal+ biotin, 20 ° C, anaerobe, dark, barrage zone

18: Oatmeal (Quicker)+ 5 mM inositol+ biotin, 20 ° C, anaerobe, dark, barrage zone

19: Oatmeal (Quicker)+ 10 mM inositol+ biotin, 20 ° C, anaerobe, dark, barrage zone

20: Oatmeal (Quicker)+ biotin, 20 ° C, anaerobe, 12 light-dark cycle, barrage zone

21: Oatmeal (Quicker)+ biotin, 20 ° C, sealed after 7 days of growth, 12 light-dark cycle, barrage zone

22: CYA, 20 ° C, anaerobe, dark, barrage zone

23: CYA+ biotin, 20 ° C, anaerobe, dark, barrage zone IX. Supplementary Data 77

24: CYA+ biotin, 20 ° C, sealed after 10 days and anaerobe for 2 month, dark, barrage zone

25: CYA+ biotin, 20 ° C, sealed after14 days and anaerobe for 2 month, dark, barrage zone

26: V8-agar, 20 ° C, anaerobe, dark, barrage zone

27: V8, 20 ° C, sealed after 7 days of growth, dark, barrage zone

28: Oatmeal (Quicker)+ biotin, 10 ° C, anaerobe, dark; fertilization

29: Oatmeal (Quicker)+ biotin, 20 ° C, anaerobe, dark; fertilization

30: Oatmeal (Quicker)+ biotin, 23 ° C, anaerobe, dark; fertilization

31: Oatmeal (Quicker)+ biotin, 10 ° C, aerobe, dark; fertilization

32: Oatmeal (Quicker)+ biotin, 20 ° C, aerobe, dark; fertilization

33: Oatmeal (Quicker)+ biotin, 23 ° C, aerobe, dark; fertilization

34: Murashige and Skoog (MS) medium + biotin, 20 ° C, anaerobe, dark; barrage zone

35: Murashige and Skoog (MS) medium + biotin, 20 ° C, anaerobe, dark; fertilization X. Eigenanteil an Publikationen 78

X. Eigenanteil an Publikationen

Mahmoudjanlou Y, Hoff B and Kück U (2019) Construction of a codon-adapted nourseotricin-resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum. Journal of Fungi (Basel) 10;5(4). pii: E96. doi: 10.3390/jof5040096.

Planung (P): 60%

Experimentelle Durchführung (E): 90 %

Verfassen des Manuskripts (M): 50 %

Mahmoudjanlou Y, Dahlmann TA, Kück U (2020) Molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum: Phylogeny and MAT protein characterization suggest a cryptic sexual life cycle. submitted to Fungal Biology (under review)

Planung (P): 50 %

Experimentelle Durchführung (E): 80 %

Verfassen des Manuskripts (M): 60 % XI. Curriculum Vitae 79

XI. Curriculum Vitae

Personal data

Yasaman Mahmoudjanlou

Email: [email protected] Date of birth: 25-04-1987 Place of birth: Tehran, Iran

Education

Since 09/2016 Ruhr-Universität Bochum, Bochum, Germany Doctoral studies of biology at the Department of General and Molecular Botany Title of the PhD thesis: Molecular biology of the mycophenolic acid producing fungus Penicillium brevicompactum

04/2013-04/2016 Ruhr-Universität Bochum, Bochum, Germany Masters degree in biology Title of the master thesis: The role of aryl hydrocarbon receptor in an animal model for multiple sclerosis

09/2005-08/2009 Islamic Azad University of Tehran, Tehran, Iran Bachelors degree in molecular and cellular biology

Publications

Mahmoudjanlou Y, Dahlmann TA, Kück U. (2020) Molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum: Phylogeny and MAT protein characterization suggest a cryptic sexual life cycle. Fungal Biol (under review)

Mahmoudjanlou Y, Hoff H, Kück U. (2019) Construction of a codon-adapted nourseotricin- resistance marker gene for efficient targeted gene deletion in the mycophenolic acid producer Penicillium brevicompactum. J. Fungi.

Berg J, Mahmoudjanlou Y, Duscha A, Massa M, Thöne J, Esser C, Gold R, Haghikia A (2016) The immunomodulatory effect of laquinimod in CNS autoimmunity is mediated by the aryl hydrocarbon receptor. J Neuroimmunol.

Forootanfar H, Moezzi A, Aghaie-Khozani M, Mahmoudjanlou Y, Ameri A, Niknejad F, Faramarzi MA (2012) Synthetic dye decolorization by three sources of fungal laccase. Iranian J Environ Health Sci Eng. 9-27. doi: 10.1186/1735-2746-9-27.

Hosseinzadeh N, Hasani M, Foroumadi A, Nadri H, Emami S, Samadi N, Faramarzi MA, Saniee P, Siavoshi F, Abadian N, Mahmoudjanlou Y, Sakhteman A, Moradi A, Shafiee A (2012) 5-Nitro-heteroarylidene analogs of 2-thiazolylimino-4-thiazolidinones as a novel series of antibacterial agents. Med Chem Re. 2293-2302. doi: 10.1007/s00044-012-0224-6.

XI. Curriculum Vitae 80

Workshop and congress attendance

Mahmoudjanlou Y, Frisvad JC, Kück U (2019) genetic engineering of the mycophenolic acid producing fungus. 13th Symposium of the VAAM Special Group Biology and Biotechnology of Fungi, Göttingen, Germany. Poster.

Mahmoudjanlou Y, Kück U (2018) Mating-type loci can be used for identification of strains of mycophenolic acid producer Penicillium brevicompactum. 3rd International & 15th Iranian Genetics Congress, Tehran, Iran. Presentation.

Mahmoudjanlou Y, Dahlmann TA, Kück U (2017) Usage of mating-type loci to identify strains from mycophenolic acid producer Penicillium brevicompactum. Annual Conference of the German Genetics Society (GFG). Poster.

Black Forest Summer School on NGS and phylogenetics, Herzogenhorn, Black Forest, Germany. 24.- 27 June 2017, Workshop

XII. Erklärung 81

XII. Erklärung Ich versichere an Eides statt, dass ich die eingereichte Dissertation selbstständig und ohne unzulässige fremde Hilfe verfasst, andere als die in ihr angegebene Literatur nicht benutzt und dass ich alle ganz oder annähernd übernommenen Textstellen sowie verwendete Grafiken und Tabellen kenntlich gemacht habe. Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten oder eine eindeutige Dokumentation von Art und Umfang der inhaltsverändernden Bildbearbeitung vorliegt. Außerdem versichere ich, dass es sich bei der von mir vorgelegten Dissertation (elektronische und gedruckte Version) um völlig übereinstimmende Exemplare handelt und die Dissertation in dieser oder ähnlicher Form noch nicht anderweitig als Promotionsleistung vorgelegt und bewertet wurde.

Es wurden keine anderen als die angegebenen Hilfsmittel verwendet.

Die Dissertation wurde gemäß der Promotionsordnung und der Betreuungsvereinbarung angefertigt.

Bochum, den 04.05.2020

Yasaman Mahmoudjanlou