MINI-REVIEW

The biotechnological use and potential of plant pathogenicsmut fungi

Michael Feldbrügge & Ronny Kellner & Kerstin Schipper

Received: 31 October 2012 /Revised: 12 February 2013 /Accepted: 13 February 2013 /Published online: 2 March 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Plant pathogens of the family Ustilaginaceae par-asitise mainly on grasses and cause smut disease. Amongthe best characterised members of this family are the cov-ered smut fungus Ustilago hordei colonising barley and oatas well as the head smut Sporisorium reilianum and the cornsmut Ustilago maydis, both infecting maize. Over the pastyears, U. maydis in particular has matured into a modelsystem for diverse topics like plant–pathogen interaction,cellular transport processes or DNA repair. Consequently, abroad set of genetic, molecular and system biologicalmethods has been established. This set currently serves asa strong foundation to improve existing and establishnovel biotechnological applications. Here, we review fourpromising aspects covering different fields of appliedscience: (1) synthesis of secondary metabolites producedat fermenter level. (2) Lipases and other hydrolytic en-zymes with potential roles in biocatalytic processes. (3)Degradation of ligno-cellulosic plant materials for bio-mass conversion. (4) Protein expression based on uncon-ventional secretion, a novel approach inspired by basicresearch on mRNA transport. Thus, plant pathogenicUstilaginaceae offer a great potential for future biotech-nological applications by combining basic research andapplied science.

Keywords Smut fungi . Secondary metabolite .

Bioconversion . Biofuel . Protein expression

Introduction

Ustilaginaceae comprise diverse plant pathogenic smut fun-gi that parasitise on grasses (Poaceae; Stoll et al. 2005;Begerow et al. 2006). The family currently includes 12genera of which Ustilago and Sporisorium constitute themost speciose groups (Vánky 2011). All parasiticUstilaginaceae species share a biotrophic growth mode thatrelies on a systemic infection without killing the host plant(Mendgen and Hahn 2002). This intimate interaction in-volves a highly specialised set of secreted effector proteinsto allow fungal proliferation and to concomitantly evade theplant defence system (Kämper et al. 2006; Schirawski et al.2010; Laurie et al. 2012; Hemetsberger et al. 2012; Djameiet al. 2011). During biotrophic growth, the fungus spreadswithin the plant tissue until massive production of thick-walled diploid spores occurs. Because disease symptoms areoften restricted to the inflorescences, some Ustilaginaceaespecies account for economically relevant losses in agriculture,like for example the covered smut of barley and oats (Ustilagohordei; Laurie et al. 2012). With respect to its biology, the cornsmut fungus Ustilago maydis represents the best characterisedmember within the Ustilaginaceae (see below).

Besides being investigated in basic research for varioustopics like DNA recombination and cell signalling (Holliday2004; Brefort et al. 2009), the Ustilaginaceae are currentlyrevealing a promising potential in terms of biotechnologicalquestions. In this regard, the production of secondary me-tabolites like glycolipids is the most advanced application(Bölker et al. 2008). The elucidation of the first smut ge-nome sequence and its detailed manual annotation (Kämperet al. 2006) has been a great step forward and allowed, for

Electronic supplementary material The online version of this article(doi:10.1007/s00253-013-4777-1) contains supplementary material,which is available to authorized users.

M. Feldbrügge :K. Schipper (*)Institute for Microbiology, Heinrich Heine University Düsseldorf,40204 Düsseldorf, Germanye-mail: kerstin.schipper@hhu.de

R. KellnerMax Planck Institute for Terrestrial Microbiology,Fungal Biodiversity, Karl-von-Frisch-Str. 10,35043 Marburg, Germany

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example, to propose the underlying biosynthesis routes.Two additional smut genomes were recently published(Schirawski et al. 2010; Laurie et al. 2012). Subsequently,genome mining and comparative genomics have been dem-onstrated to constitute powerful tools for the identificationof novel enzymes and the prediction of conserved proteindomains, helping to assign functional regions (Doehlemannet al. 2009; Stock et al. 2012).

The scope of this review is to summarise the biotechno-logical potential of plant pathogenic Ustilaginaceae species.We will cover four aspects with potential applications main-ly in red and white biotechnology: (1) synthesis of second-ary metabolites, (2) identification of enzymes forbiocatalysis, (3) application of smuts in biomass degradationand (4) as protein expression platform. For essential back-ground information, we briefly introduce the basic biologyof the Ustilaginaceae using the best investigated member,the corn smut U. maydis, as a blueprint.

Biology of plant pathogenic Ustilaginaceae

A characteristic scheme of the plant pathogenicUstilaginaceaeis their dimorphic lifecycle: on the one hand, the cells are ableto grow in a saprotrophic yeast-like form. On the other hand,they can grow as filaments, a growth mode that is directlylinked to pathogenic development (Bauer 2001; Begerow et al.2006). During the yeast-like growth haploid cells multiply bybudding (Fig. 1a). In this developmental stage, cells can beeasily cultivated in the laboratory (Brefort et al. 2009;Bakkeren and Kronstad 1993; Schirawski et al. 2005; Raboinet al. 2007). For U. maydis, duplication time during exponen-tial growth in liquid cultures is about two hours and is thereforecomparable to the Ascomycete fungus Saccharomycescerevisiae (Becht et al. 2005).

During plant infection two compatible yeast cells mate onthe leaf surface to form a dikaryotic filament which enters theplant tissue and subsequently induces the characteristic smutsymptoms (Fig. 1c). Cell–cell recognition and subsequent cellfusion is mediated by a pheromone/pheromone-receptor sys-tem encoded by the biallelic a locus, assuring that only cells ofdifferent mating type can fuse (Kahmann and Schirawski2007). Subsequently, filamentous growth is triggered by theheterodimeric key transcription factor bE/bW (Kämper et al.1995). To constitute an active heterodimer, subunits must bederived from different alleles provided after plasmogamy of thetwo compatible mating partners. In nature, the filamentousform is strictly connected to the parasitic stage (Kämper et al.2006). However, the detailed knowledge of the underlyingmolecular processes in U. maydis enabled the generation ofstrains in which filamentous growth can be mimicked in thelaboratory (e.g. strain AB33; Fig. 1a, b; Brachmann et al. 2001).This allows reproducible filament formation in liquid cultureunder simple laboratory conditions (Fig. 1b). Importantly,

similar strains could easily be generated for related speciesdue to the conserved molecular principle of the mating systemswithin the Ustilaginaceae (Kellner et al. 2011).

U. maydis serves as a eukaryotic model organism in basicresearch covering diverse topics like DNA recombination,signalling, RNA biology, cell biology and biotrophic plant–pathogen interactions (Bölker 2001; Holliday 2004; Steinbergand Pérez-Martin 2008; Brefort et al. 2009; Dean et al. 2012;

Fig. 1 The dimorphic corn smut fungus U. maydis. a Haploid yeast-likecells cultivated in liquid culture. Cells multiply by budding. b Filaments ofthe laboratory strain AB33 containing the bE/bW transcription factor undercontrol of the nitrate-inducible nar1 promoter (Brachmann et al. 2001). bE/bW expression is necessary and sufficient for filamentation (Kämper et al.1995; Brachmann et al. 2001) and was induced by a medium shift for 6 haccording to Baumann et al. (2012). cMaize plant infected withU.maydis.The resulting tumour localises to the cob where dark spore replace thekernels. d Tinned corn smut galls, “huitlacoche”, served as delicacy inLatin America and the USA. e Traditional Chinese dish prepared withJiaobai, a vegetable prepared from sliced shoots of Z. latifolia that areinfected with U. esculenta. Scale bars, 10 μm

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Vollmeister et al. 2012a). This broad research interest resultedin the development of a technically mature set of genetic,biochemical, cell biological, as well as system biologicalmethods (Brachmann et al. 2004; Scherer et al. 2006;Böhmer et al. 2007; Steinberg and Pérez-Martin 2008;Koepke et al. 2011). Sophisticated protocols for gene deletionand insertion mutants are available, and a FLP recombinase-based system allows the generation of multiple gene deletionsby resistance-marker recycling (Brachmann et al. 2004;Kämper et al. 2006; Khrunyk et al. 2010). Various inducibleand constitutive promoters have been described that allowcontrolled gene expression (Spellig et al. 1996; Hartmann etal. 1999; Brachmann et al. 2001; Zarnack et al. 2006).Furthermore, tagging of proteins with various epitopes andmarker proteins for detection (e.g. Gfp, Rfp and mCherry)and purification (e.g. Tap tag) can be performed (Brachmannet al. 2004; König et al. 2009; Baumann et al. 2012; http://www.mikrobiologie.hhu.de/ustilago-community/types-of-genetic-engeneering.html). The genome is sequenced, manu-ally annotated and transcriptomics as well as proteomics arewell established (Kämper et al. 2006; Scherer et al. 2006;Heimel et al. 2010; Koepke et al. 2011; MUMDB, http://mips.helmholtz-muenchen.de/genre/proj/ustilago). Moreover,besides cultivation in baffled shaking flasks, different fermen-tation protocols up to the range of a few litres culture volumehave been developed in the last years (Drews and Kraume2005; Drews and Kraume 2007; Klement et al. 2012). Morespecifically, the submerged fermentation of U. maydis andrelated Ustilaginaceae in the batch and fed-batch mode,chemostats as well as membrane bioreactors with biomassretention have been described (Drews and Kraume 2005,2007; Liu et al. 2011; Morita et al. 2011). Intriguingly, duringcultivation of yeast cells in bioreactors, this fungus combineskey advantages of yeasts and filaments, displaying a highhydromechanic stress resistance and the robustness againstculture impurities comparable to filamentous fungi (Klementet al. 2012). Furthermore, although glucose is the most effi-cient nutrient, U. maydis is able to grow on various carbonsources including arabinose, xylose, xylan, cellulose,polygalacturonic acid, homogenised plant tissues and biologi-cal (waste) oils (Cano-Canchola et al. 2000; Cortes-Sánchez etal. 2011; Klement et al. 2012; Spoeckner et al. 1999).

U. maydis displays a very narrow host range and infectsonly corn and its ancestor teosinte (Banuett 1992; Basse andSteinberg 2004). In Mexico, the resulting corn smut gallscalled “huitlacoche” are served as traditional dishes that havebeen consumed by humans for centuries (Fig. 1d). Nowadays,there is an increasing industrial market for this delicacy withcustomers in Latin America and the USA (Valverde et al.1995; Kämper et al. 2006; Vollmeister et al. 2012a). In addi-tion, “huitlacoche” is regarded as high-valued functional food-containing diverse bioactive and anti-mutagenic substances(Juárez-Montiel et al. 2011; Valdez-Morales et al. 2010).

Comparably, the aquatic perennial grass Zizania latifoliainfected with Ustilago esculenta has been domesticated inChina as a vegetable about 1,500 years ago. Nowadays, it iswidely cultivated in Asian countries for its nutritional valueand has gained substantial economic importance (Xu et al.2008). Different dishes are prepared from the infected plantshoot material called “Jiaobai” or “makomotake” (inChinese or Japanese, respectively; Fig. 1e; Xu et al. 2008;Suzuki et al. 2012). These two examples clearly indicate theinnocuousness of these fungi for humans.

Synthesis of secondary metabolites

Secondary metabolites are used in various applications, e.g.as antibiotics, UV protectants or biosurfactants (Vaishnavand Demain 2011). Interestingly, some of these moleculeshave also been discovered in U. maydis and otherUstilaginaceae species (Bölker et al. 2008). Here, we willfocus on the most promising substances, namely surface-active glycolipids, iron-chelating siderophores and trypto-phan derivatives. Importantly, genome mining allowed theelucidation of the corresponding biosynthesis routes withthe respective genes localising mostly in gene clusters(Hewald et al. 2005, 2006; Winterberg et al. 2010; Zutheret al. 2008).

U. maydis synthesises two glycolipids, a cellobiose lipidtermed ustilagic acid (Fig. 2) and a mannosylerythritol lipid(ustilipid/MEL; Bölker et al. 2008). In general, glycolipidsfind broad industrial applications in textile, paper, polymer,plastic, cosmetics, pharmaceuticals, food and machinery man-ufacture (Kitamoto et al. 2002). Ustilagic acids display potentsurface activity and act antagonistic to pro- and eukaryoticmicroorganisms (Haskins and Thorn 1951; Reed and Holder1953). Unfortunately, this medical resource has not been usedto date as the stability of ustilagic acids in oral application inhumans is low (Bölker et al. 2008). Nevertheless, with itsstrong surface activity the substance could still find appli-cations as a biosurfactant. Glycolipids of the ustilagic acidtype are only synthesised by a few basidomycete fungi,such as Pseudozyma flocculosa, a natural inhabitant of thephyllosphere representing an anamorphic member of theUstilaginaceae (Teichmann et al. 2011; Vánky 2011; seebelow). Flocculosin, the P. flocculosa glycolipid, is knownas a biocontrol agent acting against different fungal species,in particular human-pathogenic yeasts (Mimee et al. 2005).In contrast to flocculosin that is synthesised as an isomer,U. maydis produces a mixture of four ustilagic acid variants(Fig. 2a; Teichmann et al. 2011; Hewald et al. 2005). Thecorresponding biosynthetic enzymes are encoded in a genecluster consisting of 12 genes, including the central tran-scriptional regulator gene rua1 (Bölker et al. 2008;Teichmann et al. 2007; Teichmann et al. 2010). By con-trast, U. hordei and Sporisorium reilianum seem to lack

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similar gene clusters for ustilagic acid synthesis (Teichmannet al. 2011). Recent studies demonstrated that besides theirknown surface active properties ustilagic acids generated byU. maydis suppress plant infections with the grey mouldfungus Botrytis cinerea, indicating their potential as bio-control agents (Teichmann et al. 2007). Broader studiesincluding other plant-pathogenic microorganisms may pro-vide further insights into the application of these com-pounds in biocontrolling.

Even without further biotechnological optimisation, U.maydis yeast cultures produce ustilagic acids under nitrogen-limiting conditions at levels up to 20 g/L. Intriguingly,overexpression of rua1 resulted in ustilagic acid productioneven with a good nitrogen source (Teichmann et al. 2010).Based on the detailed knowledge of their biosynthesis furthergenetically engineered strains could be tested for improvedbiocontrol abilities through an even higher production orslightly altered structural compositions (Bölker et al. 2008).

MELs display several promising medical and biotechno-logical features like for example the ability to induce celldifferentiation, anti-tumour activity or binding affinity to-wards human immunoglobulin G (Singh and Cameotra2004). They could find further applications in skin and haircare, in moisturisers, oil-in-water-type emulsifiers and wash-ing detergents (Morita et al. 2010b; Morita et al. 2011). Likethe cellobiose lipids, also conventional MEL glycolipids aremainly produced by Ustilaginaceae species of the generaPseudozyma and Ustilago (Morita et al. 2009, 2011; Hewald

et al. 2006; Arutchelvi et al. 2008). MELs are oily, surface-active glycolipids consisting of mannosylerythritol disaccha-rides that are acetylated with short or medium chain fatty acidsat the mannosyl moiety (Hewald et al. 2006). Four variants,differing in the number of acetyl groups, have been described(MELA-D; Kitamoto et al. 1990) and their production patternstrongly depends on the individual organism (Morita et al.2011). U. maydis produces huge amounts of the three MELlipids A, B and C; whereas, MEL D is only secreted in minoramounts (Hewald et al. 2006). MEL biosynthesis is regulatedby a gene cluster consisting of five genes which are transcrip-tionally up-regulated upon nitrogen limitation. As for theustilagic acid biosynthesis cluster, the transcriptional regulatorcontrolling MEL production is known (Hewald et al. 2006).MEL production has also been reported for related smutsbesides U. maydis. Ustilago cynodontis, the Bermuda grasssmut fungus, predominantly produces MEL C-type glyco-lipids (Morita et al. 2008). In Sporisorium scitamineum(renamed from Ustilago scitaminea), the sugarcane smut fun-gus, MEL B is gained from sugarcane juice at levels up to25 g/L in bioreactors (Morita et al. 2009, 2011). The strongbiotechnological potential of glycolipid production in U.maydis and other smuts was recently further demonstrated ina fed-batch bioprocess that efficiently generated glycolipidsfrom crude glycerol, a by-product from the biodiesel industry.Using an optimised protocol, about 32 g/L total glycolipidswere obtained during this procedure (Liu et al. 2011).Furthermore, the use of vegetable and fish oils as carbon sourcefor glycolipid production has been reported (Spoeckner et al.1999; Cortes-Sánchez et al. 2011), showing that biologicalwaste products could be used as cheap and renewable growthsubstrates. Elimination of ustilagic acid that is toxic to the U.maydis cells when accumulating in high concentrations(Haskins and Thorn 1951; Teichmann et al. 2010) by deletingthe respective transcription factor could further stimulate pro-duction of the higher valued MEL compounds in a pure form(Hewald et al. 2006; Liu et al. 2011). A mutant which success-fully produces pure fully deacetylated MELs while simulta-neously being defective in ustilagic acid biosynthesis haspreviously been generated (Hewald et al. 2006). Thus, althoughmore established producers like Pseudozyma species are capa-ble of accumulating up to 140 g/L pure MELs in the culturesupernatant (Morita et al. 2010a), smut fungi comprise prom-ising organisms as a novel source of this secondary metabolite.

Although biotechnological glycolipid production is themost advanced application in Ustilaginaceae, some othersecondary metabolites also display the potential for use atindustrial level. Siderophores are chelating agents that re-cruit iron, an essential metal for almost every organism. Inbiotechnology, these iron chelators are used, for example, inclinical applications and in agriculture (Neilands 1995). Thefirst siderophore described in literature was identified in thesmut Ustilago sphaerogena (Neilands 1952). U. maydis

Fig. 2 Ustilagic acid production by U. maydis. a Molecular structureof ustilagic acid. b Ustilagic acid production (visible as needle-shapedcrystals) in the haploid U. maydis strain FB1 induced by nitrogenstarvation (Hewald et al. 2005). The culture was incubated in nitro-gen-starvation medium containing 5 % (w/v) glucose and 0.17 % (w/v)yeast nitrogen base without ammonium sulfate for 4 days at 28 °C(Hewald et al. 2005). Asterisks depict yeast-like cells; arrows indicateustilagic acid crystals. Scale bar, 10 μm

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produces two small cyclic hexapeptide siderophores,ferrichrome and ferrichrome A, by non-ribosomal peptidesynthesis (Wang et al. 1989; Mei et al. 1993; Winterberg etal. 2010; Yuan et al. 2001). Interestingly, siderophore produc-tion has even served as a model process in optimising mem-brane bioreactor cultivation (Drews and Kraume 2005, 2008;Drews and Kraume 2007). Thus, there seems the potential touse U. maydis for the large-scale production of siderophores.Tryptophan-derived indole pigments are of medical impor-tance displaying anti-inflammatory and UV-protectant capac-ities. U. maydis produces the compounds pityrianhydride, thepityriarubins A, B and C and pityriacitrin. However, thebiotechnological and economic relevance of pigment produc-tion in smut fungi is rather questionable, as it was demonstrat-ed that those compounds can be generated spontaneously anddo not need enzymatic conversion (Zuther et al. 2008).

The full range of applicable secondary metabolites ofsmut fungi has probably not yet been discovered. Furtherpromising substances are UV-protective melanin or N-glycosids with anti-proliferative activities (saponins orglycoalkaloids; Suzuki et al. 2012). Also secreted β-D-glycans may represent compounds that can be gained fromUstilaginaceae and may find application, e.g. as thickeningagents in food industry (Forseca-García et al. 2011).However, additional research in this direction needs to beconducted to elucidate the full potential. This will alsoinclude novel strategies like the identification of crypticmetabolic pathways that are silent under standard conditionsby overexpression of putative transcriptional activators(Bergmann et al. 2007).

Identification of enzymes with novel properties

Microbial enzymes with novel properties are promisingcandidates for applications in life sciences and industry,for example as biocatalysts. In the rapidly growing field ofbiocatalysis, enzymes are utilised for the conversion oforganic compounds. In industrial manufacturing, this pro-cess is often preferred over chemical transformation, be-cause it is powerful, highly selective and in most casesmore efficient and economical in conducting key reactionsteps (Nestl et al. 2011). The produced enantiopure com-pounds are subsequently being used as building blocks formore complex substances, such as drugs and agrochemicals.

Nowadays, along with metagenome libraries sequencedspecies are the most promising pools for the exploitation ofnovel enzymes by genome mining. As mentioned before,the genome of U. maydis was the first smut genome publiclyavailable. More than 6,900 protein-encoding genes wereidentified (Kämper et al. 2006). This has already led to theidentification of several enzymes that may find a futureapplication in biotechnology. More recently, the genomesof two related biotrophic crop pathogens, the head smut

fungus S. reilianum and the covered smut U. hordei, werereleased (Schirawski et al. 2010; Laurie et al. 2012;MUHDB, http://mips.helmholtz-muenchen.de/genre/proj/MUHDB; MSRDB, http://mips.helmholtz-muenchen.de/genre/proj/sporisorium). Both genomes display a highsynteny in comparison to the genome of U. maydis, whichfacilitates genome mining and thereby, will greatly acceler-ate the elucidation of the enzymatic potential of smut fungi.

Prime examples for high-valued biocatalysts are lipases.These carboxylesterases catalyse the hydrolysis and synthesisof long-chain acylglycerols with trioleoylglycerol at the inter-face of a lipid and an aqueous phase. In industry, lipases arefrequently applied in the production of enantiopure com-pounds, for example fine chemicals or biopolymers (Jaegerand Eggert, 2002). A well-known example for fungal lipaseswith industrial applications is CalB from the basidiomyceteousyeast Pseudozyma aphidis (renamed from Candida antarctica;Fig. 3a). CalB exhibits high enantioselectivity towards second-ary alcohols and thus constitutes a valuable catalyst in manyindustrial processes (Bornscheuer and Kazlauskas 1999). Thissecreted enzyme has been industrially produced and sold inlarge-scale for years (Vakhlu JK 2006). Some years ago, thephylogenetic position of P. aphidis has been revised and it hasbeen placed into the anamorphic genus Pseudozyma in thefamily of the Ustilaginaceae, thus linking it closely to thepathogenic members of this family (Fig. 3a; Begerow et al.2000). In line with this phylogenetic relationship, the threesequenced members of the Ustilaginaceae harbour close ho-mologs of CalB. TheU.maydis CalB homolog Uml1 has beenproduced in the Ascomycete Pichia pastoris and characterisedwith respect to its hydrolytic activity towards (phospho)lipidsand esters. Despite their sequence homology, distinct differ-ences in the enzyme specificities were detected, e.g. a phos-pholipase activity that is absent in P. aphidis CalB (C. Bürth,K.-E. Jäger, J. Ernst and D. Tielker, personal communication).It will be interesting to additionally determine the activityprofiles of the orthologs of the other two species and to analysewhether observed differences correspond to specific sequencedissimilarities in comparison to the P. aphidis lipase. Hence,we speculate that the availability of sequence information fromthree close relatives will allow the identification of divergentand conserved regions that may account for differences orsimilarities in specificity, respectively. Thus, in the futuredomain mapping based on phylogenetic sequence compari-sons could help to assign specific activities as a basis foroptimisation of enzyme functions.

Bioinformatic predictions indicate that the genome of U.maydis encodes at least ten other secreted lipases with poten-tially interesting activities (Müller et al. 2008). In line with this,an early study from 1995 described that culture supernatantscontain lipases that display interesting characteristics withrespect to hydrolysis and ester synthesis (Katsivela et al.1995). Crude enzyme extracts were shown to mediate the

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preferential hydrolysis of fatty acid ethylesters (fatty acid chainlength C8, C10 and C18:2), whereas the enzyme mix exhibitedonly low specificity towards simple triacylglycerols (Katsivelaet al. 1995). Furthermore, a CalA-type lipase (Um03410) hasbeen characterised in detail (Brundiek et al. 2012). Comparableto CalB, this enzyme belongs to the closest homologs of CalAfrom the basidiomycetous yeast P. aphidis (Fig. 3b). P. aphidisCalA shows the unique property of accepting very bulky sub-strates as well as being selective towards trans- over thecorresponding cis-fatty esters (Kirk and Christensen 2002).Although CalA-type lipases are mostly unexplored with re-spect to industrial use, they constitute promising biocatalystsdue to their unusual selectivity (Brundiek et al. 2012).Interestingly, the trans-fatty acid activity could be con-firmed for a functional truncated version of U. maydisCalA expressed in P. aphidis, resembling the only otherlipase with this property until now (Brundiek et al. 2012).As already speculated by the authors based on sequencecomparisons (Brundiek et al. 2012), the initial automaticannotation of U. maydis CalA turned out to be inaccurateand the coding region was manually corrected, resulting ina shortened secreted protein version (now called Um11070;MUMDB) that displays 67 % sequence identity to P.aphidis CalA. This underlines the need to manuallyrecheck automatically annotated sequences stored inNCBI. Orthologous genes for lipases are also present inS. reilianum and U. hordei (Sr14401 and UHOR_05268;identities of 65 % each to the P. aphidis enzyme; Fig. 3b)but have not been studied yet.

Further enzymes with promising catalytic properties willlikely be discovered in the next years. Interesting candidatesfor which bioinformatic or experimental evidence exists couldbe, for example, phytases, multi-copper oxidases or β-glucosidases (Müller et al., 2008; Kües and Rühl 2011;Floudas et al. 2012). Phytases play an important role in indus-try as they are used for phosphate liberation (Lei and Porres2003). The main application of these enzymes is animal andhuman food processing. U. maydis encodes three as yetunexplored proteins with putative phytase activities (Mülleret al. 2008). The enzyme family of the multi-copper oxidasescomprises different subgroups and its members could findmultiple industrial applications (Hoegger et al. 2006; Küesand Rühl 2011). U. maydis harbours six proteins with amino-acid similarities to the members of different multi-copperoxidase subgroups (Floudas et al. 2012; Kües and Rühl2011). β-glucosidases have multiple applications like, i.e.flavour enhancement, detoxification and glucose production(Nakajima et al. 2012a). A β-glucosidase and a glycosidehydrolase of U. esculenta were recently shown to have theability to efficiently hydrolyse laminarin and other β-glucans(Nakajima et al. 2012a, b).

In conclusion, a few interesting enzymes have already beenidentified in U. maydis. The discovery of other candidates by

genome mining and their subsequent experimental characteri-sation remains an important future task.

Degradation of ligno-cellulosic biomass

Ligno-cellulosic biomass, like, e.g. non-food plant parts,woody crops, sugarcane bagasse or agricultural residues, isgaining increasing interest as a source for second generationbiofuel production. However, the current bottleneck is thecomplex cost- and energy-consuming step of converting thelignin-rich plant matter to simple sugars or platform chemicals(i.e. itaconic acid or glycerol; Margeot et al. 2009; Jäger andBüchs 2012). Lignin acts as the connecting compound be-tween the cellulose and hemi-cellulose components and thus,needs to be degraded to access the high-valued carbohydratesfor subsequent fermentation to ethanol. Right now, harshchemical treatments are used to remove the lignin part. For abiological conversion, identification of organisms that are ca-pable of efficiently degrading lignin-rich plants to generatecrude supplies is crucial. This would allow a sustainable andeconomic production of biofuels (Sanchez 2009; Chundawatet al. 2011). Filamentous fungi have a great potential in plant-cell wall degradation as they encounter similar substrates intheir environments. Currently, the main research focus is di-rected toward Ascomycete fungi, like, e.g. Trichoderma reeseior Aspergillus species as well as toward Agaricomycetes com-prising for example the brown andwhite rot fungi (Couturier etal. 2012; Floudas et al. 2012). Due to the nature of theirbiology, basidiomycetous plant pathogens may be furtherpromising repositories for such enzymes.

U. maydis is equipped with a rather limited set of lyticenzymes (Kämper et al. 2006; Floudas et al. 2012). Of 168secreted enzymes, only 33 cell-wall degrading enzymes(CWDEs) are encoded in the genome (Kämper et al. 2006).This low number of CWDE likely alludes to the biotrophicgrowth mode of smut fungi that aims at keeping host cellsalive (Mendgen and Hahn 2002; Kämper et al. 2006; Brefortet al. 2009). According to bioinformatic predictions, the se-creted CWDEs mainly function as glucanases and xylanases(Doehlemann et al. 2008; Müller et al. 2008). Most of theseenzymes are up-regulated during plant invasion, suggestingthat their activity is associated with proliferation in the leaftissue and subsequent tumour formation (Doehlemann et al.2008). Although the enzyme equipment is restricted, thisfungus has recently been described as a promising organismwith respect to an application in biomass conversion. Firstly,U. maydis has been demonstrated to efficiently release sugarsfrommicronised wheat straw. This is likely due to secretion ofa limited but strikingly potent mix of saccharification enzymesincluding a comparatively high abundance of oxidoreductasesthat could ease the depolymerisation of ligno-cellulosic mate-rial (Couturier et al. 2012). Thus, in synergy with potenthydrolytic enzymes of other fungi, CWDEs of U. maydis

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and other Ustilaginaceae species might constitute good sup-plements in efficient enzymatic release of sugars from bio-mass—especially material that is derived from monocots likemaize or wheat (Couturier et al. 2012). Furthermore, due to itssuperb genetic tractability, additional enzymes could be easilyintroduced into the genome to generate an even more potentenzyme cocktail and enhance the capacity of U. maydis indegrading ligno-cellulosic biomass.

In addition, U. maydis was shown to synthesise itaconicacid, a biomass-derived platform chemical, from glucose orxylose (Haskins et al. 1955; Klement et al. 2012). In thefuture, this acid may deal as raw material for the synthesis ofbiofuel components like 3-methyltetrahydrofuran or 2-methylbutanediol (Geilen et al. 2010). Moreover, the studydemonstrated that U. maydis is relatively robust with respect

to impurities in the pretreated biomass used for fermentation(Klement et al. 2012). Hence, U. maydis is now beingregarded as a potent organism to support the industrialbioconversion process.

Establishment of novel protein expression platforms

Research over the past decades demonstrated that eachprotein seems to have its own specific needs with respectto expression conditions. At present, none of the existingexpression systems fulfils the requirement to produce alldesired proteins (Demain and Vaishnav 2009). Instead ofoptimising poor expression of the protein of interest in aninappropriate system, it is more efficient to choose a moresuitable expression platform. Therefore, there is an urgent

Fig. 3 Homology of P. aphidis CalA to smut lipases. a, Multi-genephylogeny of biotechnological relevant basidiomyceteous andascomyceteous fungi. The three sequenced Ustilaginaceae and theclosely related biotechnological important species P. aphidis are indi-cated in red and by enlarged fonts. Circles next to branches indicatebootstrap support values and a posteriori probabilities of maximumlikelihood and Bayesian analyses, respectively. Branch lengths corre-spond to substitutions per site. Species names in brackets representsynonyms of respective species. For a detailed description of the

phylogenetic analysis see Electronic supplementary material. b, Aminoacid sequence alignment of CalA-type lipases from P. aphidis (Pa), U.maydis (Um), U. hordei (Uh) and S. reilianum (Sr). Conserved aminoacids are shaded black. The predicted signal peptide cleavage site isindicated by a red arrowhead (SignalP 3.0). Red asterisks indicate thecatalytic triade; grey triangles mark the position of the four conservedcysteine residues forming disulphide bridges. The Kex2 cleavage sitefor removal of the pro-peptide is boxed

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need to establish alternative expression platforms to fillthese gaps (Schmidt 2004; Sodoyer 2004).

The main current platforms for the expression of re-combinant proteins for industrial and pharmacologicalpurposes are bacterial and eukaryotic microorganisms aswell as eukaryotic cell cultures (Schmidt 2004; Demainand Vaishnav 2009; Graumann and Premstaller 2006).Produced molecules either accumulate cytoplasmically,in an intracellular organelle or are secreted to the cellbroth. In general, the latter is of higher commercialrelevance since simple and fast downstream processingprocedures can be applied and costly cell rupture, dena-turation and refolding processes are avoided (Schmidt2004). Moreover, synthesis of proteins harbouring di-sulphide bonds largely relies on secretory systems asthe oxidative environment outside the cells is crucial forproper folding (Moir and Mao 1990). Among the bestexamples for eukaryotic microorganisms used for indus-trial protein production are on the one hand yeasts, such as S.cerevisiae, P. pastoris or Hansenula polymorpha and on theother hand filamentous fungi like Aspergillus or Trichoderma(Demain and Vaishnav 2009; Ward 2012). Filamentous fungioffer the clear advantage that they possess a highly efficientsecretory machinery that is streamlined to secrete a number ofdifferent enzymes such as hydrolases and proteases (Ward2012).

Recently, basic research on mRNA transport in themodel organism U. maydis has inspired the idea for anovel protein expression system based on unconventionalsecretion (Koepke et al. 2011; Stock et al. 2012). Infungal filaments, mRNA transport is mainly achievedby the RNA-binding protein Rrm4, which hitchhikes onendosomes shuttling along microtubules (Becht et al.2005, 2006; König et al. 2009; Baumann et al. 2012;Vollmeister et al. 2012b, Göhre et al. 2012). Apart fromits important role in the establishment of unipolar growthlikely mediated through local translation of transportedmRNAs, this post-transcriptional process has been shownto be crucial for the secretion of an endochitinase, Cts1(Fig. 4a; Koepke et al. 2011). Interestingly, Cts1 secre-tion occurs through a yet unknown unconventional secre-tory pathway, as it lacks an N-terminal secretion signaland its N-terminal domain is dispensable for export(Stock et al. 2012). This hypothesis is further supportedby data obtained with a β-glucuronidase (Gus) reportersystem (Fig. 4b). Upon secretion via the conventionalsecretory pathway involving passage of the endoplasmicreticulum and the Golgi apparatus this bacterial enzymegets inactivated by N-glycosylation (Iturriaga et al. 1989).By contrast, active Gus could be detected in culturesupernatants of U. maydis strains expressing a fusionprotein of Gus and Cts1, suggesting that this pathwaycircumvents N-glycosylation (Fig. 4b; Stock et al. 2012).

The exploitation of this unique feature for the export ofheterologous proteins constitutes the basic idea for thenovel protein expression platform. This will be advanta-geous, e.g. for the production of sugar-free proteins formedical purposes to avoid immune reactions in humanapplications.

During the further investigation of the unconventional Cts1secretion pathway, it was demonstrated that unconventionalsecretion of Cts1 does not only occur in filamentous but alsoin yeast-like cells (Stock et al. 2012). Hence, the novel path-way permits using both cell types for protein expression.Successful secretion of foreign proteins as Cts1 hybrid pro-teins could be shown, e.g. for a single-chain antibody directedagainst the c-Myc epitope. Besides the advantage of avoidingN-glycosylation, the system is capable of secreting huge pro-teins (with a 173-kDa fusion protein being the largest mole-cule tested until now; Stock et al. 2012). Furthermore, theexisting broad range of molecular and biochemical tools aswell as its robustness during fermentation renderU.maydis anideal organism for secretory protein production. A combina-tion of genetic manipulation and fermentation may eliminatethe current drawback of the system, the quite low amount ofsecreted protein (Stock et al. 2012).

Despite the application of the mechanism of uncon-ventional secretion, it is conceivable that U. maydiswould also allow intracellular protein production orsecretion through the conventional pathway. To thisend, the N-glycosylation machinery constitutes a keyproperty as hyperglycosylation is a major drawback inproducing some proteins of, for example, medical im-portance. In a recent paper, glycosylation in U. maydishas been studied in detail by means of bioinformaticpredictions, revealing the seeming absence of enzymesfor the elongation and extension of N-linked core glycosyla-tion (Fernandez-Alvarez et al. 2010). This could indicate thatsecreted U. maydis proteins may possess a simpler core sugarstructure than, for example, S. cerevisiae proteins. Hence, thisfungus may represent a promising alternative to humanisingthe glycosylation pathway of other organisms used for proteinexpression (Fernandez-Alvarez et al. 2010; De Pourcq et al.2010).

In all potential applications as protein expressionsystem, secreted but also intracellular proteases maynegatively affect the yields since they are potentiallyinvolved in degrading the produced proteins. Based oncurrent bioinformatics predictions, the U. maydis ge-nome encodes a limited set of proteases (e.g. about 30proteases annotated in MUMDB). The presence and theactivity of several U. maydis proteases have been prov-en experimentally, including for example an acid and aneutral protease as well as an aminopeptidase and adipeptidylaminopeptidase (Mercado-Flores et al. 2003a, b).However, the exact nature of these enzymes has not

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been elucidated to date. The generation of proteasedeletion mutants and the subsequent analysis of down-stream effects on protein secretion will be an interestingfuture task. Deleting key proteases has for example beenshown to increase yields of protease-sensitive proteinssignificantly in fission yeast or Aspergillus oryzae (Idiriset al. 2006, 2010; Jin et al. 2007). The marker-recyclingsystem provides the possibility of generating multipledeletions in a single U. maydis strain (Khrunyk et al.2010). In this respect, the relatively low number ofproteases in U. maydis in comparison to other expres-sion hosts (e.g. 135 in A. oryzae; Machida et al. 2005)constitutes a further advantage.

In summary, U. maydis offers the potential to functionas an expression system for cytoplasmic production ofproteins on the one hand or for either conventional orunconventional secretion of proteins on the other hand,depending on the specific requirements of the product. Inthe far future, it is even conceivable to use smut-inducedtumours occurring in plant infections with U. maydis orU. esculenta, e.g. for the generation of oral vaccines(Daniell et al. 2001; Mason et al. 2002).

Conclusions

In recent years, great progress has been made with respect tobasic research on different plant pathogenic Ustilaginaceae

species. In addition, first studies on applied aspects cov-ering diverse fields are accumulating. Now, it is impor-tant to push these new research directions forward. Tothis end, genetic engineering of known pathways usingthe strong advantages of detailed sequence informationand genetic tractability will be the key to increasingproduct yields and quality. Subsequently, up-scaling tothe bioreactor level may allow for the commercial ex-ploitation of a product.

Until now, research mainly focussed on the sequencedmodel smuts U. maydis, S. reilianum and U. hordeiwhereas other species of the Ustilaginaceae did not re-ceive much attention. Sequence comparisons using theavailable genomes indicate that it would be advantageousto enlarge the focus and sequence additional smuts. In thefuture, we foresee that comparable to the 1,000 fungalgenomes project initiated by the Joint Genome Institute(US Department of Energy), it will soon be feasible andhighly valuable to sequence an equal number of smutgenomes. Since sequencing costs are constantly dropping,the limiting step will be gathering of environmentalsamples (Vánky 2011). It is easily conceivable that onlya small repertoire of the secondary metabolite and en-zyme capacity buried in the genomes of Ustilaginaceaespecies has been extracted to date. Because transferringestablished methods to other smuts will in all likelihoodbe simple, much more smut species could soon disclosetheir biotechnological potential.

Fig. 4 Applying unconventional secretion in U. maydis to exportactive foreign proteins. a Model depicting the mRNA-transport-medi-ated secretion of the endochitinase Cts1 at the filament tip. Motorproteins drive the shuttling of endosomes along microtubules. Thekey mRNA-binding protein Rrm4 co-localises with these endosomesand is thought to distribute the mRNAs through the cell. The exactmolecular link to Cts1 secretion is not clear. Symbols are explained inthe inlay. The model was slightly modified from Göhre et al. (2012). bLeft panel, secretion of active Gus was assayed using the specificsubstrate 4-methylumbelliferyl β-D-galactopyranoside (MUG). Strainswere grown to the exponential phase, dropped on MUG containing

(final concentration, 2 mM) complete medium plates supplementedwith 1 % (w/v) glucose (Holliday 1974) and incubated for 1 day at28 °C. Generation of the fluorescent product 4-methylumbelliferonewas detected using UV light. (1) AB33 expressing the unconvention-ally secreted Gus-Cts1 fusion protein, (2) AB33 expressing Gus fusedto a conventional signal peptide (inactive due to N-glycosylation), (3)AB33 producing cytoplasmic Gus (lysis control). Right panel, sche-matic representation of the open reading frames: blue bar, Gus; greenbar, Cts1, red bar, signal peptide for conventional secretion; asteriskindicates N-glycosylation of Gus during ER/Golgi passage

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Acknowledgements We acknowledge all lab members for valuablediscussion and critical reading of the manuscript. Special thanks forproviding the photos and the chemical structure displayed in Figs. 1, 2,3 and 4 to Dr. Longjang Fan, Hanne Horn, Dorothee Schipper,Thorsten Langner, Janpeter Stock and Sebastian Schulz, respectively.We further acknowledge the excellent technical assistance by BettinaAxler. Our applied work was supported by the Ministry of Innovation,Science and Research of North Rhine-Westphalia and the HeinrichHeine University Düsseldorf (HHUD) through funding within theCLIB-Graduate Cluster Industrial Biotechnology and by a grant fromthe Strategic Research Fund of the HHUD to KS. Basic research in thelaboratory was funded by the Deutsche Forschungsgemeinschaft(DFG) as part of the German/Mexican research group FOR1334 (FE448/5-1), DFG grant FE448/3-2 as well as the HHUD graduate schoolsiGRAD-MOI and iGRAD-Plant.

Conflict of interest None

References

Arutchelvi JI, Bhaduri S, Uppara PV, DobleM (2008)Mannosylerythritollipids: a review. J Ind Microbiol Biotechnol 35:1559–1570.doi:10.1007/s10295-008-0460-4

Bakkeren G, Kronstad JW (1993) Conservation of the b mating-typegene complex among bipolar and tetrapolar smut fungi. Plant Cell5:123–136. doi:10.1105/tpc.5.1.123

Banuett F (1992) Ustilago maydis, the delightful blight. Trends Genet8:174–180

Basse CW, Steinberg G (2004) Ustilago maydis, model system foranalysis of the molecular basis of fungal pathogenicity. Mol PlantPathol 5:83–92. doi:10.1111/j.1364-3703.2004.00210.x

Bauer R, Begerow D, Oberwinkler F, Piepenbring M, Berbee ML(2001) Ustilaginomycetes. In: McLaughlin DJ, McLaughlin EG,Lemker PA (eds) The mycota VII. Systematics and evolution, partB. Springer, Berlin, pp 57–83

Baumann S, Pohlmann T, Jungbluth M, Brachmann A, Feldbrügge M(2012) Kinesin-3 and dynein mediate microtubule-dependent co-transport of mRNPs and endosomes. J Cell Sci 125:2740–2752.doi:10.1242/jcs.101212

Becht P, Vollmeister E, Feldbrügge M (2005) Role for RNA-bindingproteins implicated in pathogenic development of Ustilago maydis.Eukaryot Cell 4:121–133. doi:10.1128/EC.4.1.121-133.2005

Becht P, König J, Feldbrügge M (2006) The RNA-binding protein Rrm4is essential for polarity in Ustilago maydis and shuttles along mi-crotubules. J Cell Sci 119:4964–4973. doi:10.1242/jcs.03287

Begerow D, Bauer R, Boekhout T (2000) Phylogenetic placements ofustilaginomycetous anamorphs as deduced from nuclear LSUrDNA sequences. Mycol Res 104:53–60

Begerow D, Stoll M, Bauer R (2006) A phylogenetic hypothesis ofUstilaginomycotina based on multiple gene analyses and morpho-logical data. Mycologia 98:906–916

Bergmann S, Schürmann J, Scherlach K, Lange C, Brakhage AA,Hertweck C (2007) Genomics-driven discovery of PKS-NRPShybrid metabolites from Aspergillus nidulans. Nat Chem Biol3:213–217

Böhmer M, Colby T, Böhmer C, Bräutigam A, Schmidt J, Bölker M(2007) Proteomic analysis of dimorphic transition in the phyto-pathogenic fungus Ustilago maydis. Proteomics 7:675–685.doi:10.1002/pmic.200600900

Bölker M (2001)Ustilago maydis—a valuable model system for the studyof fungal dimorphism and virulence. Microbiology 147:1395–1401

Bölker M, Basse CW, Schirawski J (2008) Ustilago maydis secondarymetabolism-from genomics to biochemistry. Fungal Genet Biol45:S88–93. doi:10.1016/j.fgb.2008.05.007

Bornscheuer UT, Kazlauskas RJ (1999) Hydrolases in organic synthe-sis: region- and stereoselective biotransformations, 2nd edn.Wiley, Weinheim

Brachmann A, Weinzierl G, Kämper J, Kahmann R (2001) Identificationof genes in the bW/bE regulatory cascade in Ustilago maydis. MolMicrobiol 42:1047–1063

Brachmann A, König J, Julius C, Feldbrügge M (2004) A reversegenetic approach for generating gene replacement mutants inUstilago maydis. Mol Genet Genom 272:216–226. doi:10.1007/s00438-004-1047-z

Brefort T, Doehlemann G, Mendoza-Mendoza A, Reissmann S,Djamei A, Kahmann R (2009) Ustilago maydis as a pathogen.Annu Rev Phytopathol 47:423–445. doi:10.1146/annurev-phyto-080508-081923

Brundiek H, Saß S, Evitt A, Kourist R, Bornscheuer UT (2012) The shortform of the recombinant CAL-A-type lipase UM03410 from thesmut fungus Ustilago maydis exhibits an inherent trans-fatty acidselectivity. Appl Microbiol Biotechnol 94:141–150. doi:10.1007/s00253-012-3903-9

Cano-Canchola C, Acevedo L, Ponce-Noyola P, Flores-Martínez A,Flores-Carreón A, Leal-Morales CA (2000) Induction of lytic en-zymes by the interaction of Ustilago maydis with Zea mays tissues.Fungal Genet Biol 29:145–151. doi:10.1006/fgbi.2000.1196

Chundawat SP, Beckham GT, Himmel ME, Dale BE (2011)Deconstruction of lignocellulosic biomass to fuels and chemicals.Annu Rev Chem Biomol Eng 2:121–145. doi:10.1146/annurev-chembioeng-061010-114205

Cortes-Sánchez A, Hernández-Sánchez H, Jaramillo-Flores M (2011)Production of glycolipids with antimicrobial activity by Ustilagomaydis FBD12 in submerged culture. Afr J Microbiol Res5:2512–2523

Couturier M, Navarro D, Olivé C, Chevret D, Haon M, Favel A,Lesage-Meessen L, Henrissat B, Coutinho PM, Berrin JG(2012) Post-genomic analyses of fungal lignocellulosic biomassdegradation reveal the unexpected potential of the plant path-ogen Ustilago maydis. BMC Genomics 2:57. doi:10.1186/1471-2164-13-57

Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming:production of antibodies, biopharmaceuticals and edible vaccinesin plants. Trends Plant Sci 6:219–226

De Pourcq K, De Schutter K, Callewaert N (2010) Engineering ofglycosylation in yeast and other fungi: current state and perspec-tives. Appl Microbiol Biotechnol 87:1617–1631. doi:10.1007/s00253-010-2721-1

Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di PietroA, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, FosterGD (2012) The Top 10 fungal pathogens in molecular plantpathology. Mol Plant Pathol 13:414–430. doi:10.1111/j.1364-3703.2011.00783.x

Demain AL, Vaishnav P (2009) Production of recombinant proteins bymicrobes and higher organisms. Biotechnol Adv 27:297–306.doi:10.1016/j.biotechadv.2009.01.008

Djamei A, Schipper K, Rabe F, Ghosh A, Vincon V, Kahnt J, Osorio S,Tohge T, Fernie AR, Feussner I, Feussner K, Meinicke P, StierhofYD, Schwarz H, Macek B, Mann M, Kahmann R (2011) Metabolicpriming by a secreted fungal effector. Nature 478:395–398.doi:10.1038/nature10454

Doehlemann G, Wahl R, Vranes M, de Vries RP, Kämper J, KahmannR (2008) Establishment of compatibility in the Ustilago maydis/maize pathosystem. J Plant Physiol 165:29–40. doi:10.1016/j.jplph.2007.05.016

Doehlemann G, van der Linde K, Assmann D, Schwammbach D, HofA, Mohanty A, Jackson D, Kahmann R (2009) Pep1, a secretedeffector protein of Ustilago maydis, is required for successfulinvasion of plant cells. PLoS Pathog 5:e1000290. doi:10.1371/journal.ppat.1000290

3262 Appl Microbiol Biotechnol (2013) 97:3253–3265

Drews A, Arellano-Garcia H (2008) Model-based determination ofchanging kinetics in high cell density cultures using respirationdata. Chem Eng Sci 63:4789–4799

Drews A, Kraume M (2005) Process improvement by application ofmembrane bioreactors. Chem Eng Res Design 83:276–284

Drews A, Kraume M (2007) On maintenance models in severely andlong-term limited membrane bioreactor cultivations. BiotechnolBioeng 96:892–903. doi:10.1002/bit.21211

Fernández-Alvarez A, Elías-Villalobos A, Ibeas JI (2010) Proteinglycosylation in the phytopathogen Ustilago maydis: From coreoligosaccharide synthesis to the ER glycoprotein quality controlsystem, a genomic analysis. Fungal Genet Biol 47:727–735.doi:10.1016/j.fgb.2010.06.004

Floudas D, Binder M, Riley R, Barry K, Blanchette RA, Henrissat B,Martínez AT, Otillar R, Spatafora JW, Yadav JS, Aerts A, BenoitI, Boyd A, Carlson A, Copeland A, Coutinho PM, de Vries RP,Ferreira P, Findley K, Foster B, Gaskell J, Glotzer D, Górecki P,Heitman J, Hesse C, Hori C, Igarashi K, Jurgens JA, Kallen N,Kersten P, Kohler A, Kües U, Kumar TK, Kuo A, LaButti K,Larrondo LF, Lindquist E, Ling A, Lombard V, Lucas S, LundellT, Martin R, McLaughlin DJ, Morgenstern I, Morin E, Murat C,Nagy LG, Nolan M, Ohm RA, Patyshakuliyeva A, Rokas A,Ruiz-Dueñas FJ, Sabat G, Salamov A, Samejima M, Schmutz J,Slot JC, St John F, Stenlid J, Sun H, Sun S, Syed K, Tsang A,Wiebenga A, Young D, Pisabarro A, Eastwood DC, Martin F,Cullen D, Grigoriev IV, Hibbett DS (2012) The Paleozoic originof enzymatic lignin decomposition reconstructed from 31 fungalgenomes. Science 336:1715–1719. doi:10.1126/science.1221748

Fonseca-García C, López MG, Aréchiga-Carvajal ET, Ruiz-Herrera J(2011) A novel polysaccharide secreted by pal/rim mutants of thephytopathogen fungus Ustilago maydis. Carbohydr Polym86:1646–1650

Geilen FMA, Engendahl B, Harwardt A, Marquardt W, KlankermayerJ, Leitner W (2010) Selective and flexible transformation ofbiomass-derived platform chemicals by a multifunctional catalyticsystem. Angew Chem, Int Ed Engl 49:5510–5514

Göhre V, Vollmeister E, Bölker M, Feldbrügge M (2012) Microtubule-dependent membrane dynamics in Ustilago maydis: traffickingand function of Rab5a-positive endosomes. Commun Integr Biol5:485–490

Graumann K, Premstaller A (2006) Manufacturing of recombinanttherapeutic proteins in microbial systems. Biotechnol J 1:164–186. doi:10.1002/biot.200500051

HartmannHA, Krüger J, Lottspeich F, KahmannR (1999) Environmentalsignals controlling sexual development of the corn smut fungusUstilago maydis through the transcriptional regulator Prf1. PlantCell 11:1293–1306

Haskins RH, Thorn JA (1951) Biochemistry of the Ustilaginales. VII.Antibiotic activity of ustilagic acid. Can J Bot 29:585–592

Haskins RH, Thorn JA, Boothroyd B (1955) Biochemistry of theUstilaginales. XI. Metabolic products of Ustilago zeae in sub-merged culture. Can J Microbiol 1:749–756

Heimel K, Scherer M, Vranes M, Wahl R, Pothiratana C, Schüler D,Vincon V, Finkernagel F, Flor-Parra I, Kämper J (2010) Thetranscription factor Rbf1 is the master regulator for b-mating typecontrolled pathogenic development in Ustilago maydis. PLoSPathog 6:e1001035. doi:10.1371/journal.ppat.1001035

Hemetsberger C, Herrberger C, Zechmann B, Hillmer M, DoehlemannG (2012) The Ustilago maydis effector Pep1 suppresses plantimmunity by inhibition of host peroxidase activity. PLoS Pathog8:e1002684. doi:10.1371/journal.ppat.1002684

Hewald S, Josephs K, Bölker M (2005) Genetic analysis of biosurfactantproduction in Ustilago maydis. Appl Environ Microbiol 71:3033–3040. doi:10.1128/AEM.71.6.3033-3040.2005

Hewald S, Linne U, Scherer M, Marahiel MA, Kämper J, Bölker M(2006) Identification of a gene cluster for biosynthesis of

mannosylerythritol lipids in the basidiomycetous fungus Ustilagomaydis. Appl Environ Microbiol 72:5469–5477. doi:10.1128/AEM.00506-06

Hoegger PJ, Kilaru S, James TY, Thacker JR, Kües U (2006)Phylogenetic comparison and classification of laccases and relat-ed multicopper oxidase protein sequences. FEBS J 273:2308–2326

Holliday R (1974) Ustilago maydis. In: King RC (ed) Handbook ofgenetics, vol 1. Plenum Press, New York, pp 575–595

Holliday R (2004) Early studies on recombination and DNA repairin Ustilago maydis. DNA Repair 3:671–682. doi:10.1016/j.dnarep.2004.02.002

Idiris A, Tohda H, Bi KW, Isoai A, Kumagai H, Giga-Hama Y (2006)Enhanced productivity of protease-sensitive heterologous proteins bydisruption of multiple protease genes in the fission yeastSchizosaccaromyces pombe. ApplMicrobiol Biotechnol 73:404–420

Idiris A, Tohda H, Sasaki M, Okada K, Kumagai H, Giga-Hama Y(2010) Enhanced protein secretion from multiprotease-deficientfission yeast by modification of its vacuolar protein sorting path-way. Appl Microbiol Biotechnol 85:667–677

Iturriaga G, Jefferson RA, Bevan MW (1989) Endoplasmic reticulumtargeting and glycosylation of hybrid proteins in transgenic to-bacco. Plant Cell 1:381–390. doi:10.1105/tpc.1.3.381

Jaeger KE, Eggert T (2002) Lipases for biotechnology. Curr OpinBiotechnol 13:390–397

Jäger G, Büchs J (2012) Biocatalytic conversion of lignocellulose toplatform chemicals. Biotechnol J 7:1122–1136. doi:10.1002/biot.201200033

Jin FJ, Watanabe T, Juvvadi PR, Maruyama J, Arioka M, Kitamoto K(2007) Double disruption of the proteinase genes, tppA and pepE,increases the production level of human lysozyme by Aspergillusoryzae. Appl Microbiol Biotechnol 76:1059–1068

Juárez-Montiel M, Ruiloba de León S, Chávez-Camarillo G,Hernández-Rodríguez C, Villa-Tanaca L (2011) Huitlacoche(corn smut), caused by the phytopathogenic fungus Ustilagomaydis, as a functional food. Rev Iberoam Micol 28:69–73

Kahmann R, Schirawski J (2007)Mating in the smut fungi: from a to b tothe downstream cascades. In: Heitman J, Kronstad JW, Taylor JW,Casselton LA (eds) Sex in fungi: molecular determination andevolutionary implications. ASM Press, Washington, pp 377–387

Kämper J, Reichmann M, Romeis T, Bölker M, Kahmann R (1995)Multiallelic recognition: nonself-dependent dimerization of thebE and bW homeodomain proteins in Ustilago maydis. Cell81:73–83

Kämper J, Kahmann R, Bölker M, Ma LJ, Brefort T, Saville BJ,Banuett F, Kronstad JW, Gold SE, Müller O, Perlin MH,Wösten HA, de Vries R, Ruiz-Herrera J, Reynaga-Peña CG,Snetselaar K, McCann M, Pérez-Martin J, Feldbrügge M, BasseCW, Steinberg G, Ibeas JI, Holloman W, Guzman P, Farman M,Stajich JE, Sentandreu R, González-Prieto JM, Kennell JC,Molina L, Schirawski J, Mendoza-Mendoza A, Greilinger D,Münch K, Rössel N, Scherer M, Vranes M, Ladendorf O,Vincon V, Fuchs U, Sandrock B, Meng S, Ho EC, Cahill MJ,Boyce KJ, Klose J, Klosterman SJ, Deelstra HJ, Ortiz-CastellanosL, Li W, Sanchez-Alonso P, Schreier PH, Häuser-Hahn I, VaupelM, Koopmann E, Friedrich G, Voss H, Schlüter T, Margolis J,Platt D, Swimmer C, Gnirke A, Chen F, Vysotskaia V, MannhauptG, Güldener U, Münsterkotter M, Haase D, Oesterheld M, MewesHW, Mauceli EW, DeCaprio D, Wade CM, Butler J, Young S,Jaffe DB, Calvo S, Nusbaum C, Galagan J, Birren BW (2006)Insights from the genome of the biotrophic fungal plant pathogenUstilago maydis. Nature 444:97–101. doi:10.1038/nature05248

Katsivela E, Kleppe F, Lang S, Wagner F (1995) Ustilago maydislipase I. Hydrolysis and ester-synthesis activities of crude enzymepreparation. Enzyme Microb Tech 17:739–745. doi:10.1016/0141-0229(94)00127-d

Appl Microbiol Biotechnol (2013) 97:3253–3265 3263

Kellner R, Vollmeister E, Feldbrügge M, Begerow D (2011) Interspecificsex in grass smuts and the genetic diversity of their pheromone-receptor system. PLoS Genet 7:e1002436. doi:10.1371/journal.pgen.1002436

Khrunyk Y, Münch K, Schipper K, Lupas AN, Kahmann R (2010) Theuse of FLP-mediated recombination for the functional analysis of aneffector gene family in the biotrophic smut fungus Ustilago maydis.New Phytol 187:957–968. doi:10.1111/j.1469-8137.2010.03413.x

Kirk O, Christensen MW (2002) Lipases from Candida antarctica:unique biocatalysts from a unique origin. Org Process Res Dev6:446–451. doi:10.1021/op0200165

Kitamoto D, Akiba S, Hioki C, Tabuchi T (1990) Extracellular accu-mulation of mannosylerythritol lipids by a strain of Candidaantarctica. Agric Biol Chem 54:31–36

Kitamoto D, Isoda H, Nakahara T (2002) Functions and potential appli-cations of glycolipid biosurfactants – from energy-saving materialsto gene delivery carriers. J Biosci Bioeng 94(3):187–201

Klement T, Milker S, Jäger G, Grande PM, Domínguez de María P,Büchs J (2012) Biomass pretreatment affects Ustilago maydis inproducing itaconic acid. Microb Cell Fact 11:43. doi:10.1186/1475-2859-11-43

Koepke J, Kaffarnik F, Haag C, Zarnack K, Luscombe NM, KönigJ, Ule J, Kellner R, Begerow D, Feldbrügge M (2011) TheRNA-binding protein Rrm4 is essential for efficient secretionof endochitinase Cts1. Mol Cell Proteomics 10:M111.011213.doi:10.1074/mcp.M111.011213

König J, Baumann S, Koepke J, Pohlmann T, Zarnack K, FeldbrüggeM (2009) The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs. EMBO J 28:1855–1866. doi:10.1038/emboj.2009.145

Kües U, Rühl M (2011) Multiple multi-copper oxidase families inBasidiomycetes—what for? Curr Genet 12:17–94

Laurie JD, Ali S, Linning R, Mannhaupt G, Wong P, Güldener U,Münsterkotter M, Moore R, Kahmann R, Bakkeren G, SchirawskiJ (2012) Genome comparison of barley and maize smut fungireveals targeted loss of RNA silencing components and species-specific presence of transposable elements. Plant Cell 24:1733–1745. doi:10.1105/tpc.112.097261

Lei XG, Porres JM (2003) Phytase enzymology, applications, andbiotechnology. Biotechnol Lett 25:1787–1794

Liu Y, Koh CM, Ji L (2011) Bioconversion of crude glycerol toglycolipids in Ustilago maydis. Bioresour Technol 102:3927–3933. doi:10.1016/j.biortech.2010.11.115

Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, KusumotoK, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H,Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE,Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D,Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, HosoyamaA, IchinomiyaM, Igarashi R, Iwashita K, Juvvadi PR, KatoM, KatoY, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J,Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, SakamotoK, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR,Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T,Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S,Ogasawara N, Kikuchi H (2005) Genome sequencing and analysisof Aspergillus oryzae. Nature 438:1157–1161

Margeot A, Hahn-Hagerdal B, Edlund M, Slade R, Monot F (2009)New improvements for lignocellulosic ethanol. Curr OpinBiotechnol 20:372–380. doi:10.1016/j.copbio.2009.05.009

Mason HS, Warzecha H, Mor T, Arntzen CJ (2002) Edible plantvaccines: applications for prophylactic and therapeutic molecularmedicine. Trends Mol Med 8:324–329

Mei B, Budde AD, Leong SA (1993) sid1, a gene initiatingsiderophore biosynthesis in Ustilago maydis: molecular charac-terization, regulation by iron, and role in phytopathogenicity. ProcNatl Acad Sci USA 90:903–907

Mendgen K, Hahn M (2002) Plant infection and the establishment offungal biotrophy. Trends Plant Sci 7:352–356

Mercado-Flores Y, Guerra-Sánchez G, Villa-Tanaca L, Hernández-Rodríguez C (2003a) Purification and characterization of an ex-tracellular non-aspartyl acid protease (pumAe) from Ustilagomaydis. Curr Microbiol 47:408–411

Mercado-Flores Y, Hernández-Rodríguez C, Ruiz-Herrera J, Villa-Tanaca L (2003b) Proteinases and exopeptidases from thephytopathogenic fungus Ustilago maydis. Mycologia 95:327–339

Mimee B, Labbé C, Pelletier R, Bélanger RR (2005) Antifungal activityof flocculosin, a novel glycolipid isolated from Pseudozymaflocculosa. Antimicrob Agents Chemother 49:1597–1599

Moir DT, Mao JI (1990) Protein secretion systems in microbial andmammalian cells. Bioprocess Technol 9:67–94

Morita T, Konishi M, Fukuoka T, Imura T, Kitamoto D (2008)Identification of Ustilago cynodontis as a new producer of glyco-lipid biosurfactants, mannosylerythritol lipids, based on ribosom-al DNA sequences. J Oleo Sci 57:549–556

Morita T, Ishibashi Y, Fukuoka T, Imura T, Sakai H, Abe M, Kitamoto D(2009) Production of glycolipid biosurfactants, mannosylerythritollipids, by a smut fungus, Ustilago scitaminea NBRC 32730. BiosciBiotechnol Biochem 73:788–792

Morita T, Ito E, Kitamoto HK, Takegawa K, Fukuoka T, Imura T,Kitamoto D (2010a) Identification of the gene PaEMT1 for bio-synthesis of mannosylerythritol lipids in the basidiomycetousyeast Pseudozyma antarctica. Yeast 27:905–917. doi:10.1002/yea.1794

Morita T, Kitagawa M, Yamamoto S, Sogabe A, Imura T, Fukuoka T,Kitamoto D (2010b) Glycolipid biosurfactants, mannosylerythritollipids, repair the damaged hair. J Oleo Sci 59:267–272

Morita T, Ishibashi Y, Hirose N, Wada K, Takahashi M, Fukuoka T,Imura T, Sakai H, Abe M, Kitamoto D (2011) Production andcharacterization of a glycolipid biosurfactant, mannosylerythritollipid B, from sugarcane juice by Ustilago scitaminea NBRC32730. Biosci Biotechnol Biochem 75:1371–1376

Müller O, Kahmann R, Aguilar G, Trejo-Aguilar B, Wu A, de Vries RP(2008) The secretome of the maize pathogen Ustilago maydis.Fungal Genet Biol 45:S63–70. doi:10.1016/j.fgb.2008.03.012

Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T (2012a)Identification, cloning, and characterization of beta-glucosidasefrom Ustilago esculenta. Appl Microbiol Biotechnol 93:1989–1998. doi:10.1007/s00253-011-3538-2

Nakajima M, Yamashita T, Takahashi M, Nakano Y, Takeda T (2012b)A novel glycosylphosphatidylinositol-anchored glycoside hydro-lase from Ustilago esculenta functions in β-1,3-glucan degrada-tion. Appl Environ Microbiol 78:5682–5689. doi:10.1128/AEM.00483-12

Neilands JB (1952) A crystalline organo-iron pigment from a rustfungus (Ustilago sphaerogena). J Am Chem Soc 74:4856–4847

Neilands JB (1995) Siderophores: structure and function of microbialiron transport compounds. J Biol Chem 270:26723–26726

Nestl BM, Nebel BA, Hauer B (2011) Recent progress in industrialbiocatalysis. Curr Opin Chem Biol 15:187–193. doi:10.1016/j.cbpa.2010.11.019

Raboin LM, Selvi A, Oliveira KM, Paulet F, Calatayud C,Zapater MF, Brottier P, Luzaran R, Garsmeur O, Carlier J,D'Hont A (2007) Evidence for the dispersal of a uniquelineage from Asia to America and Africa in the sugarcane fungalpathogen Ustilago scitaminea. Fungal Genet Biol 44:64–76.doi:10.1016/j.fgb.2006.07.004

Reed RW, Holder MA (1953) The antibacterial spectrum of ustilagicacid. Can J Med Sci 31:505–511

Sanchez C (2009) Lignocellulosic residues: biodegradation and bio-conversion by fungi. Biotechnol Adv 27:185–194. doi:10.1016/j.biotechadv.2008.11.001

3264 Appl Microbiol Biotechnol (2013) 97:3253–3265

Scherer M, Heimel K, Starke V, Kämper J (2006) The Clp1 protein isrequired for clamp formation and pathogenic development ofUstilago maydis. Plant Cell 18:2388–2401. doi:10.1105/tpc.106.043521

Schirawski J, Heinze B, Wagenknecht M, Kahmann R (2005) Matingtype loci of Sporisorium reilianum: novel pattern with three a andmultiple b specificities. Eukaryot Cell 4:1317–1327. doi:10.1128/EC.4.8.1317-1327.2005

Schirawski J, Mannhaupt G, Münch K, Brefort T, Schipper K,Doehlemann G, Di Stasio M, Rössel N, Mendoza-Mendoza A,Pester D, Müller O, Winterberg B, Meyer E, Ghareeb H,Wollenberg T, Münsterkotter M, Wong P, Walter M, StukenbrockE, Güldener U, Kahmann R (2010) Pathogenicity determinants insmut fungi revealed by genome comparison. Science 330:1546–1548. doi:10.1126/science.1195330

Schmidt FR (2004) Recombinant expression systems in the pharma-ceutical industry. Appl Microbiol Biotechnol 65:363–372.doi:10.1007/s00253-004-1656-9

Singh P, Cameotra SS (2004) Potential applications of microbial sur-factants in biomedical sciences. Trends Biotechnol 22:142–146.doi:10.1016/j.tibtech.2004.01.010

Sodoyer R (2004) Expression systems for the production of recombi-nant pharmaceuticals. BioDrugs 18:51–62

Spellig T, Bottin A, Kahmann R (1996) Green fluorescent protein(GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis. Mol Gen Genet 252:503–509

Spoeckner S, Wray V, Nimtz M, Lang S (1999) Glycolipids of the smutfungus Ustilago maydis from cultivation on renewable resources.Appl Microbiol Biotechnol 51:33–39

Steinberg G, Pérez-Martin J (2008) Ustilago maydis, a new fungalmodel system for cell biology. Trends Cell Biol 18:61–67.doi:10.1016/j.tcb.2007.11.008

Stock J, Sarkari P, Kreibich S, Brefort T, Feldbrügge M, Schipper K(2012) Applying unconventional secretion of the endochitinaseCts1 to export heterologous proteins in Ustilago maydis. JBiotechnol 161:80–91. doi:10.1016/j.jbiotec.2012.03.004

Stoll M, Begerow D, Oberwinkler F (2005) Molecular phylogeny ofUstilago, Sporisorium, and related taxa based on combined anal-yses of rDNA sequences. Mycol Res 109:342–356

Suzuki T, Choi JH, Kawaguchi T, Yamashita K, Morita A, Hirai H,Nagai K, Hirose T, Omura S, Sunazuka T, Kawagishi H (2012)Makomotindoline from Makomotake, Zizania latifolia infectedwith Ustilago esculenta. Bioorg Med Chem Lett 22:4246–4248.doi:10.1016/j.bmcl.2012.05.021

Teichmann B, Linne U, Hewald S, Marahiel MA, Bölker M (2007) Abiosynthetic gene cluster for a secreted cellobiose lipid with anti-fungal activity from Ustilago maydis. Mol Microbiol 66:525–533.doi:10.1111/j.1365-2958.2007.05941.x

Teichmann B, Liu L, Schink KO, Bölker M (2010) Activation of theustilagic acid biosynthesis gene cluster in Ustilago maydis by theC2H2 zinc finger transcription factor Rua1. Appl EnvironMicrobiol 76:2633–2640. doi:10.1128/AEM.02211-09

Teichmann B, Labbé C, Lefebvre F, Bölker M, Linne U, Bélanger RR(2011) Identification of a biosynthesis gene cluster for flocculosin

a cellobiose lipid produced by the biocontrol agent Pseudozymaflocculosa. Mol Microbiol 79:1483–1495. doi:10.1111/j.1365-2958.2010.07533.x

Vaishnav P, Demain AL (2011) Unexpected applications of second-ary metabolites. Biotechnol Adv 29:223–229. doi:10.1016/j.biotechadv.2010.11.006

Vakhlu JK, A. (2006) Yeast lipases: enzyme purification, biochemicalproperties and gene cloning. Electron J Biotechnol 9. doi:10.225/vol9-issue1fulltext-9

Valdez-Morales M, Barry K, Fahey GC Jr, Domínguez J, Gonzalez deMeja E, Valverde ME, Paredes-López O (2010) Effects of maizegenotype, developmental stage, and cooking process on the nu-traceutical potential of huitlacoche (Ustilago maydis). Food Chem119:689–697

Valverde ME, Paredes-López O, Pataky JK, Guevara-Lara F (1995)Huitlacoche (Ustilago maydis) as a food source—biology, com-position, and production. Crit Rev Food Sci Nutr 35:191–229.doi:10.1080/10408399509527699

Vánky K (2011) Smut fungi of the world. APS press, St PaulVollmeister E, Schipper K, Baumann S, Haag C, Pohlmann T, Stock J,

Feldbrügge M (2012a) Fungal development of the plant pathogenUstilago maydis. FEMS Microbiol Rev 36:59–77. doi:10.1111/j.1574-6976.2011.00296.x

Vollmeister E, Schipper K, Feldbrügge M (2012b) Microtubule-dependent mRNA transport in the model microorganism Ustilagomaydis. RNA Biol 9:261–268. doi:10.4161/rna.19432

Wang J, Budde AD, Leong SA (1989) Analysis of ferrichrome bio-synthesis in the phytopathogenic fungus Ustilago maydis: cloningof an ornithine-N5-oxygenase gene. J Bacteriol 171:2811–2818

Ward OP (2012) Production of recombinant proteins by filamentousfungi. Biotechnol Adv 30:1119–1139

Winterberg B, Uhlmann S, Linne U, Lessing F, Marahiel MA,Eichhorn H, Kahmann R, Schirawski J (2010) Elucidation ofthe complete ferrichrome A biosynthetic pathway in Ustilagomaydis. Mol Microbiol 75:1260–1271. doi:10.1111/j.1365-2958.2010.07048.x

Xu XW, Ke WD, Yu XP, Wen J, Ge S (2008) A preliminary study onpopulation genetic structure and phylogeography of the wild andcultivated Zizania latifolia (Poaceae) based on Adh1a sequences.Theor Appl Genet 116:835–843. doi:10.1007/s00122-008-0717-3

Yuan WM, Gentil GD, Budde AD, Leong SA (2001) Characterizationof the Ustilago maydis sid2 gene, encoding a multidomain peptidesynthetase in the ferrichrome biosynthetic gene cluster. J Bacteriol183:4040–4051. doi:10.1128/JB.183.13.4040-4051.2001

Zarnack K, Maurer S, Kaffarnik F, Ladendorf O, Brachmann A,Kämper J, Feldbrügge M (2006) Tetracycline-regulated gene ex-pression in the pathogen Ustilago maydis. Fungal Genet Biol43:727–738. doi:10.1016/j.fgb.2006.05.006

Zuther K, Mayser P, Hettwer U, Wu W, Spiteller P, Kindler BL,Karlovsky P, Basse CW, Schirawski J (2008) The tryptophanaminotransferase Tam1 catalyses the single biosynthetic stepfor tryptophan-dependent pigment synthesis in Ustilagomaydis. Mol Microbiol 68:152–172. doi:10.1111/j.1365-2958.2008.06144.x

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