Thermoascus Aurantiacus Is an Intriguing Host for the Industrial Production of Cellulases

Send Orders for Reprints to [email protected] 89 Current Biotechnology, 2017, 6, 89-97

REVIEW ARTICLE

ISSN: 2211-5501 eISSN: 2211-551X Thermoascus aurantiacus is an Intriguing Host for the Industrial Production of Cellulases

Timo Schuerg1, Raphael Gabriel1,2, Nora Baecker3,4, Scott E. Baker5 and Steven W. Singer1,*

1Biological and Systems Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; 2Department of Microbiology and Ecosystem Science, University of Vienna, Vienna, Austria; 3Physical Biosciences Di- vision, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; 4Faculty of Biotechnology, University of Applied Sciences Mannheim, Mannheim, Germany and 5Environmental Molecular Sciences Laboratory, Pacific North- west National Laboratory, Richland, WA, USA

Abstract: Background: The conversion of biomass to fuels and chemicals is an important technology to replace petroleum as a transportation fuel which will ease climate effects of burning fossil fuels. Recent advances in cellulosic ethanol production have enabled the establishment of commercial scale plants that produce ethanol for transportation fuel. Thermotolerant cellulase enzymatic mixtures from thermophilic fungi are an attractive alternative to currently available commercial cellulase cocktails.

A R T I C L E H I S T O R Y Methods: Thermoascus aurantiacus is a thermophilic ascomycete fungus within the order of Eurotiales Received: January 17, 2016 Revised: May 18, 2016 that was first isolated by Miehe in 1907. Strains of T. aurantiacus have been isolated from a variety of Accepted: May 18, 2016 terrestrial environments, which all have been shown to be homothallic and produce large amounts of

DOI: ascopores with an optimal growth temperature at ~50C. T. aurantiacus secretes high titers of cellulases 10.2174/2211550105666160520123504 (>1 g/L) when grown in the presence of plant biomass substrates and produces a remarkably simple cellulase mixture consisting of GH7 cellobiohydrolase, GH5 endoglucanase, AA9 lytic polysaccharide monooxygenase and GH3 beta-glucosidase.

Results: In this mini-review, the biology and enzymology underlying cellulase production are described and an approach to developing T. aurantiacus strains for industrial cellulase production is outlined.

Conclusion: The properties of T. aurantiacus and the thermotolerant cellulase mixture it produces may be the basis for new enzymatic cocktails to produce sugars from plant biomass that can be converted to biofuels.

Keywords: Cellulase, thermophilic, fungi, Thermoascus aurantiacus, lytic polysaccharide monooxygenase.

1. INTRODUCTION fuels and chemicals [4]. Trichoderma reesei (syn. Hypocrea jecorina) was originally identified as a copious producer of The conversion of biomass to fuels and chemicals is an enyzmes for cellulose hydrolysis [5]. Sustained efforts in important technology to replace petroleum as a transporta- government, academia and industry have developed strains tion fuel and as a feedstock for the chemical industry [1]. of Trichoderma reesei that produce ~100 g/L of cellulase Reducing the dependence on petroleum will ease climate enzymes and have enabled the establishment of the cellulosic effects of burning fossil fuels. Recent advances in cellulosic ethanol industry [6]. T. reesei protein engineering efforts ethanol production have enabled the establishment of com- have focused on improving the thermotolerance of the cellu- mercial scale plants that produce ethanol for transportation lases to increase the efficacy of industrial bioprocessing [7]. fuel [2] Among these advances, the production of large amounts of cellulase enzymes for saccharification of pre- An alternative approach to obtaining thermotolerant cel- treated biomass has enabled commercialization of cellulosic lulase enzymatic mixtures is to develop thermophilic fungi ethanol [3]. Ascomycete fungi are the predominant produc- as platforms for cellulase production [8]. Though thermo- ers of commercial enzymes to convert plant biomass to philic fungi have been identified for over 100 years and their complement of secreted cellulase enzymes is well-known, it *Address correspondence to this author at the Lawrence Berkeley National is only recently that they have been identified as potential Laboratory, 5885 Hollis Street, Emeryville, CA 94608, USA; Tel: 510-486- commercial producers of cellulase mixtures for biomass to 5556; E-mail: [email protected] biofuel conversion [9]. The genome sequences of Myceli- Current Bio technology 2211-551X/17 $58.00+.00 © 2017 Bentham Science Publishers 90 Current Biotechnology, 2017, Vol. 6, No. 2 Schuerg et al. ophthora thermophila and Thielavia terrestris, thermophilic and contain sack-like asci with eight unsorted ascospores members of the Sordariales, indicated a broad potential for (Fig. 1B). T. aurantiacus ascospores are of elliptical shape cellulose hydrolysis. Additionally, a strain of M. thermophi- and relatively small (5-7 x 4-5 μm) [17] They are released la, the C-1 strain, has been optimized by mutagenesis and upon disintegration of the asci and walls of the ripe cleisto- scaled for commercial production of cellulases [10]. Cellu- thecia [13] Although it has been shown in our laboratory that lases produced from the C-1 strain are capable of high sac- T. aurantiacus is capable of producing large amounts of as- charification conversion at higher temperature (60 C) and cospores on PDA plates at 50 C (Schuerg et al., un- higher pH (pH 6), than T. reesei-based commercial enzymes published data), Miehe did not observe any spore production [11]. Rasamsonia emersonii (syn. Talaromyces emersonii), a or pigmentation at 50C during liquid culture surface cultiva- thermophilic member of the Eurotiales, was identified as a tions on glucose/asparagine medium [13]. copious cellulase producer and has been developed into an Since T. aurantiacus is a homothallic fungus which pro- industrial production strain whose enzymes were capable of saccharifying biomass at 65C [12]. duces masses of uncommonly small sexual ascospores in a short amount of time on rich medium, general caution is ad- Thermoascus aurantiacus is a thermophilic fungus affili- vised to not confuse these sexual spores with asexual conidi- ated with the Eurotiales that was isolated in 1907 and is fre- ospores. A dominant asexual mass propagation pattern, quently recovered from terrestrial habitats [13]. Multiple which is common for most Ascomycete fungal species is reports indicate a substantial ability to secrete cellulases that apparently absent in T. aurantiacus. Miehe described cystoid may be exploited to develop a thermophilic platform for cel- blown-up structures, which he discovered terminally or in lulase production [14-16]. In this review, work describing T. between thinner sections. It can be hypothesized that these aurantiacus strains will be reviewed as well as their ability cysts might be considered as chlamydospores or chla- to produce cellulases for biomass deconstruction. Analysis of mydoconidia, which are rare asexual propagation structures the genome of T. aurantiacus ATCC 26904 (Fig. 1) will be [18]. Interestingly, Miehe was only able to observe these described to identify cellulase genes and the genes responsi- structures at 50C and not at 40C. It is important to note ble for regulation of cellulase production. Finally, strategies that germination of these putative chlamydoconidial struc- to improve T. aurantiacus strains to develop an industrial tures has never been reported. This lack of observation, to- thermophilic cellulase producer will be outlined. gether with a minor role and occurrence in lower abundance of these structures, has led to the frequently expressed notion 2. ISOLATION AND DESCRIPTION OF THERMOAS- that an anamorph is unknown for T. aurantiacus. However, CUS AURANTIACUS Salar and Aneja use the ability of producing chlamydospores Thermoascus aurantiacus was originally isolated from as a critical characteristic in their classification key to distin- guish between T. aurantiacus and Paecilomyces (Polypaeci- self-heating hay and described by German mycologist Hugo lum) [19]. Therefore, the question if T. aurantiacus has an Miehe [13]. Since Miehe was not able to assign the isolated species to any of the know fungal genera, he created the ge- anamorph is still not fully answered and should be addressed in the future. nus Thermoascus (thermos (greek) = hot; askos (greek) = tube). Based on the golden/orange fruiting bodies Miehe T. aurantiacus strains that match the description provided discovered, he selected the species name aurantiacus (lat., by Miehe have been isolated from a variety of different habi- orange-colored) (Fig. 1A). Miehe described T. aurantiacus tats, predominantly soil, compost and agricultural residues. as a true thermophile with a growth optimum around 50C T. aurantiacus Miehe strains are found in multiple culture and no growth lower than 30C or lower. He observed fruit- collections; an example of the range of T. aurantiacus iso- ing body formation after two days of incubation at 40C and lates found in the American Type Culture Collection is de- subsequent pigmentation and ripening thereof. The so-called picted in Table 1. The type strain of T. aurantiacus Miehe is cleistothecia are round-shaped, of 0.25-1 mm in diameter IMI 91781, which was isolated from alluvial soils in Not-

Fig. (1). A, T. aurantiacus ATCC 26904 on PDA agar plates incubated for 4 days (left) and 11 days (right) at 50 °C. B, T. aurantiacus ascus with eight unsorted ascospores. Scale bar = 10 µm. Thermoascus aurantiacus is an Intriguing Host for the Industrial Production of Cellulases Current Biotechnology, 2017, Vol. 6, No. 2 91 tingham, UK and described by Apinis [17]. A second variety that the putative T. aurantiacus strain reported by Cooney of T. aurantiacus Miehe, T. aurantiacus Miehe var. levispo- and Emerson was actually Thermoascus crustaceus. T. au- rus, was isolated from soil in Honduras [18]. This strain was rantiacus ATCC 46993, isolated from compost heaps in distinguished from the Miehe strains by having smooth ra- Christchurch, New Zealand, was grown in submerged cul- ther than echinulate ascospores and having a uniform layer ture with filter paper as the carbon source and the cellulases of the cleistothecial wall. were purified from culture supernatants [14]. Four cellulases were purified from the culture supernatant: a beta-glucosi- Phylogenetic profiling of T. aurantiacus has demonstrated dase (~87 kDa), an endoglucanase (~78 kDa), a cellobiohy- that T. aurantiacus is affiliated with the Eurotiales order, in the drolase (~49 kDa) and a second endoglucanase (~34 kDa). family Thermoascaceae [20]. Currently two genera, Thermo- Comparable cellulases were purified from a T. aurantiacus ascus and Byssochlamys, are classified in the family Thermo- strain isolated in Bangalore, India, with the exception of the ascaceae, whereas both differ in the form of fruiting body for- 78 kDa endoglucanase, which was not observed in this strain mation. Thermoascus produces firm, sclerotioid cleistothecia, [25]. T. aurantiacus ATCC 26904 was cultivated on intact surrounded by a dense pseudoparenchymatous tissue. Bysso- and pretreated switchgrass and cellulases were identified by chlamys, on the contrary, produces almost naked ascomata. A mass-spectrometry based proteomics [16]. The supernatants multi-protein phylogenetic tree established that one strain of from these switchgrass-grown cultures produced cellulases Byssochlamys, Byssochlamys verrucosa, is more closely affiliat- with similar measured molecular weights to previous studies ed with the Thermoascus strains (T. aurantiacus, T. crustaceus, (cellobiohydrolase (GH7), ~54 kDa; endoglucanase (GH5), T. thermophilus), than the other Byssochlamys strains. Due to ~33 kDa). In addition, a prominent band was observed at their phylogenetic distance, it is unclear if the two genera repre- ~25 kDa that was identified as a GH61 protein, which has sent separate families. However, common characteristics like been reclassified as a polysaccharide monooxygenase (see asci development pattern and the formation of smooth or finely below). A xylanase (GH10) was also identified that over- roughened ascospores lacking a furrow or slit are shared between lapped with the GH5 protein at ~33 kDa. Subsequent inves- Thermoascus and Byssochlamys [20, 21]. tigations of supernatants generated from T. aurantiacus ATCC 26904 cultures grown on pretreated corn stover iden- 3. PRODUCTION OF GLYCOSIDE HYDROLASES tified a beta-glucosidase (GH3) at ~92 kDa (Fig. 2). A num- AND LYTIC POLYSACCHARIDE MONOOXYGE- NASE BY T. AURANTIACUS ber of studies have examined the thermostability of T. aurantiacus cellulases, establishing that Topt for individual ac- Initial reports on the cellulolytic activity of thermophilic tivities (endoglucanase, beta-glucosidase) were >70C and fungi suggested that T. aurantiacus [22], as described by that the mixture of cellulases produced by T. aurantiacus Cooney and Emerson [23], was unable to grow by hydrolyz- was capable of the saccharification of crystalline cellulose ing filter paper. Subsequent investigations demonstrated that and chemically pretreated plant biomass at temperatures authenticated strains of T. aurantiacus Miehe that matched >60C [26, 27]. Conditions for induction of cellulases by T. the original description were capable of growing vigorously aurantiacus have also been tested, and induction of cellulas- on acid-swollen cellulose in an agar diffusion assay [24] and es has been observed in the presence of Solka Floc and sugar

Table 1. Thermoascus aurantiacus strains in American Type Culture Collection (ATCC).

Thermoascus aurantiacus Strain Habitat References

ATCC 16179 Peanuts, Texas and Oklahoma [53]

ATCC 208821 Unknown [26]

ATCC 26904 Soil, Japan [54]

ATCC 26905 Soil, Japan [54]

ATCC 26196 Soil, Japan [54]

ATCC 28082 Soil, Japan [54]

ATCC 34115 Peanuts, Texas and Oklahoma [53]

ATCC 461972 Soil, Honduras [18]

ATCC 46993 Compost, New Zealand [14][57]

ATCC 58156 Compost, Hungary Not cited

ATCC 64510 Poplar bark, Italy [55]

ATCC 204492 Eucalyptus wood chips, Brazil [56]. 1This strain has been reclassified as Thermoascus crustaceus (https://curve.carleton.ca/system/files/theses/23103.pdf), but this reclassification has not been confirmed by molecular methods. 2This strain is the type of T. aurantiacus var. levisporus 92 Current Biotechnology, 2017, Vol. 6, No. 2 Schuerg et al. cane bagasse [15, 28]. Use of xylooligomers has suggested enabled the construction of chimeric T. aurantiacus cellulas- that they may be responsible for the induction of both cellu- es with CBMs from T. reesei analogs [29, 30]. Comparison lase and xylanase activtity in T. aurantiacus [28]. of saccharification efficiencies between heterologous enzymatic mixtures containing T. aurantiacus GH7, GH5, GH10 xylanase and GH3 beta-glucosidase illustrated that while CBMs increase saccharification efficiency at low substrate loadings, at 25% substrate loading (steam pretreated wheat straw) the cellulases lacking CBMs were as efficient as the CBM containing enzymes and could be recovered from high solids loading for enzyme recycle [30]. As mentioned above, growth of T. aurantiacus on switchgrass substrates produced large amounts of a GH61 protein [16]. This family, renamed auxiliary activity family 9 (AA9), has been characterized as copper-containing lytic polysaccharide monooxygenases that oxidize the C-1 and C- 4 positions of polysaccharides, including cellulose, xylan, starch and chitin [31, 32]. All genes encoding AA9 family proteins are found in fungal genomes; however, a companion set of lytic polysaccharide monooxygenases, the AA10 family, has been characterized from multiple bacterial genomes [33]. Thermoascus aurantiacus has been a key organism in the discovery and characterization of the AA9 family. Mix- ing supernatants from thermophilic fungi, including T. aurantiacus, with Celluclast enhanced saccharification activities compared to Celluclast alone [34]. The enhancement produced by the thermophilic fungi was shown to be a AA9 (GH61) protein, previously characterized as endoglucanase with low specific activity [35]. Expression of a T. aurantiacus GH61 protein in a T. reesei cellulase production strain decreased the amount of total protein from the T. reesei supernatant required to saccharify dilute-acid pretreated corn stover. Initial studies indicated the enhancement effect of T. aurantiacus AA9 was only observed during saccharification of complex biomass and AA9 did not enhance the saccharification of crystalline cellulose. Subsequent investigations established that a reductant such as gallate or ascorbate was required to enhance saccharification of crystalline cellulose [36]. Cellobiose dehydrogenase also promoted enhanced saccharification of crystalline cellulose when it was added to mixtures containing T. aurantiacus AA9 and Celluclast [37]. Saccharification of lodgepole pine steam pretreated to different levels of delignification demonstrated that lignin frag- ments in complex biomass provide reductants for AA9 and complete delignification of the lodgepole pine substrate required addition of exogenous gallate to recover the AA9 enhancement effect [38]. The T. aurantiacus AA9 has also been instrumental in defining the molecular structure and mechanism of cellulose oxidation by LPMOs. The T. au- Fig. (2). Cellulase production in the supernatant of T. aurantiacus 2+ rantiacus AA9 was shown to tightly bind Cu with a KD grown on AFEX-pretreated corn stover. T. aurantiacus ATCC estimated at <1 nM. The crystal structure of T. aurantiacus 26904 was incubated for four days at 50C in 50 mL of minimal AA9 revealed a unique N-methylated histidine residue that medium with 1% AFEX-pretreated corn stover and supernatant bound the Cu2+ in its active site [31] Spectroscopic and com- collected by filtration. Proteins in supernatant were visualized by putational studies of the Cu active site of T. aurantiacus SDS-PAGE (8-16%, Tris-Glycine). Lane 1, Molecular weight AA9 indicated that the reduced form of the enzyme, which markers: 8, 13, 20, 30, 40, 65, 90 kDa. Lane 2: 1) AA9 (~25 kDa); adopts a three coordinate T-shaped structure, reacts with 2) GH5/GH10 (~33 kDa); 3) GH7 (~54 kDa); 4) S10 peptidase oxygen to form a Cu-superoxide species, which may be the (~62 kDa); 5) GH3 (~92 kDa). intermediate that oxidizes the cellulose chain [39]. An unusual feature of the T. aurantiacus cellulases compared to other fungal cellulases (e.g. Trichoderma reesei) is 4. GENOMIC SEQUENCING OF T. AURANTIAUCS that they lack cellulose binding module (CBMs). Heterolo- The genome sequence of T. aurantiacus ATCC 26904 gous expression of T. aurantiacus GH7 and GH5 in T. reesei was obtained to determine the cellulase genes present in the Thermoascus aurantiacus is an Intriguing Host for the Industrial Production of Cellulases Current Biotechnology, 2017, Vol. 6, No. 2 93 isolates as well as regulatory elements that determine cellu- Homologs of a number of regulatory elements identified lase production (http://genome.jgi.doe.gov/Theau1/Theau1. in ascomycete fungi have been identified in the genome of T. home.html). In the genome of T. aurantiacus ATCC 26904, aurantiacus (Table 3) [40]. A homolog of a gene expressing genes coding for a GH7, four GH5s, six GH3s and three Cre-1, a zinc finger transcription factor broadly present in AA10s were identified (Table 2). As noted above, individual filamentous fungi, was identified that likely controls cellu- GH7, GH5, GH3 and AA10 proteins were identified as high- lase expression by repressing cellulase genes in the presence ly abundant in supernatants grown on switchgrass substrates of sugars and other readily metabolizable carbon sources. A [16]. GH6, which hydrolyzes cellulose chains to cellobiose XlnR homolog, which has been shown to be a transcriptional at the non-reducing end, and cellobiose dehydrogenase, regulator in Trichoderma reesei and Aspergillus spp., was which has been proposed to be a physiological electron do- also detected in the T. aurantiacus genome [41]. Both the nor for LPMO [37], were both absent from the T. aurantia- Cre-1 and XlnR homologs were most closely related to cus genome. homologs from Rasamsonia emersonii, a related thermo-

Table 2. Putative cellulases in Thermoascus aurantiacus ATCC 26904 genomes.

Thermoascus aurantiacus Protein Annotated Closest Proteomics ID (Mycocosm)1 Function Homolog (GenBank) Detection2

Theau_41785 GH7 (cellobiohydrolase) Talaromyces leycettanus (81%) Yes

GH5 Theau_44560 Neosartorya fischeri NRRL 181 (82%) No (glucanase)

GH5 Theau_63321 Rasamsonia emersonii CBS 393.64 (78%) No (endo-beta-1,4-endoglucanase)

GH5 Theau_63225 Rasamsonia emersonii CBS 393.64 (83%) No (glycosyl hydrolase)

GH5 Theau_65156 Aspergillus fumigatus Af293 (77%) Yes (endo-beta-1,4-endoglucanase)

GH3 Theau_62557 Rasamsonia emersonii CBS 393.64 (82%) No (beta-glucosidase)

GH3 Byssochlamys spectabilis No. 5 Theau_34576 No (beta-glucosidase) (82%)

GH3 Byssochlamys spectabilis No. 5 Theau_47220 No (beta-glucosidase) (77%)

GH3 Byssochlamys spectabilis No. 5 Theau_46699 Yes (beta-glucosidase) (83%)

GH3 Byssochlamys spectabilis No. 5 Theau_47220 No (beta-glucosidase) (77%)

GH3 Theau_62557 Rasamsonia emersonii CBS 393.64 (82%) No (beta-glucosidase)

AA9 Byssochlamys spectabilis No. 5 Theau_1859 No (lytic polysaccharide monooxygenase) (68%)

GH61 Theau_3070 Talaromyces stipitatus ATCC 110500 (74%) Yes (lytic polysaccharide monooxygenase)

GH61 Byssochlamys spectabilis No. 5 Theau_39139 No (lytic polysaccharide monooxygenase) (77%) 1Found at http://genome.jgi.doe.gov/Theau1/Theau1.home.html 2Proteomics data was obtained from SDS-PAGE gel slices from supernatants grown on ammonia-fiber expansion (AFEX)-pretreated corn stover. AFEX- pretreated corn stover was provided by Professor Bruce Dale, Michigan State University. 94 Current Biotechnology, 2017, Vol. 6, No. 2 Schuerg et al. philic, cellulolytic fungus [42]. Homologs of Clr-1 and Clr- Theau_16404 do not share significant identities with MAT1- 2, transcriptional cellulase activators recently identified in 2 proteins of close relatives of T. aurantiacus. The only ho- Neurospora crassa and found in an number of Aspergillus mologous region which is shared between Theau_59447 and genomes, were also identified in the T. aurantiacus genome Theau_16404 and the MAT1-2 proteins constitutes the [43]. Finally, a CblR homolog, which has been shown to HMG-box region (Fig. 4). Thus, T. aurantiacus is one of the participate in a XlnR-independent cellulase induction path- rare examples of sexually reproducing fungi lacking one of way in Aspergillus aculeatus, was found in the T. aurantia- the two mating type genes. cus genome [44]. This unusual form of homothallism in fungi has been first The observed homothallic sexual cycle indicates that described for Neurospora africana and has later been de- mating genes should be present in the T. aurantiacus genome fined as unisexual reproduction [47, 48]. It has been pro- [45]. A mat1-1 gene (mating type alpha box protein) was posed that such a mating strategy provides these species with identified in cluster with three other genes: a gene coding for the benefits associated with sexual reproduction like out- a DNA lyase (APN-2), a hypothetical protein (Theau_55954) crossing of deleterious mutations while minimizing the costs and actin binding protein (SLA-2) (Fig. 3). Both APN-2 and associated with locating a mating partner [49]. Further stud- SLA-2 are typical flanking genes observed at the mat loci of ies of the T. aurantiacus sexual cycle will further elucidate ascomycete fungi (Lee et al. 2010). Often, homothallic fungi the mechanisms underlying this type of unisexual homothal- have both the mat genes, the mating type alpha box and the lism occurring in this species. HMG box, co-located in the genome, as exemplified by Sor- daria macrospora [46]. However, sequence alignment and 5. FUTURE PERSPECTIVES phylogenetic analysis showed that Theau_55954 is unrelated The high level of cellulase secretion, the simplicity of the to known HMG-box proteins found in other ascomycete fun- cellulase mixture and the thermotolerance of the cellulases gi (Fig. 4). A search of the T. aurantiacus genome was una- indicate that T. aurantiacus is a promising thermophilic fun- ble to identify an HMG box protein homologous to MAT1-2. gus for commercial production of cellulase enzymes. A A ClustalW alignment and phylogenetic analysis showed number of issues must be addressed to enable the scale-up of that the two closest MAT1-2 BLAST hits Theau_59447 and

Table 3. Putative regulatory elements for cellulase production in T. aurantiacus ATCC 26904 genome.

Transcription Thermoascus aurantiacus protein ID Proposed Closest Factor (Mycocosm) Function Homolog (GenBank)

Cre-1 Rasamsonia emersonii CBS 393.64 Theau_44762 Carbon catabolite repression (Zn finger) (78%)

XlnR Regulation of cellulase/xylanase Rasamsonia emersonii CBS 393.64 Theau_38177 (Zn finger) gene expression (72%)

Clr-1 Aspergillus Theau_37408 Induction of cellulase genes (Zn finger) fumigatus Af293 (71%)

Clr-2 Aspergillus Theau_50720 Induction of cellulase genes (Zn finger) lentulus (56%)

ClbR Byssochlamys spectabilis No. 5 Theau_41281 Induction of cellulase genes (Zn finger) (74%)

Fig. (3). Mating type locus in Thermoascus aurantiacus ATCC 26904 genome. The genes in this locus, located on scaffold 5 of the assem- bled genome, encode for: APN-2 (DNA lyase), Theau_55954 (hypothetical protein), MAT1-1 (mating type alpha box protein), SLA-2 (actin binding protein).

Thermoascus aurantiacus is an Intriguing Host for the Industrial Production of Cellulases Current Biotechnology, 2017, Vol. 6, No. 2 95 A B

Theau_55954 ------DVRNMFYSRLPEEART---ASEASPDS Theau_16404 DDYGSSSAYHTSGGDYPHILPVQHSAFTHPVQVNTPPAVADEGIVARKGRSIPPPSSPLA Theau_59447 ------PSKVAVADRADGGSNGPSSSKYGANPATPST mat1-2_RE ------LLPLNIVDLIGNKNIEEIKARLCALIHAPVV mat1-2_AN ------LLPTNVVDIIGQDNVEKIKSRLSALIGAPVV mat1-2_AF ------LLPINVTDMIGQDNVDKIKTRLGALIGAPVV mat1-2_NF ------LLPINVTDMIGQDNVDKIKTRLSALIGAPVV * Theau_55954 AFQTLPVGAMALLGEWRARARGLERKCREMSHIYWTSKFEDASSHSILLQPAVPENMQQ- Theau_16404 ATRNMGVDKVRNIKTARSRRRNKQSKADRGASPKITAPLSELTKNLTHIPIRDMEAWVNR

Theau_59447 ALSQAHLSSANREKVALNEISSSASLTRKRVAPLLTEDISLQCRSSSSSPSKSMPSKEPV mat1-2_RE GFVDNSMNSFRIM-----RSPPFTGSTNDAASHTLVPMIPNDTQQIAVEPENKTSGKKG- AF MAT1-2 MAT1-2 NF mat1-2_AN SFVDESINALRVL-----RTPTFSGSSISVASPSRALD------SWPSEPPNKPRPA--- Theau 55954 mat1-2_AF AFVDETIKALRVM-----RTPAFSGTAVSVASHGEAVKTNKVTVTESFAPRGKPVG---- mat1-2_NF AFVDKSIEALRVM-----RTPAFSGRAISVASHGAALNADKVAATESFKPRGKPAG---- MAT1-2 AN HMG box Theau_55954 ------HDSTNTFTYDPNIWF----PRQPKIPNTSSTVRLEDLSLMIDQIEELQPRR Theau_16404 PIEVRRKEVEQKNGKIARPMNSFMLYRSAYAERTKEWCAQNNHQVVSRASGQSWPLEPPE Theau_59447 QFCL-----CQPDPKIPRPRNAFILYRQHYQAAVVAQNPGLANPEISKIIGEQWRALPNE MAT1-2 RE mat1-2_RE ------SGKKAKIPRPPNAFILYRQHYHPIIKEAHPEFHNNDISVTLGKQWNNEPEH mat1-2_AN ------SMKPAKIPRPPNAFILYRQHHYPKVKEARPDLSNNEISVIIGKKWRAEPEE mat1-2_AF ------PLKAPKVPRPPNAFILYRQHHHPKIKEAYPDYSNNDISVMLGKQWKDENEE mat1-2_NF ------PMKAPKVPRPPNAFILYRQRHHPKIKEEYPDFSNNDISIMLGKQWKDEPEE HMG box * * Theau_55954 AYAMSHAYGVIIRTECSSDACD-HYIPSL------Theau_16404 IREKFELLAIIERDNHQKAHPGYKFAPNK------SHTPPKKKRAVEE--- Theau_59447 TKSEWKALAEEEKARHQQQYPDYRYQPRRYGRDGSSRGASSSGISHNPPGATVCNRCGGR mat1-2_RE VKAHFKALANEAKRKHAEDYPDYQYSPRK------PSEKKRRILSHC--- mat1-2_AN GKLHFKNLAEEFKKKHAEEYPDYQYTPRK------PSEKKRRAASRI--- mat1-2_AF IKTQFRNLAEELKKKHAEDHPDYHYTPRK------PSERKRRTSSRQ--- mat1-2_NF VKAQFRNLAEELKKKHAEDHPNYYYTPRK------PSERKRRASSRQ--- Theau 16404 * Theau_55954 ------LQYLYLLSP------Theau_16404 ------DEPSDLDDPEYNLRSSPANFRKRARSS------Theau_59447 IMNPPSTPDTPFPGSGQATSSTRSPNSSSVPARSLPSRGSKDAVRSPSPGKPCSSMDPRA mat1-2_RE ------SPGNDKHDVYQ------mat1-2_AN ------SPKNSKRTVALENPGSMTAPSSNV------0.3 mat1-2_AF ------FSKNTKPAALRDTPASMNI-SSDV------59447 Theau mat1-2_NF ------FSKNTKSAAVLDIPASMNV-ASDV------

Fig. (4). Sequence alignment of Theau_55954 with MAT1-2 proteins from close relatives of T. aurantiacus and the two closest MAT1-2 BLAST hits, Theau 59447 and Theau 16404. A, ClustalW alignment of protein sequences done with MUSCLE (3.8) online tool of EMBL-EBI. Shades indicate the presence of at least 3 equal amino acid residues per column. HMG-box region is indicated with dashed box. Figure shows only a section of the alignment around the HMG- box. B, Phylogenetic tree is based on a ClustalV (PAM250) alignment of protein sequences. MAT1-2 AF, Aspergillus fumigatus (XM_749896); MAT1-2 AN, Aspergillus nidulans (XM_657246); MAT1-2 NF, Neosartorya fischeri (XM_001263956); MAT1-2 RE, Rasamsonia emersonii (XM_013470233); Theau 55954, T. aurantiacus hypothetical protein; Theau 59447 and Theau 16404, T. aurantiacus HMG-box proteins closest to MAT1-2. Gene bank accession numbers of related fungi in parenthesis. The alignment was done with MegAlign (DNAStar V10.1.2; Lasergene). The tree was exported to and manipulated with FigTree (http:// tree.bio.ed.ac.uk/software/figtree/).

T. aurantiacus for industrial cellulase production. Currently, gineering strategy, plasmid constructs are being designed to the mechanisms of induction that lead to the production of delete or overexpress genes involved in cellulase regulation cellulases in T. aurantiacus are not known. Previous studies that will derepress or activate the transcriptional response, have suggested that cellulases are produced when the fungal respectively [51, 52]. With this approach different genomic culture enters starvation or when the cells sense xylan hydro- alterations will be combined in a single strain by successive lytic products [28]. Focused studies that examine the tran- transformation and sexual recombination adding up to signif- scriptional response of T. aurantiacus to carbon limitation icant cellulase overproduction. and potential small molecule inducers of cellulase activity In conclusion, the combination of strain development and will be important in designing strategies to scale-up T. au- scale-up strategies, together with focused studies on cellulase rantiacus cultivation. Identification of small molecule inducers for T. aurantiacus will be essential for designing large induction, bear an enormous potential for T. aurantiacus to become a high-performance next-generation production host scale cultivations for economical production of cellulases. of thermostable cellulase enzymes. A second critical element of T. aurantiacus strain development is the identification of strategies to improve cellulase CONFLICT OF INTEREST production through genetic modification. Two complement- The authors confirm that this article content has no con- ing approaches are being pursued for cellulase strain im- flict of interest. provement, a classical random mutagenesis strategy and a genetic engineering strategy. Classical strategies for random ACKNOWLEDGEMENTS mutagenesis using UV radiation or chemical mutagens combined with screening for cellulase hyperproduction are being This work was funded by the U.S. Department of Energy implemented to identify strains that hyperproduce cellulases Bioenergy Technology Office. Portions of this work were and express cellulases in the presence of simple sugars like performed as part of the DOE Joint BioEnergy Institute glucose and xylose [6, 50]. Screening of progeny from sexu- (http://www.jbei.org) supported by the U.S. Department of al crosses between mutant hyperproduction strains of T. au- Energy, Office of Science, Office of Biological and Envi- rantiacus and the parent strains will be used to remove dele- ronmental Research, through contract DE-AC02-05CH1- terious mutations and to identify genomic alterations that 1231 between Lawrence Berkeley National Laboratory and confer the hyperproduction phenotype. With the genetic en- the U.S. Department of Energy. Genome sequencing of 96 Current Biotechnology, 2017, Vol. 6, No. 2 Schuerg et al.

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