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 ~50C. 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 technology 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 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 50C 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 65C [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 50C and not at 40C. 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 50C T. aurantiacus Miehe strains are found in multiple culture and no growth lower than 30C or lower. He observed fruit- collections; an example of the range of T. aurantiacus iso- ing body formation after two days of incubation at 40C 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

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