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Current Biotechnology, 2017, 6, 178-193

REVIEW ARTICLE

ISSN: 2211-5501 eISSN: 2211-551X

Chitin and N-acetylglucosamine Metabolism in Fungi - A Complex Ma- chinery Harnessed for the Design of -Based High Value Products

Romana Gaderer1, Verena Seidl-Seiboth1 and Lisa Kappel2,*

1Research Division Biotechnology and Microbiology, Institute of Chemical Engineering, TU Wien, Vienna, Austria and 2Institute of Microbiology, University of Innsbruck, Innsbruck, Austria

Abstract: Background: Chitin is one of the world’s most abundant biopolymers, but in contrast to cellulose its potential has so far been fairly underrated. Compared to cellulose, chitin features an additional amino-group on the hex- ose sugar ring that renders it a promising substrate for functional biopolymers in biomedical applications as well as biochemical synthesis of specialty and fine chemicals. Fungi with specialized life styles, such as mycopara- sites and entomopathogenic fungi, have evolved an effective machinery to utilize chitin derived from other fun- A R T I C L E H I S T O R Y gi and , respectively.

Received: January 15, 2016 Revised: March 16, 2016 Objective: Fungal chitin degradation and chitin synthesis have been subject to extensive investigation in Accepted: March 29, 2016 the past years to gain insight into these intricately regulated processes, which render fungi capable of

DOI: harnessing the recalcitrant chitin polymer as nutritional source and building block for growth. For the 10.2174/2211550105666160330205801 development of methods to exploit chitin from these origins, the design of new products and their sus- tainable production through enzymatic action, identifying the regulators of the chitin anabolic and cata- bolic pathways will be essential. In this review our current knowledge on chitin metabolism in fungi is presented; new sources for chitin production and new products derived from enzymatic processing will be introduced.

Keywords: Chitin, chitosan, N-acetylglucosamine, biopolymer, filamentous fungi, regulation, transcription factor, bio-based chemicals.

1. CHITIN – A BIOPOLYMER OF UNEXHAUSTED partially deacetylated derivative of chitin, is a constituent of POTENTIAL many ascomycete spore walls, but occurs in high amounts only in some specialized fungi (see also section 2 and [7]). It Chitin is a linear amino- that is present in can be generated via chemical or enzymatic deacetylation. high amounts in arthropods, crustaceans and, in varying The partial deacetylation leads to free amino groups with amounts (0.5-20%), in cell walls of fungi. It is composed of positive charges at slightly acidic pH values, rendering it β-(1, 4) linked N-acetylglucosamine (GlcNAc; 2-acetamido- soluble in dilute aqueous acidic solutions and the only 2-deoxy-D-glucopyranose) subunits and these GlcNAc known polycationic natural polysaccharide. Further deacety- chains adopt a 2-fold helical conformation, similar to that lation increases solubility and antibiotic effectivity ([8] and observed in other homopolymers, such as cellulose [1]. references therein). These helical chains of chitin can be arranged in antiparallel (α-chitin) or parallel (β-chitin) order to form fibers of a high Chitin is the second most abundant polysaccharide after tensile strength and crystallinity. α-chitin, the prevalent form, cellulose [9, 10] and probably the most abundant biopolymer in is more rigid and strong due to extensive hydrogen bonding the aquatic environment [11, 12]. Six to eight million tons of [2], whereas β-chitin has a higher affinity to solvents due to chitinous waste are produced annually only by the fishing in- its weaker hydrogen-bonding and has so far only been identi- dustries, 1.5 million tons of which are generated in south Asia fied in squid pens and other specialized parts of mollusks alone [13]. This corresponds to roughly 75% of the total weight and arthropods [3-5]. A third form, γ-chitin, is assembled in of shellfish (such as shrimp, crab and krill shells), currently stacks of one antiparallel and two parallel chains, and seems disposed of in landfills, where uncontrolled decomposition by to occur very rarely, e.g., in the cocoon fibers of Ptinus bee- microbes leads to environmental and human health concerns, tle and the stomach of Loligo squid [3, 6]. Chitosan, a and thus increases costs for waste processing. This shellfish

Current Biotechnology ‘waste’ contains around 20-58% chitin [14], but only little *Address correspondence to this author at the University of Innsbruck, more than 100,000 tons of chitin are estimated to be obtained Institute of Microbiology, c/o FH Campus Wien Bioengineering, Muthgasse from these marine by-products annually by 2018 [13, 15]. Con- 11/2, 1190 Vienna, Austria; Tel.: +43-1-58801-166522, Fax: +43-1-58801- sidering the still underexploited use of chitin, it is interesting to 17299. E-mail: [email protected]

2211-551X/17 $58.00+.00 © 2017 Bentham Science Publishers Fungal Chitin Degradation for the Generation of High-Value Products Current Biotechnology, 2017, Vol. 6, No. 3 179 note that the global market for chitin and its derivatives was hydrogen bonding along the chitin chains [20, 21]. On the estimated to reach up to US$ 63 billion by the end of 2015, one hand, these appealing properties have furthered the bi- with Japan being the biggest player in chitin processing opolymer research on chitin and made it attractive for a vari- worldwide. Moreover, chitin derivatives such as chitosan, N- ety of applications, but on the other hand, harvesting chitin acetylglucosamine and glucosamine (GlcN; the monomers of and its by-products is impeded by its recalcitrant nature. chitin and chitosan, respectively), were considered to have the Chitin is an important constituent of fungi that confers greatest potential on the global chitin market by 2010 [16]. strength and rigidity to the fungal . Chitosan is a Conventional production of chitin and chitosans from shell- partially deacetylated (DA: lower than 50%, [22]) derivative fish waste is carried out in a chemical process with a deprotein- of chitin, which occurs in high amounts predominantly in the ation and a demineralization step by harsh treatment with order of Mucorales, and is produced by chitin deacetylases NaOH (around 50%) at high temperatures (up to 160 °C) fol- in the fungal cell wall [7, 23]. Chitosan has also been found lowed by an acid treatment step with organic acids. This cre- in ascospore cell walls of many fungi and in some fungi also ates large amounts of hazardous waste by-products. Further- at certain other growth stages, such as in e.g., Zygomycota more, for chitosan production a further alkali treatment results [24, 25], Cryptococcus neoformans [26], and even in low in ill-defined deacetylation grades. Poor reproducibility is an- amounts in [27]. However, due to other drawback of this process for chitin-based products. Fur- the presence of genes encoding for chitin deacetylases in the ther research on chitin properties, structure-function relation- genomes of a wide range of filamentous fungi (www.cazy. ships, and the development of strategies for the analysis of chi- org) chitosan presumably also occurs in most fungi at certain tin and chitosan polymerization and deacetylation grades led to growth stages. Chitin deacetylases are members of the Car- fairly reproducible and also marketable chitin derivatives in the bohydrate Esterase family 4 (www.cazy.org/CE4) and ex- last years (for a review on current procedures for preparation of tract the acetamido group from GlcNAc units of the nascent chitin and chitosan from marine sources see also [5]). Although chitin chain while it is secreted into the cell wall. They can chitin and chitosan are also present in fungi (basidiomycetes be grouped into two classes, one class representing the above and ascomycetes), the industrial production from these sources mentioned deacetylases, designated periplamatic deacetylas- is still in fledgling stages, due to higher costs and still remarka- es, and the second class, which includes the extracellular bly low knowledge of the interplay and regulation of chitin deacetylases that are secreted into the environment. The degrading and processing . Major efforts are currently deacetylation pattern varies considerably among chitin deac- underway to increase the knowledge on chitin and its enzymat- etylases, and is also thought to help in disguising chitin in ic decay and to develop new methods for the production of the cell wall during penetration of plants, as shown for the well-defined chitin (-oligomers) and chitosans, to be able to fungal parasite Coletrichum lindemuthianum [28]. Expres- harness their qualities for the generation of high value products. sion of deacetylases in general occurs very specifically at certain growth stages, e.g., in S. cerevisiae only during spor- Fungi have proven to be feasible tools for a variety of bi- ulation, or in M. rouxii only during cell wall formation (for a otechnological applications in the past decades. An econom- detailed review on chitin deacetylases see also [29] and ref- ically important field that evolved over the past century is erences therein). The fungal cell wall consists of an alkali- secondary metabolite production, with examples such as insoluble fraction, which contains chitin and β - (1, 3) glu- generation of citric acid from Aspergillus niger, antibiotics can, and an alkali-soluble fraction composed of α-glucans, from Penicillium spp. or carotenes from yeasts [17]. Moreo- galactomannans and other carbohydrate polymers. Masked ver, recombinant enzymes/proteins for the food (food addi- by layers of other carbohydrates and proteins, chitin and chi- tives), medical (e.g. antibodies) and textile (e.g. ) tosan are therefore not easily accessible from the outer side industry are produced with fungi. More recently, filamentous of the cell wall. The harsh chemical treatments, used to ex- fungi have proven to be potent producers for enzymes in- tract the sugars from the fungal cell wall for compositional volved in biomass degradation (advanced biofuels from cel- analysis, also lead to partial deacetylation of GlcNAc. There- lulosic waste material; [18, 19]) and production/modification fore, the reported amounts of GlcNAc and GlcN cannot al- of new biorefinery products. In the following sections the ways be reliably related to the occurrence of chitin or chi- fascinating possibilities appearing with the new knowledge tosan in the fungal cell wall. So far, there is no evidence for on enzymatic chitin processing and generation in fungi are targeted deacetylation of chitin within fungal cell walls, alt- hough it has been speculated that chitin deacetylation may matched with ideas and strategies of chitin production from increase the elasticity of the polymer and protect it from hos- alternative sources and the design of new products. We first tile chitinases [30, 31]. In fungi, the amount of chitin and/or give an overview of the current knowledge on chitin degra- chitosan differs considerably among various classes. Where- dation, including the last steps for chitin catabolism, as well as yeasts contain only 0.5-5% chitin, mainly in septa, con- as a brief overview of chitin synthesis in fungi and then illus- striction rings and budding scars, filamentous fungi harbor trate how these insights can be exploited to design biochemi- up to 20% chitin in the inner layers of the cell wall, located cal approaches for chitin hydrolysis and new high end prod- close to the plasma membrane [32, 33], which is interesting- ucts of great biotechnological potential. ly also reflected in the number of chitinases (see section 3.1). 2. CHITIN AND CHITOSAN IN FUNGI These findings show that mycoparasitic filamentous fun- gi in particular were required to evolve a very diverse net- Chitin is a crystalline, extremely strong biopolymer, with work of enzymes and auxiliary proteins to attack and nourish a tensile strength that exceeds that of many artificial materi- on other fungal cell walls and, at the same time, be able to als, which stems from extensive intra- and intermolecular 180 Current Biotechnology, 2017, Vol. 6, No. 3 Gaderer et al. distinguish between self- and non-self and remodel chitin monomers from chitin oligosaccharides, corresponding to an during certain growth stages. Exo-N-acetylglucosaminidase activity [54]. This is due to its protein architecture, because a CBM 18 is inserted in the GH 3. ENZYMATIC DECOMPOSITION OF CHITIN AND 18 module of CfcI. Bacterial chitinases have been shown to CHITOSAN IN FUNGI exhibit enzymatic activity on chitosan, as well, but this is strongly dependent on the degree of acetylation, due to the 3.1. Chitinases and Chitosanases requirement of chitinases for the acetyl residue in the -1 sub- Chitin has an estimated natural turnover of 10 9 tons per site. Most likely this finding can also be extrapolated to fun- year so that it is very efficiently decomposed by microorgan- gal chitinases. isms. It is degraded by two main groups, designated In contrast to chitinases, which prefer highly acetylated chitinases and N-acetylglucosaminidases (NAGases). In chitin (DA: higher than 50%, [22]), chitosanases favor high- analogy to that, chitosan, the N-deacetylated derivative of ly deacetylated chitosan (Fig. 1B). Chitosanases (EC chitin, is decomposed by chitosanases and exo-β-D- 3.2.1.132) are glycosyl that cleave the β- (1, 4) glucosaminidases, but so far both have not been studied as glycosidic linkage of GlcN-GlcN and GlcNAc-GlcN, but extensively as chitinases and NAGases. These enzymes hy- only chitinases are able to split GlcNAc-GlcNAc linkages. drolyse the glycosidic bonds between the complex sugars, To date, many chitosanases have been identified in a variety and thus, fall into the enzyme class of glycoside hydrolases of organisms, including fungi [55-58], bacteria [59-62], (GH) [34, 35]. Chitinases occur in organisms in all kingdoms plants [63], and viruses [64]. In general, fungal chitosanases of life, such as bacteria [36], fungi [37], plants [38], viruses belong to GH family 75 [65-68]. Chitosanases act on the [39], and animals, including insects [40] and vertebrates reducing end of the chitosan molecule and thereby produce [41]. Low numbers of chitinases are found in yeast and glucosamine oligosaccharides. They are classified according yeast-like fungi, for example, S. cerevisiae and the dimor- to their ability to hydrolyze various types of linkages in the phic basidiomycete Ustilago maydis have two, and Candida chitosan molecule [69, 70]. Enzymes that belong to subclass albicans four [42]. By contrast, filamentous fungi usually I are able to split the linkages of GlcNAc-GlcN and GlcN- possess 10 to 20 chitinases and the number of chitinases can GlcN, whereas subclass II chitosanases are characterized by rise up to 35 in fungi that have adapted to a particular life- their ability to cleave only GlcN-GlcN linkages. Subclass III style, i.e., mycoparasitic (fungi that parasitize other fungi) enzymes are able to hydrolyze GlcN-GlcNAc and GlcN- and insect-pathogenic growth (entomopathogens) [43, 44]. GlcN linkages. The extraction of Glucosamine from the non- Chitinases (EC.3.2.1.14) are hydrolytic enzymes, which reducing end of the chitosan (-oligomers) is performed by so catalyze the hydrolysis of the β- (1, 4) linkages in chitin and called Exo-β-D-glucosaminidases (Fig. 1B) [65, 71]. chitooligomers, releasing short-chain chitooligosaccharide Classification of the chitinases and chitosanases into products with a minimum chain length of 2 GlcNAc mole- groups and subgroups alone is not necessarily indicative for cules [45]. In general, chitinases belong to GH 18 and 19, their biological function. Furthermore, chitinases may play a but while in bacteria and plants chitinases from both of these role in more than one process: they are not only important families are present, in fungi exclusively GH 18 family chi- for degradation of extracellular chitin, but also in self- tinases are found (Fig. 1A). Chitinases are phylogenetically digestion, such as cell wall remodeling during fungal growth, divided into subgroups A, B and C, based on the amino acid as well as in cell wall degradation during autolysis and apop- sequences of their GH 18 modules [42, 46, 47]. These sub- tosis [72]. groups differ in their modular protein architecture, as well as in the shape of their substrate binding clefts, and thus, their As already stated above, the number of chitinases reflects predicted enzymatic activities. According to their cleavage the fungal lifestyle. For example the mycoparasites T. atro- pattern, they are further classified as endochitinases, which viride and T. virens have 29 and 36 GH 18 proteins, while cleave chitin at random positions within the chitin chain and the unicellular yeasts S. cerevisiae and Schizosaccharomyces have a more shallow and open substrate-binding region pombe, have only 2 and 1 [72-74]. A versatile array of chi- (subgroup B), or as exochitinases, which act on either end of tinases, on the other hand, might not simply be an indicator the polymer and have deep, or even tunnel-shaped catalytic for higher chitinolytic efficiency, but may rather mirror the clefts (subgroups A and C) and more likely cleave chitobiose competitive environment that the encounters and might render it less susceptible to chitinase inhibitors or pro- (GlcNAc)2 from the reducing ends of chitin chains [45, 48- 50]. Furthermore, the chitinases from different subgroups teases that are secreted from other organisms in this habitat can contain carbohydrate binding modules (CBMs) that are [72]. Furthermore, the high degree of variability among dif- characteristic for individual subgroups and influence enzy- ferent chitinases seems to reflect the adaption to various en- matic processivity (i.e. consecutive cleavage steps on the vironmental stimuli, leading to the diversification of some polymer chain; for reviews on this topic see [31, 51]). An subgroups, e.g. the subgroup C chitinases in mycoparasites. extraordinary class of GH 18 proteins in the subgroup B chi- With the 1-2 GH 20 N-acetylglucosaminidases present in tinases are the so called endo-β-N-acetylglucosaminidases fungi, the 2-5 GH 75 chitosanases and chitin deacetylases, (ENGase, EC.3.2.1.96). These enzymes cleave the (Glc- which both unfortunately have not been studied extensively in fungi yet, this confusing bundle of chitin active enzymes NAc)2 linkages that anchor N-glycan antennae to proteins in glycosylated proteins, and therefore act as deglycosylation certainly must be subject to tight and purposeful regulation enzymes [52, 53]. Furthermore, Cfcl from A. niger, which is that lacks any evidence of transcriptional activators, so far. produced during autolysis, is an interesting extension to the On the other hand, external factors regulating the expression enzymatic repertoire of GH 18 proteins, because it releases of these enzymes have already earned a lot of attention and a Fungal Chitin Degradation for the Generation of High-Value Products Current Biotechnology, 2017, Vol. 6, No. 3 181 general overview on the current findings will be given be- other stimuli, such as starvation and autolysis, or may also be low. induced during growth on chitin as carbon source, which contradicts the clear pattern. The conditions tested for chitinase expression can rough- ly be grouped into (myco-) parasitism or cell wall remodel- Chitinases involved in processes generally associated ing related stimuli, as well as to chitin as carbon source for with cell wall remodeling (including starvation and autoly- nutritional purposes. atroviride Chi18-5/Ech42 sis) can come from all three subgroups, but so far for A or B (T. reesei Chi18-5, T. harzianum Chit42 and T. virens more examples have been found [42, 50, 82, 87-89]. Some Cht42/Tv-Ech1, Neurospora crassa gh18-4, Aspergillus nid- subgroup B chitinase encoding genes, such as T. atroviride ulans ChiB, A. fumigatus ChiB1) is one of the best studied chi18-13/ech30, show a carbon source inducible regulation, chitinases. It belongs to subgroup A and is secreted during hinting rather at a catabolic role. Moreover, Chi18- the mycoparasitic attack [46, 75-77]. However, Chi18- 13/ECH30 has an unusual substrate binding cleft, underlin- 5/ECH42 is not only a mycoparasitism specific chitinase, but ing the diversity and biotechnological potential of chitinases also involved in self- and non-self cell wall degradation, [42, 51]. Additionally, subgroup B contains chitinases with since it was also found expressed during autolysis and star- other specificities, such as the ENGases (B5), which seem to vation. The role of Chi18-5/ECH42 seems to be conserved in be involved mainly in post-secretional processes or ERAD other species and even genera with a similar lifestyle [78- [52, 53]. Furthermore, one GPI-anchored subgroup B chi- 82]. tinase per species is present, such as N. crassa Chit-1 or A. nidulans ChiA that is involved in cell wall synthesis and Subgroup C chitinases were found to be partially associ- remodeling at the tips and branching points of hyphae. ated with (parasitic) fungal-fungal interactions. In T. atro- Therefore, Subgroup B again is a very diverse group of chi- viride, for example, all subgroup C chitinases were induced tinases [31, 90, 91]. Thus, despite the categorization into the during interactions with B. cinerea, but not R. solani or it- three architectonically different subgroups A, B and C, and self, except for tac6 that does not have an intact the further classification into smaller phylogenetic groups, [83]. In contrast, in the closely related T. virens the regula- which helps to allocate the vast number of chitinases present tion of the subgroup C chitinases could not clearly be as- in fungal species, their regulation can only partially be in- signed to only one fungal host or growth condition [84]. In ferred from GH18 module-based phylogenetic clustering A. nidulans expression of subgroup C-II chitinases was in analyses. general strong upon contact with some (including B. cinerea and R. solani), but not all of the tested fungal interactors, 3.2. N-Acetylglucosaminidases however, a response to intra-species interactions or chitin was not observed [85]. From the available data the subgroup As mentioned above, fungi have a multitude of chi- C chitinases have the strongest implication in inter-species tinases, which convert chitin into chitobiose. Interestingly, interactions, due to the fact that many chitinases are upregu- for the next step of the chitin degradation pathway – splitting lated under mycoparasitic growth conditions. Also the strong the dimer chitobiose into two GlcNAc monomers – fungi diversification and the increase in number in fungi with my- possess only one or two N-acetylglucosaminidases, which coparasitic lifestyle hint at their involvement in the attack belong to the GH 20 family proteins [92] (Fig. 1A). N. cras- [31, 42, 51, 72, 86]. On the other hand, chitinases that are sa has one NAGase, NAG-1, which is located in the cell wall expressed during mycoparasitic growth may also react to and is associated with fungal-fungal interactions [93]. Fur-

Fig. (1). Schematic representation of chitin and chitosan and their degradation into monomers in fungi. (A) Chitin is a linear polysaccharide, composed of β- (1, 4) linked N-acetylglucosamine subunits. GH family 18 endochitinases subgroup B (sgB) cleave the chitin chains at random positions, to produce chitooligomeres. They have a flat substrate binding cleft and can, depending on the species, contain CBM module 1, 5, 19, etc. GH 18 exochitinases, subgroup A/C (sgA/C), cleave off chitobiose from the chitin chain. They both have a narrow, tunnel shaped binding cleft, but in contrast to subgroup C, which possess multiple CBMs (CBM 18 and CBM 50), sg A chitinases are devoid of CBMs. The β-N-acetylglucosaminidases (GH 20, NAGase) break the released chitobiose into two GlcNAc monomers, which can enter the cell for GlcNAc metabolism. (B) GH family 75 chitosanases cleave highly deacetylated chitosan chains at random positions to generate chitosan-oligomers. Exo- β- D- glucosaminidases (GlcNase, GH 2) extract glucosamine from the chitosan chains. 182 Current Biotechnology, 2017, Vol. 6, No. 3 Gaderer et al. thermore, the A. nidulans nagA gene encodes a NAGase, Due to their substantial variation in substrate specificity, which additionally plays an important role during autolysis CBMs have been classified into 73 classes [80, 94]. The yeast C. albicans also harbors only one NA- (www.cazypedia.org). Chitin-binding properties have been Gase, Hex1, whereas S. cerevisiae does not possess any [95]. described for a number of different CBM families that are Two functional NAGases were identified in T. atroviride and found in fungi, including CBM 1 and 18, which usually oc- designated NAG1 and NAG2. A very distantly related third cur as separate protein domains in enzymes. CBM family 1 GH 20 protein was also found, but is rather associated with typically exhibits cellulose-binding properties, but chitin- N-acetylgalactosaminidase function and lacks any further binding characteristics have also been reported [101]. CBMs characterization (JGI accession number: 293914). The pres- of family 18 are often found in fungal chitinases (GH family ence of either of these enzymes is sufficient but essential for 18), chitin synthases (GT family 2) and chitin deacetylases the use of chitin or chitobiose as carbon source in T. atro- (CE family 4), and therefore members of CBM family 18 are viride. Although nag1 and nag2 expression was found to be probably typical chitin-binding modules. strongly inducible by GlcNAc, the double knockout strain LysM motifs belong to the CBM family 50 and have could still utilize GlcNAc as carbon source, indicating that general GlcNAc binding properties recognizing chitin, chito- the GlcNAc monomer could still enter the cell to be metabo- oligosaccharides and peptidoglycan. LysM motifs are found lized in absence of the NAGases [96]. Extracellular localiza- in prokaryotes and eukaryotes. They occur in fungal sub- tion of NAG1 and NAG2 is different - NAG1 is secreted into group C chitinases and interestingly, in contrast to other the medium, whereas NAG2 stays (non-covalently) attached CBMs, also in secreted fungal proteins that contain multiple to the cell wall [96]. In addition, NAG1 was shown to be LysMs, but lack a catalytic domain (effector proteins). In involved in autolytic processes, and its expression was also fungi, LysM motifs are phylogenetically divided into two induced by mycoparasitic growth conditions and by chitin clades, a fungal-specific and a fungal/bacterial subclass, [75, 77, 97]. NAGase activity has been reported from a based on their amino acid sequences [102]. LysMs of the number of other fungi, but a clear transcriptional regulator latter class are involved in interactions between fungi and had not been identified up to now ([72, 93, 98], see section plants. Members of this fungal/bacterial subclass are report- 4.1 for new findings on that topic). ed from the tomato pathogen Cladosporium fulvum, Mag- The presented knowledge on the regulation of chitin de- naporthe grisea, and the hemi-biotrophic wheat pathogen grading enzymes already reveals the flaw in the expression Mycosphaerella graminicola. These LysM effector proteins studies performed so far. It seems there is quite some inter- are specifically upregulated during the plant-pathogen inter- play of growth conditions, lifestyle and growth stage/age, action and associated with suppression of chitin-induced which makes it seemingly impossible to look at the expres- plant immune responses and protection against plant chi- sion of a single chitin degrading enzyme at a certain condi- tinases [103-106]. A representative for a member from the tion. From the available data, including the phylogenetic fungal-specific subclass is T. atroviride TAL6, which inhib- analysis, only a rough categorization with notable exceptions its germination from Trichoderma spp., but not from other can be drawn. Since evidence is lacking for regulation by fungi, however, the main function is still unknown [72, 107]. transcriptional or post-transcriptional regulators so far, a If addition of LysM effectors to chitinous substrates enhanc- es or decreases the accessibility for chitinases poses an inter- complete characterization is not possible at the moment. esting question for future applications. Therefore, identifying these regulators and understanding their role in chitin degradation represents a major future ob- Another auxiliary with chitin-binding jective, so that we will be able to reprogram chitin degrada- properties is the cerato-platanin protein (CPP) family, which tion for optimized biotechnological processes. is exclusively found in fungi [108-112]. Previous studies for several CPPs (for T.atroviride EPL1, C. platani CP, Cerato- 3.3. Auxiliary Proteins/ Carbohydrate Binding Modules cysis populicola Pop1 and M. perniciosa MpCP1-5) showed that they bind polymeric chitin and/or chitin oligomers [13, In addition to the catalytic domain, carbohydrate-active 110, 113]. CPPs are small secreted proteins, which are im- enzymes, such as cellulases or chitinases, often harbor one or portant factors in interaction of fungi with other organisms more carbohydrate binding modules. CBMs are devoid of and they are able to alter the polarity of solutions [110, 112, any catalytic activity, consist of up to 200 amino acids and 114]. It has been reported that CPPs can exert expansin-like multiples of the same or different CBMs can appear in one features on cellulosic materials. Expansins are small proteins protein in tandem. The topography of the CBMs is designed that show carbohydrate binding and loosening properties, such that it facilitates tight binding to an insoluble substrate, which is similar to the recent findings for CPPs, and there is and thereby, supports the penetration of a crystalline struc- also a structural similarity between CPPs and plant expansins ture. The efficient recognition and binding to the substrate is [108-110]. Expansins contain an N-terminal (D1) and a C- facilitated by an arrangement of conserved residues in the terminal (D2) domain. The expansin D1 domain forms a of the CBMs, which mirror the specific polysac- double β-barrel fold, which is similar to the CPPs single do- charide structure. Minute changes in the topology of the main named certo-platanin domain [109, 113]. Some CPPs binding sites alter the specificity towards the ligand. Thus, were found to have weakening activity on cellulose materials CBMs increase specific binding of an enzyme to and can without enzymatic activity. Interestingly, an effect on chitin prevent the dissociation from a substrate after successful has not been detected yet, however, the binding affinity of cleavage, enabling the protein to slide along the carbohydrate CPPs to chitin is stronger than to cellulose [13, 110, 113, chain for the next cleavage step, and thereby positively affect 115]. Therefore, CPPs are an attractive target for future stud- the processivity of these enzymes [99, 100]. ies to assist the industrial chitinolytic processes. It will be Fungal Chitin Degradation for the Generation of High-Value Products Current Biotechnology, 2017, Vol. 6, No. 3 183 interesting to see if CPPs help in making chitin more acces- olism pathway for chitin breakdown and anabolism to gener- sible, due to their amphiphilic character, or if they stabilize ate UDP-GlcNAc, as well as chitin synthesis will briefly be chitin, such that chitinases can more easily access the chitin introduced and the mechanisms regulating these processes chains for catalytic cleavage. will be discussed.

An additional group of enzymes, more recently described, 4.1. N-Acetylglucosamine Catabolism are lytic polysaccharide monooxygenases (LPMOs) that intro- duce random chain breaks into cellulose, hemicellulose and The monomer of chitin, GlcNAc, can serve as carbon and chitin. In contrast to the hydrolytic cleavage by GH enzymes, nitrogen source, due to the presence of the acet-amido group the copper-dependent LPMOs insert molecular oxygen into the on the hexose ring structure. Microorganisms, using chitin as C-H bonds adjacent to the glycosidic linkage, which results in nutritional source, have evolved a simple but effective three oxidative cleavage of the substrate [116-118]. LPMOs exhibit step process to feed GlcNAc into glycolysis with the benefit increased enzymatic cellulose degradation properties, but evi- of ammonium sequestration. GlcNAc catabolism gained dence for the importance in utilization of chitin is now also considerable attention mostly in bacteria, due to the potential accumulating. They offer an alternative to typical chitinases, to produce enough enzymes to effectively degrade GlcNAc due to their ability to cleave oxidatively and [133, 134]. In eukaryotes, however, GlcNAc catabolism was may also be used to increase chitinolytic activity of chitinases investigated mainly in C. albicans, in the past years [98, [116, 117, 119-121]. LPMOs are registered as “Auxiliary Ac- 135]. In S. cerevisiae, which lacks the enzymes for GlcNAc tivities” (AAs) family 9 (formerly GH61) in the CAZy data- catabolism, introduction of the GlcNAc catabolic genes from base (www.cazy.org). The first LPMO that was reported is C. albicans was tested for bioethanol production [136]. CBP21 [117], a bacterial chitin active enzyme from Serratia C. albicans is a dimorphic ascomycete that can switch marcescens (AA family 10; formerly CBM 33), but an between a unicellular budding yeast form and a multicellular, orthologue does not seem to exist in fungi. However, recently pathogenic filamentous form, which show different charac- the first chitin-active LPMO, AoAA11 from A. oryzae was teristics like a white versus opaque growth phenotype at cer- detected and is now listed as AA family 11 [122]. tain temperatures, respectively. The switch to filamentous Testing these LPMOs in chitin degradation, with respect growth is induced by human/animal serum and, interestingly, to synergistic effects with chitinases, represents a promising also by the presence of GlcNAc and renders C. albicans a task to increase chitooligomer production from the recalci- highly pathogenic threat for transplant patients, as well as a trant chitin sources. cumbersome nuisance as oral and vaginal candidiasis [132, 137, 138]. This switch can also be induced by GlcNAc in 4. CHITIN AND GLCNAC - NUTRITIONAL SOURCE other yeasts, e.g., C. lusitaniae [139] and Yarrowia liplytica OR BUILDING BLOCK FOR GROWTH [126], and only recently, also the thermally dimorphic yeast Histoplasma capsulatum was shown to efficiently switch to Besides its role in chitin and chitosan, GlcNAc plays an filamentous form upon concurrent induction by GlcNAc and important role in a broad range of mechanisms throughout a switch to room temperature [140]. In C. albicans the all kingdoms of life [115]. It also occurs in heterogeneous mechanism of GlcNAc signaling earned substantial attention polysaccharides, such as peptidoglycan/murein, and a β- (1, and led also to the discovery of a highly specific GlcNAc:H+ 6) linked poly-N-acetylglucosamine is the major constituent symporter from the major facilitator group of transporters of bacterial biofilms [123, 124]. Apart from its important (MFS), Ngt1, and a cluster of three catabolic genes that are role in cell structure, GlcNAc itself may mediate cellular involved in the well-defined GlcNAc catabolism pathway signaling. In dimorphic yeasts, e.g. C. albicans, GlcNAc [141]. Via consecutive action of Hxk1 (GlcNAc-hexokinase; induces the switch from budding to filamentous growth [125, EC 2.7.1.59), Dac1 (GlcNAc-6-PO -deacetylase; EC 126]. In bacteria extracellular GlcNAc provokes production 4 3.5.1.33) and Nag1 (GlcN-6-PO -deaminase; EC 3.5.99.6) of CURLI (= curled pili) fibers that promote biofilm for- 4 GlcNAc is transformed to fructose-6-PO , which can enter mation [127]. Furthermore, GlcNAc is an essential part of 4 glycolysis [131, 142] (Fig. 2A). This catabolic pathway is the N- and O-linked attachment on glycoproteins or in GPI dispensable for the switch to filamentous growth in C. albi- anchors (glycosylphosphatidylinositol) on plasma membrane cans. Deletion of either of the catabolic enzymes rendered C. attached proteins. Even in mammals (including humans) albicans incapable of utilizing GlcNAc, but the switch to GlcNAc plays a pivotal role. Hyaluronic acid (HA) is an filamentous growth could still be performed [131, 135]. indispensable component of the connective, epithelial and Moreover, it could be shown, that in a homozygous hxk1 neuronal tissue, and consists of repeating D-glucuronic acid knockout strain signaling was still induced by GlcNAc [98]. and GlcNAc residues [128]. In some cases GlcNAc exists in In fact, it was proposed that GlcNAc serves as signal rather free form, e.g. in human milk (at 600–1500 mg/mL) [129, than GlcNAc-6-PO , since the cell thereby can distinguish 130]. 4 between the external GlcNAc that enters the cell and the Thus, GlcNAc can serve as carbon source, but can also native GlcNAc-6-PO4 generated by anabolic processes in the be recycled and used as building block to generate new cell cell (see also section 4.2). It was further shown that GlcNAc material or contribute to cell signaling in free form. Regulat- needs to enter the cell, to signal availability of the carbon ing the fate of GlcNAc in the fungal cell, by coordinating its source for nutritional purposes and that Ngt1 is the major distribution, must be subject to tight control. The balance and high affinity transporter for GlcNAc [141]. In contrast to between the anabolic and catabolic pathways is critical, since a hxk1 knockout strain, deletion of dac1 or nag1 caused se- disproportional coordination of the metabolism can severely vere growth defects when GlcNAc served as carbon source, impair growth [131, 132]. In the following section the catab- even in the presence of other sugars (D-glucose, D-fructose, 184 Current Biotechnology, 2017, Vol. 6, No. 3 Gaderer et al.

D-galactose), suggesting that excess GlcNAc-6-phosphate is indispensable. Therefore, also the enzymatic machinery, deleterious [131]. In our recent study analysis of a broad needed to produce these universally present structures, is taxonomic range of ascomycetous fungi (Pezizomycotina) conserved in eukaryotes down to yeast S. cerevisiae and Sz. identified the presence of a GlcNAc cluster in many line- pombe as well as in prokaryotes. Gene deletion of any of the ages, although with variations in cluster organization [143]. involved enzymes is deleterious unless GlcN or GlcNAc are The three catabolic genes (designated hxk3, dac1 and dam1) present in the growth medium, emphasizing their detrimental are conserved in filamentous fungi and are essential for role in growth processes [157-159], but also pointing at pos- growth of T. reesei on GlcNAc as sole carbon source and to sible redundancies with the catabolic enzymes. some extent also on chitin (Fig. 2C). The GlcNAc trans- porter NGT1, identified in this study, is also important for Hyphae of C. albicans harbor around five-fold amounts growth on GlcNAc [143]. Another gene, identified in the of chitin (with around 4-5% chitin in their cell walls) com- clusters, is a GH3-family gene of yet unclassified function. pared to the yeast like form, while in the thermally dimor- The gene, which was termed nag3, exhibits similarities to phic H. capsulatum the opposite is the case [159, 161]. The bacterial GH 3 β-N-acety-lhexosaminidases ([144-149] and anabolic enzymes for chitin synthesis were extensively stud- www.cazy.org/GH3.html). In fungi, N-acetylglucosamini- ied mainly in the two yeasts, C. albicans and S. cerevisiae, dase function has so far only been assigned to GH family 20 and a brief overview of the generation of UDP-GlcNAc and [42, 96], but the gene is essential for growth on GlcNAc chitin is given here (for more details on that topic the reader [143]. Recently, a GH3 family protein, Nag3, from Cellulo- is referred to [160]; for an overview on other GlcNAc modi- monas fimi was identified as a GlcNAc-phosphorylase, using fications the reader is referred to [162, 163] for these topics). phosphate rather than water as nucleophile [149]. Important- The conversion of sugar into sugar nucleotides was discov- ly, in many filamentous fungi a transcription factor with an ered by Luis F. Leloir in the 1950s, and therefore the biosyn- Ndt80-like DNA-binding domain (PFAM family PF05224) thetic reaction to create UDP-GlcNAc represents one version is also included in the cluster, which was designated RON1 of the Leloir pathway (Fig. 2B). The first two steps are an (regulator of N-acetylglucosamine catabolism 1) in Tricho- exact reversion of the last two catabolic steps conferred by derma spp. RON1 is a member of a rare family of exclusive- CaNag1/TrDAM1 and CaDac1/TrDAC1, respectively. Fruc- ly fungal transcription factors, which were first described in tose-6-PO4 is converted to GlcN-6-PO4 by Glucosamine-6- S. cerevisiae [150-153]. In Trichoderma spp. RON1 highly PO4 synthase (EC 2.6.1.16), which is designated Gfa1 in S. induces transcription of all three (hxk3, dac1, dam1) catabol- cerevisiae and C. albicans. Gfa1 catalyses the transfer of ic genes in the presence of GlcNAc, as well as the GlcNAc ammonium from L-glutamine to fructose-6-PO4. In homozy- transport protein NGT1 [144] (Fig. 2C). In C. albicans a gous deletions of gfa1 in diploid C. albicans strains growth gene for an orthologous Ndt80-like transcription factor, was only possible upon supplementation with GlcNAc [157]. REP1 (orf 19.7521) has been described that plays a role in The second step is generation of GlcNAc-6-PO4 by GlcN-6- the regulation of a multiple drug resistance efflux pump PO4 acetyltransferase/Gna1 (EC 2.3.1.4), which was charac- [154]. In filamentous fungi, Ndt80-family proteins were also terized in detail in S. cerevisiae, C. albicans, and Sz. pombe, studied extensively and XprG from Aspergillus spp. [155] and involves Acetyl-CoA as a co-factor. S. cerevisiae gna1 and NCU04729 from N. crassa [156] were identified as the deficient mutants cannot grow, even in the presence of Glc- closest orthologs in our study. An involvement of these can- NAc [159], but homozygous C. albicans gna1 null mutants didates in GlcNAc catabolism may be inferred but still needs may grow when supplemented with GlcNAc, although they to be confirmed. dramatically swell upon growth and are not able to undergo Importantly, in T. reesei, induction of gene expression of cell separation any more [164]. The differences in response all GlcNAc cluster genes and ngt1 was completely abolished to gna1 deletion in the two yeasts can be explained by the upon deletion of ron1. Furthermore, biomass formation on lack of GlcNAc-deacetylase and -kinase in S. cerevisiae. GlcNAc as sole carbon source was suppressed in knockout Considerable effort was made to identify regulators of the strains in submerged cultivations. Thus RON1 is essential anabolic enzyme Gfa1, although in contrast to GlcNAc ca- for growth on GlcNAc. Remarkably expression of the genes tabolism (RON1), so far, evidence for the transcriptional encoding the two NAGases, NAG1 and NAG2, was also regulator(s) of this pathway is missing. Cell wall stress and dependent on RON1 [143]. With the discovery of the tran- mating trigger S. cerevisiae GFA1 expression [165-168] and scription factor RON1 in the GlcNAc cluster of many fila- cell wall stress, as an inducer of gfaA (the GFA1 ortholog) mentous fungi, the first positive regulator of GlcNAc catabo- expression and chitin deposition in the cell wall, was con- lism genes has been pinpointed [143]. RON1, furthermore, is firmed in A. niger [169] and in C. albicans, during the switch the first identified positive regulator of chitin degradation for from the yeast-like to hyphal form of growth [170]. The nutritional purposes. catalytic activity of Gfa1 can further be post-transcriptionally increased nearly five-fold upon phosphorylation by protein 4.2. N-Acetylglucosamine Anabolism – The Chitin Bio- kinase A, as has been demonstrated for CaGfa1 [171]. A synthesis Pathway relatively strong and specific inhibitor of Gfa1 enzymatic activity is feedback inhibition by UDP-GlcNAc, the end- From the available data generated over the past years it product of the anabolic pathway, which can be alleviated can be expected that the intracellular GlcNAc pool not only when glucose-6-PO is absent [168]. undergoes catabolic recycling, but also feeds into anabolism 4 as UDP-GlcNAc, which is a building block for de novo chi- The last two synthesis steps are carried out by phospho- tin synthesis. For chitin synthesis as well as glycosylation acetylglucosamine mutase, Agm1 (EC 5.4.2.3), which ac- and anchoring of proteins in the cell wall UDP-GlcNAc is counts for the isomerization of GlcNAc-6-PO4 to GlcNAc-1- Fungal Chitin Degradation for the Generation of High-Value Products Current Biotechnology, 2017, Vol. 6, No. 3 185

Fig. (2). Metabolism of Chitin and N-acetylglucosamine exemplified by the fungi T. reesei and C. albicans. (A) The GlcNAc catabolic pathway. N-acetylglucosamine (GlcNAc) enters the cell via the MFS GlcNAc/H+ symporter T. reesei TrNGT1/ C. albicans CaNgt1. The pathway for gener- ation of fructose-6-PO4 from GlcNAc via three consecutive steps is shown. HXK3, DAC1 and DAM1 (Tr), as well as Hxk1, Dac1, and Nag1 (Ca) are the three orthologous proteins involved. The produced fructose-6-PO4 can directly enter glycolysis. (B) The GlcNAc anabolic pathway in C. albicans. The pathway for chitin synthesis, starting from fructose-6-PO4, is depicted. The involved proteins Gfa1, Gna1, Agm1, and Uap1 and co- factors needed are shown. The final product, UDP-N-acetylglucosamine, may be fed into the chitin synthesis pathway. Chitin synthases (GT family 2, CHS), located in the cell membrane (CM), catalyze the addition of UDP-N-GlcNAc to the non-reducing end of the growing acceptor oligosac- charide. Deacetylases (CE family 4), located in the periplasm, may contribute to deacetylation during generation of the chitin chain. Alternatively, UDP-N-GlcNAc may enter the pathway for N- or O-glycosylation and GPI anchoring (ER, Golgi). (C) Regulation of the catabolic pathway in T. reesei. The genes involved in GlcNAc catabolism are clustered in many filamentous fungi, as shown for T. reesei. The gene encoding the GlcNAc importer, on the other hand, often is separated from the cluster. A gene encoding for an Ndt80-like transcription factor, ron1 that is present in the cluster, is a major activator of all cluster genes as well as of ngt1.

PO4 and UDP-GlcNAc pyrophosphorylase ScQri1/ CaUap1 UDP-GlcNAc may then be fed into chitin biosynthesis (EC 2.7.7.23), that catalyzes the exchange of phosphate with and again many of the studies on chitin synthases (CHS, Uridine-5'-diphosphate (UDP). For both anabolic enzymes Chitin-UDP acetyl-glucosaminyl , EC 2.4.1.16) only limited data is available regarding their regulation [171, and their regulation were performed in S. cerevisiae and C. 172]. For ScAGM1 three putative pheromone-responsive albicans (Fig. 2B). There are 3 CHS enzymes in S. cere- elements have been identified [173] and the Uap1 enzyme visiae and 4 in C. albicans and in filamentous fungi the was effectively inhibited in vitro by uridine [174]. number is balanced around eight, but may exceed 20 chitin synthases in many mucoromycete species [20, 175]. By 186 Current Biotechnology, 2017, Vol. 6, No. 3 Gaderer et al. search in genomes with the catalytic motif Q(R/Q)XRW 5. RECENT APPROACHES FOR MINING CHITIN more than 150 synthases have been identified and were AND THE GENERATION OF NEW HIGH VALUE grouped into seven classes. Class III seems to be important PRODUCTS for the bulk chitin synthase activity in S. cerevisiae and C. albicans, accounting for 80-90% of total cellular chitin pro- 5.1. New Sources duction. Simultaneous deletion of all three chitin synthases Over the past decades chitin has proven to be present - if in S. cerevisiae causes a lethal phenotype that can only be not as major constituent, but still in measurable amounts - in alleviated when osmotically stabilizing the cells [176]. Class every eukaryotic kingdom except for plants and higher ver- V and VII are highly conserved, 1500 amino acid long chitin tebrates. Interestingly, the main source of chitin to enter pro- synthases and most of them harbor a myosin-motor like do- duction processes remains crustacean shells, derived from main [20, 177]. In N. crassa 7 chitin synthases were identi- the high amounts of crustacean wastes from the fishing in- fied, each for every class, of which NcChs-3 (class I) seems dustries. Moreover, the prevailing method used to extract to be responsible for the majority of chitin production [178, chitin is chemo-catalytic leaching, which not only results in 179]. In A. fumigatus and A. nidulans, which have 8 chitin poorly defined end-products, but also accumulates consider- synthases, AfCHSE and AnCHSB (class III) are essential for able amounts of unhealthy wastes. growth [180, 181]. Chitin synthases transfer UDP-GlcNAc In order to reduce the hazardous wastes, many projects1 in an inverting mechanism onto the non-reducing end of the were recently initiated to exploit the current knowledge on growing acceptor oligosaccharide [182]. The large enzymes chitinases and chitosanases (produced from filamentous fun- are an integral part of the membrane, with multiple domains gi, such as Trichoderma and Aspergillus, but also from bac- important for activation and subcellular localization. They teria, e.g., and Serratia) to assist the chemo- are believed to form a transport channel through the outer catalytic methods in mining GlcNAc or chitooligomers form membrane and deposit chitin at the outer surface, similar to crude chitinous sources [194]. Efforts to further optimize the the cellulose synthases [183]. In C. albicans they have been enzymes for direct GlcNAc production are being made to shown to synthesize different forms of chitin (long microfi- increase the output and reduce production steps. Chern et al., brils or short chitin rodlets), depending on their localization identified a bacterium, Chitinibacter tainanensis, in Taiwan and the life cycle [184]. that seems to be able to synthesize GlcNAc into its environ- ment, when fed with chitin [195, 196]. From the Antarctic Regulation of chitin synthesis is tightly linked to the life fungus Lecanicillium muscarium cold tolerant chitinases cycle. In yeast S. cerevisiae CHS1 (class I) transcription is could be isolated that would further reduce energy costs of increased during mating as well as after activation of the chitin hydrolysis [197]. The new methods and processes that salvage pathway, i.e. the nucleotide re-usage after recombi- have been evolving over the past years have an additional nation [185, 186]. In A. nidulans and N. crassa differential benefit: the enzymatic lysis by-products (proteins, lipids etc.) expression of chitin synthases during sexual and asexual can be used as animal food or for biogas-production as alter- development was also reported [177, 187]. For the class I native energy source. The highly hazardous chitinous waste, homolog in C. albicans induction of transcription was found generated in millions of tons every year by the fishing indus- after the switch to filamentous growth [188, 189]. The activi- try could, thereby, be completely salvaged and prevent the ty of ScCHS2 (class II) is highest before cytokinesis, where- ecological risks otherwise generated by disposal in landfills. as it is strongly decreased during mating and sporulation [190, 191]. For the homolog CaCHS1 a low, but constant Extensive overfishing and the immense demand for fish expression has so far been determined [189]. ScCHS3, which led to a dramatic decline of natural fish stock in the past cen- is responsible for the bulk chitin synthesis, seems to lack tury. Therefore, commercial production of fish and crusta- transcriptional regulation, but is expressed throughout the ceans in large scale marine farms was established. Crusta- whole cell cycle. Instead of transcriptional regulation, chap- ceans (mainly shrimps) accounted for the highest percentage erones are implicated in directing ScCHS3 chitin synthase (57%) of farmed marine animals for food production in activity to sites of action. Chs7 (close homologs have been 2010. These farms are mainly built on land (in ponds) or sea identified in C. albicans, A. fumigatus, A. gossipyi and N. close to the coast and pose a high threat to coastal areas, crassa [20]) and Chs6/5 are involved in recruiting the chitin since the effluents from these farms contain vast amounts of synthase from the ER or chitosome to the cell membrane. organic matter, nitrogen and phosphate that harm and pollute Another protein, Chs4, could act as a direct activator of the marine ecosystem [198]. The high density of crustaceans Chs3, since its overexpression increases the activity of Chs3 in the farms further raises the possibility that illnesses spread during synthesis ([20, 192, 193] and reviewed in in the colony, and therefore antibiotics are frequently admin- [20]). CHS4 has a functional homolog SHC1, which is alter- istered, raising the probability for antibiotic resistances. On natingly expressed only during ascospore formation, when the other hand, when antibiotics are omitted, species such as the chitosan layer is produced and expression of CHS4 is Vibrio spp., the most common bacterial disease in shrimp turned off, so that the proteins are functionally redundant, farms, could also be transmitted to other inhabitants of the but biologically compartmentalized by differential expres- coast [198]. Thus, the environmental concerns, caused by the sion. A recent finding also suggests an involvement of GFA1 high amounts of shrimp and crab production, require other in regulation of chitin synthase activity/expression, because sources to be considered. its expression level seems to directly alter chitin synthesis and vice versa: the need for chitin synthesis can increase 1 GFA1 levels [168]. ChiBio project: www.chibiofp7.eu; funded by the European Union's Sev- enth Framework Programme (FP7) under the grant agreement n° 289284. Fungal Chitin Degradation for the Generation of High-Value Products Current Biotechnology, 2017, Vol. 6, No. 3 187

Very recently, chitin was shown to be present in the (sub-) bisporus, leading the list of preferred fungi [8, 209]. Studies arctic coralline alga, Clathromorphum compactum, were it for chemical extraction from basidio- and ascomycetous occurs in the organic matrix together with collagen [199]. The wastes showed that fungi are an excellent source for chitin chitin content is believed to provide additional strength to the and chitosan. A. bisporus, A. niger and M. rouxii wastes con- calcified algal skeleton and might play an important role in tain 19, 12 and 20% harvestable chitin/chitosan, respectively, buffering negative impacts on skeleton formation by ocean in dry mycelia, with lower deacetylation grades than shrimp acidification. shell chitin [210, 211]. Especially low DA chitosan could be directly harvested from M. rouxii, which harbors mainly chi- Coralline algae have been dredged in high amounts in th tosan in the cell wall [212]. Pigmentation can be avoided Britain and France for soil conditioning since the 18 centu- when using white fungi such as A. bisporus or when only ry, but their slow growth and ecological importance may white parts of fungi, i.e. stalks or mycelium that is devoid of oppose any further economic use [200]. Sponges (Porifera) pigments, are processed, so that chemical extraction of pig- also contain considerable amounts of chitin [201, 202]. They ments could be bypassed or strongly reduced. Furthermore, grow comparatively fast and in many shapes and the associa- in comparison to chitin from animal sources, chitin from tion of chitin with collagen in glass sponges makes it an at- edible fungi does not pose medical risks, since allergic reac- tractive resource for chitin scaffolds in a predetermined tions and transmission of diseases are considered unlikely, shape to be used in the reconstructive surgical field [203]. facilitating certification for pharmacological and medicinal Extraction of pure chitin from sponges for other purposes, purposes. The large scale enzymatic fermentation processes though, might again be limited, due to insufficient raw mate- accumulate vast quantities of fungal biomass, which often rial generation over time even with farmed sponges [204]. pose problems as unwanted by-products. Extraction of chitin Harvesting chitin from crustacean shells is extremely en- from these industrial sources would as well be a cheap alter- ergy intensive, since the vast amount of pigments and resid- native to crustacean chitin. ual proteins in and around the chitin shell need to be extract- ed first, and so far an effective enzymatic extraction (prote- 5.2. New Products ase mixtures with maximum 20 % extraction efficiency Production of chitin, chitosan and its derivatives is now en- [205]) for these contaminants has not been explored exten- tering the third generation. When production of chitinous mate- sively. Therefore, other sources of chitin with very low ash rials was established, the so called ‘first generation’ of chitin content are interesting targets for biotechnological exploita- and chitosans sufficed only for the use as biomaterial for, e.g., tion. Moreover, obtaining chitin from crustacean shells for waste water treatment or as livestock food additives. The poor- medical purposes is challenging. This is again due to the ly defined polymer mixtures of varying purity and composition high amounts of proteins and pigments associated with chi- rendered chitin applications and production at industrial scale tin, because residual solvent from the extraction process or unappealing. A ‘second generation’ with more reliable produc- proteins/pigments may create allergic reactions or intoler- tion standards, after intense efforts to understand the structure ances [5]. Therefore, a current approach is to find and exploit and function of chitin and derivatives, led to well-defined new new, more ecologically save sources, which may yield easier products in terms of degrees of polymerization and acetylation. extractable chitin of higher purity. In an ongoing project in- 2 They were more suitable for the development of reliable, high sect farming is tested, where yellow mealworm larvae (Te- value products, due to known molecular structure-function nebrio molitor) are bred with controlled aliments. An ad- relationships, which made it possible to increase affectivity, for vantage of insects is their easy and cheap breeding and their example, in plant protection from 40kg/ha to 4g/ha. The chal- separation from the natural environment in a self-contained lenge for future studies will be the exploitation and generation system. Trials, conducted by the E.U. initiative PROteIN- 3 of new design products to enter the ‘third generation’ of chitin SECT , have found that at least 150 tons of insect protein and chitosan based products. Chitosans with non-random pat- could be produced on one hectare of land per year [206]. terns of acetylation and chitin and chitooligomers with clearly Therefore, the insects would be bred in the first place not for defined biological activities and cellular modes of action will chitin production, but to generate the high amounts of pro- be generated. The last section of this review, therefore, discuss- teins and lipids currently needed as animal feed. The chitin es new products that are already being designed or are envis- shell of insect larvae further contains lower amounts of pig- aged for future applications. ments, i.e. the percentage of ash in chitin is lower than in adults, which decreases the amount of chemicals needed to Chitosan and chitooligomers of differing acetylation purify chitin [207, 208]. Thus, chitin would only be a by- grades can be used in medical applications. They are highly product of protein and lipid production, but could more easi- antibacterial, but can be degraded by endogenous human ly be extracted with less contaminant, which would dramati- chitinases, and therefore assist regeneration of human tissue cally decrease costs and efforts for production [206]. after injury. Their current applications comprise wound dressings, separation membranes, antibacterial coatings for Mushrooms, as source for chitin, represent another alter- stents and tissue engineering scaffolds. The positive charge native to crustacean shells. Global edible fungi cultivation of chitosan raises the possibility to crosslink chitosan to oth- was estimated to 32 million tons in 2011, with Agaricus er negatively charged polymers or ions. Chitosan cross- linked to alginate or gelatin improves stability, cyto- 2 YNSECT: Genopole – Campus 3, 1 rue Pierre Fontaine Bâtiment 2, 91058 biocompatibility and provides a better environment for cell Evry CEDEX, France, www.ynsect.com 3 attachment and proliferation [213-215]. The covalent combi- PROteINSECT project: http://www.proteinsect.eu; co-financed by the nation of glycine and chitosan results in N-carboxymethyl European Commission (EC) under the seventh framework program (FP7) n° 312084 chitosan. The amphiphilic behavior of the glycine moiety 188 Current Biotechnology, 2017, Vol. 6, No. 3 Gaderer et al. enhances the solubility over a continuous and extended pH 6. OUTLOOK range and the polymer has been demonstrated to serve as Over the past decades major efforts have been made to antioxidant in cellular processes [8]. Electrospinning is fur- ther applied now to generate higher molecular weight fibers increase our knowledge on the structure and function of chi- tin/chitosan and its degradation products and how they are from chitin solutions, by pulling micron and nano-sized fi- functionalized in chitin metabolism. With the use of new bers from this polymer solution in an electric field. The higher molecular weight fibers can also serve as scaffolds in sources that are more reliable, contain less pigments and contaminants and are thus medically safer, chitin and tissue engineering [216]. The project Nano3bio4 aims at chitooligomers will be established as bio-based, renewable identifying chitinases and chitin deacetylases to obtain chi- tosans with known and defined, non-random patterns of compounds and will serve as substrate for (bio-) chemical design processes, such as heterocycle chemistry and func- deacetylation. Chitin deacetylases are capable of producing tionalized fatty acids. Moreover, a better understanding of distinct patterns of DA, and therefore will be used to gener- ate chito-oligosaccharides with defined DA grades. Hamer et the function and action of chitin metabolizing enzymes and the interplay with auxiliary proteins will help to increase al. have produced specific chitosan oligomers, which are their productivity and specificity via directed genetic engi- deacetylated with a novel and defined pattern, using two chitin deacetylases [217]. By increasing the number of dif- neering. Importantly, regulators for chitin metabolism need to be identified, so that we are able to modulate the activity ferent deacetylases (also from fungal sources), novel chi- of the enzymes and alter the time course of their action. With tosan oligomers with a fully defined architecture will be pro- duced in the near future. the identified regulators and the knowledge about the en- zymes involved in chitin anabolism and catabolism, it will GlcNAc itself was discovered to be a valuable pharmaco- soon be possible to stop reactions at any given point and logical agent in the treatment of a wide variety of maladies in modify the produced intermediate with the introduction of the past decade. GlcNAc is effective to treat joint damage, heterologous enzymes, to create new substrates for designed including arthritic diseases, cartilage- or joint injury and de- chemicals on a sustainable basis. generative joint diseases, when delivered to sites of damage. Furthermore, provident GlcNAc administration can prevent LIST OF ABBREVIATIONS joint damage. GlcNAc also inhibits elastase activity and su- AAs = Auxiliary Activities peroxide release from human polymorphonuclear leukocytes and is tested as potential candidate to treat inflammatory CBM = Carbohydrate Binding Module bowel disease [218-221]. Another field of application is CE = = Carbohydrate Esterase cosmetics. Hyaluronic acid is widely used to treat skin and mucosal damages, despite inefficient absorption. GlcNAc, CHS = Chitin Synthase however, absorbs effectively and has been shown to induce a dose-dependent increase in the production of HA in cultured CPP = Cerato-Platanin Protein keratinocytes [222, 223]. CURLI = Curled Pili N-acetylglucosamine and Glucosamine can serve as start- DA = Degree of Acetylation ing material (or platform chemical) to synthesize new poly- mers or N-containing compounds, such as isocyanates and ENGase = Endo-β-N-Acetylglucosaminidase polyamides, which have so far not been produced from re- ER = = Endoplasmatic Reticulum newable raw material. GlcNAc and GlcN can be harnessed as carbon and energy source for specialized yeast cells to ERAD = Endoplasmatic Reticulum-Associated Pro- produce functionalized fatty acids and amino-carboxylic tein Degradation acids as starting point for chemical synthesis processes. Both GH = Glycoside can be fed into the polymer production process. In a multi- enzymatic process, currently developed5, also heterocycles GlcN = Glucosamine can be produced from GlcN. An excellent example for the GlcNAc = N-Acetylglucosamine successful implementation of such a process is N- acetylneuraminic acid (Neu5Ac) production. Neu5Ac is one (GlcNAc)2 = Chitobiose of the most common sialic acids, used to produce neuramini- dase inhibitors to treat influenza infections [224]. For enzy- GlcNase = Exo-β-D-Glucosaminidase matic synthesis of Neu5Ac N-acetylmannosamine (Man- GPI = Glycosylphosphatidylinositol NAc) and pyruvate are used as substrates for recombinant Neu5Ac aldolase. ManNAc in turn can be produced inex- HA = Hyaluronic Acid pensively from GlcNAc, via epimerization with the GlcNAc LPMOs = Lytic Polysaccharide Monooxygenase 2-epimerase. Thus, from 27 kg of GlcNAc 29 kg of Neu5Ac could be obtained using recombinant GlcNAc 2-epimerase LysM = Motif and Neu5Ac as catalysts [225]. ManNAc = N-Acetylmannosamine MFS = Major Facilitator Symporter 4 www.nano3bio.eu/start funded by the European Union's Seventh Frame- work Programme (FP7) under the grant agreement n° 613931 NAGase = N-Acetylglucosaminidase 5 ChiBio project: www.chibiofp7.eu; funded by the European Union's Sev- enth Framework Programme (FP7) under the grant agreement n° 289284. Neu5Ac = N-Acetylneuraminic Acid Fungal Chitin Degradation for the Generation of High-Value Products Current Biotechnology, 2017, Vol. 6, No. 3 189

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