Anaerobic fungi, though low in abundance in rumen, play an important role in the degradation of forage for herbivores. Anaerobic fungi have been found in almost all animals that ferment in the predigestive tract, including ruminants (e.g., Bovidae, Cervidae), pseudoruminants (e.g., hippos, camels, llamas, alpacas), and nonruminants (e.g., wallabies). They are also found in many postdigestive tract fermenters that digest plant tissues in the cecum and large intestine (e.g., elephants, horses, and rhinoceroses) and in some large herbivore rodents (e.g., long-eared guinea pigs). It appears that the establishment of anaerobic fungi in the alimentary canal of herbivores may be attributed to the complex and distinct chamber with a relatively neutral pH in the digestive tract and a long lag time in the digestive process for the ingested plant tissue, which is conducive to the growth and activity of anaerobic fungi. Therefore, anaerobic fungi, which appear in the digestive tract of these herbivores, especially in those that take in a lot of roughage, must have some unique reasons and advantages to exist in such a complex environment.
Organism | Assembly Length | Genes Count | Isolation Source | Sample | Published |
---|---|---|---|---|---|
Piromyces sp. UH3-1 | 84,096,456 | 16,867 | Donkey | Feces | - |
Piromyces sp. E2 | 71,019,055 | 14,648 | Elephant | Feces | [30] |
Piromyces finnis | 56,455,805 | 10,992 | Horse | Feces | [30] |
Caecomyces churrovis A | 165,495,782 | 15,009 | Sheep | Feces | [31] |
Anaeromyces robustus | 71,685,009 | 12,832 | Sheep | Feces | [30] |
Neocallimastix sp. Gf-Ma3-1 | 209,503,801 | 28,646 | Giraffe | Feces | - |
Neocallimastix sp. WI3-B | 206,810,295 | 28,960 | Wildebeest | Feces | - |
Neocallimastix lanati | 200,974,851 | 27,677 | Sheep | Feces | [32] |
Neocallimastix californiae G1 | 193,032,486 | 20,219 | Goat | Feces | [30] |
Pecoramyces ruminantium C1A | 100,954,185 | 18,936 | Angus steer | Feces | [33] |
Pecoramyces sp. F1 | 106,834,627 | 17,740 | Goat | Rumen sample | [34] |
Cellulase catalyzes the hydrolysis of β-1, 4-glucosidase in cellulose. Three types of cellulases (endoglucanase, exoglucanase, and β-glucosidase) that have been reported in anaerobic fungi together hydrolyze cellulose and release glucose[42]. Endoglucanase is the most important component of the cellulase system, which can cleave the glucan chain inside the cellulose. High levels of endoglucanase were found in the culture supernatants of Neocallimastix frontalis[43][44], Piromyces communis[45], and OrpinomyceOrpinomyces s sp.[46]. Endoglucanase production was maximum when the fungi were grown on cellulose, whereas synthesis was totally repressed by the addition of glucose, indicating that the enzyme was subject to regulation [43]. Exoglucanase cleaves cellobiose units, the building blocks of cellulose, from the ends of the cellulose chain. Exoglucanase active against microcrystalline cellulose was also detected, but at lower levels than endoglucanase[43][46]. In addition, β-glucosidase cleaves cellobiose, which is a potent inhibitor of the former two enzymes, to yield glucose[47]. Previous studies have classified that the glycoside hydrolase enzyme classes GH1, GH2, GH3, GH5, GH6, GH8, GH9, GH38, GH45, GH48, and GH74 encode for cellulases[48]. Enzymes in different GHs have a different fiber hydrolysis activity according to their structure. The expression of these classes of enzymes varies according to fungal species and provided growth substrates. GH6 and GH5 were the two highest-expressed families in the PecoramycePecoramyces s sp. F1 according to its transcriptome analysis, whereas GH1 and GH33 were the lowest-expressed ones[49]. In the genome of Pecoramyces ruminantium C1A, GH45 and GH48 were highly expressed with glucose as substrate, while the expression of GH6 was dramatically improved by corn stover[50].
Hemicellulose is the main component of the plant primary wall. It is a heteropolysaccharide containing xylan, glucuronoxylan, arabinoxylan, glucomannan, galactomannan, and xyloglucan[51][52]. The component monosaccharides are connected to each other, forming a hard part of the cell wall to prevent microorganisms from using the plant cell contents. The hemicellulases secreted by anaerobic fungi include xylanases, mannanases, galactanases, β-glucanases and so on, which can degrade hemicellulose into various oligomers and monosaccharides. Due to xylan being the basic polymeric compound of hemicellulose and the high hydrolase activity of xylanases, xylanases are the most studied enzymes to date among the hemicellulose hydrolases from anaerobic fungi[53][54][55]. Heterologous expression of these enzymes has also been developed. Xue et al. isolated a Neocallimastir patricianun xylanase cDNA and engineered it for heterologous expression in Escherichia coli (E. coli). The modified xylanase produced in E. coli had a specific activity of 1229 U/mg protein at pH 7 and 50 °C, without purification[56]. The genes encoding xylanases from Neocallimastix sp. GMLF2[57], Orpinomyces sp. Strain 2, and Neocallimastix frontalis[58] were also cloned into E. coli, and a high expression was obtained. In addition to E. coli, Hypocrea sp.[59], Kluyveromyces sp., and Pichia sp.[58] may also be ideal heterologous expression vectors for xylanases from anaerobic fungi. Successful development of heterologous production technologies for the enzymes from anaerobic fungi constitutes a significant advancement in applying this promising source of genes towards lignocellulose bioconversion. The glycoside hydrolase classes GH10, GH11, GH30, GH31, GH38, GH39, GH43, GH47, GH53, and GH115 encode for hemicellulases, respectively[48]. Xylanases are representative enzymes of GH10 and GH11. The former has a relatively high molecular weight, while the latter has a lower molecular weight.
In addition to the free enzymes secreted outside the cell, anaerobic fungi also secrete a number of large (MDa) multiprotein cellulolytic complexes, known as cellulosomes, which can degrade crude fibers more completely and efficiently. Although most studies have reported the function of cellulosomes in anaerobic microorganisms, such as anaerobic fungi, and the bacteria Clostridium thermocellum and Ruminococcus albus, there have also been reports on the discovery of cellulosomes in aerobic bacteria[60]. Cellulosomes include carbohydrate-binding domains, noncatalytic protein domains, and many glycosyl hydrolases (cellulases, hemicellulases, pectin enzymes, chitinases, and other enzymes). The assembly of cellulosomes involves interactions between dockerin domains on different catalytic enzyme subunits and cohesin domain on noncatalytic scaffolding, and the specific interactions between them allow various enzyme proteins to bind stably in this supramolecular structure. Scaffold proteins and some enzymes contain CBMs, which promote the binding of the cellulosome enzyme to substrate cellulose. Cellulosomes are attached to the cell surface by additional anchoring domains by noncovalent bonding[61]. Many GHs of anaerobic fungi are involved in the formation of cellulosomes. It was reported that anaerobic fungal cellulosomes exhibit additional 13% higher GH activity due to the presence of GH3, GH6, and GH45 compared with bacterial cellulosomes; especially the supplementary β-glucosidase conferred by GH3 activity empowers fungal cellulosomes in converting cellulose to single simple sugars (monosaccharides) when compared with low-molecular-weight oligosaccharide-generating bacterial cellulosomes[30][62]. Because of the powerful fiber degradation capability of cellulosomes, better than commercial preparations containing noncomplexed enzymes, the efficient degradation of cheap fibrous materials is possible if the modified cellulosome genes can be inserted into appropriate host cells and expressed using genetic engineering techniques.