number of diverse enzymes, and anaerobic
fungi meet this challenge via the secretion of a
vast array of intricately organized and environmentally regulated enzymes (Haitjema et al.
2017; Solomon et al. 2016a, 2018; Youssef et al.
2013).
In this chapter, we briefly describe the phylogeny, unusual life style, isolation procedures,
and growth requirements of these uniquely
anaerobic fungi. We further discuss how
“omics” technologies facilitate the identification of useful proteins and metabolic pathways
and give examples of successful efforts to transfer gut fungal proteins to model microbes. Further, we provide an overview of current and
future biotechnological uses of anaerobic
fungi, including a survey of their intellectual
property landscape. We conclude that the
understudied Neocallimastigomycota are a
rich and largely untapped source of bioactivities that can be harnessed for diverse biotechnological applications, from animal nutrition to
bioprocessing technologies that seek to convert
renewable biomass into valuable fuels and chemicals.
A. Background and Taxonomy
The Neocallimastigomycota were first proposed
as fungi following Orpin’s seminal work in the
1970s analyzing the morphology of rumen “flagellates” that proliferated on fiber-rich diets in
the guts of ruminant animals (Orpin 1975).
Subsequent studies conclusively demonstrated
that these were true fungi with a chitinous cell
wall, which challenged the previously held
dogma that all fungi required oxygen to grow
(Orpin 1977). It is estimated that the anaerobic
fungi from the phylum Neocallimastigomycota
diverged from other fungi in the late Cretaceous
period (66 Æ 10 million years ago), around the
time when grasses and mammalian grazers
emerged (Emerling et al. 2018; Prasad et al.
2005; Wang et al. 2019). Similar to their relatives in the basal fungal phyla Chytridiomycota
and Blastocladiomycota, Neocallimastigomycota alternate between motile zoospores and
vegetative thalli (James et al. 2006) as part of
their life cycle (Fig. 17.1). In the animal gut,
fungal zoospores (Fig. 17.1a) are released from
mature sporangia and swim toward and attach
to ingested plant material, likely attracted by
soluble sugars and phenolic acids (Wubah and
Kim 1996). Following attachment, the zoospores encyst and germinate, penetrating the
plant surface and developing either to rhizoidal
or bulbous thalli (Fig. 17.1d). Anaerobic fungi
thrive in herbivores via enzymatic degradation
of plant carbohydrates, and they directly utilize
sugar-rich breakdown products from degradation—this leads to expansion of fungal rhizoids/holdfasts, and zoosporangia develop and
enlarge. Finally a new generation of zoospores
is produced and matured in the zoosporangium
(Fig. 17.1f), with the timing of zoospore release
likely triggered by ingestion of fresh forage,
though the mechanistic underpinnings remain
unclear (Orpin 1977). The full life cycle is
reported to take 8–32 h (Mountfort and Orpin
1994; Theodorou et al. 1996a).
Anaerobic fungal taxa have historically
been grouped according to thallus morphology
and the number of flagella per zoospore, as
exemplified in Fig. 17.2 (Wang et al. 2017).
For example, fungi with monocentric thalli
have one center of growth, while polycentric
fungi have multiple centers of growth—they
are readily distinguishable via light microscopy. Based on these morphological features,
six genera were originally identified: Neocallimastix (rhizoidal, monocentric, polyflagellate),
Piromyces (rhizoidal, monocentric, monoflagellate), Anaeromyces (rhizoidal, polycentric,
monoflagellate), Orpinomyces (rhizoidal, polycentric, polyflagellate), Caecomyces (bulbous,
monocentric, monoflagellate), and Cyllamyces
(bulbous, polycentric, monoflagellate) (Griffith
et al. 2010).
Recently multiple additional genera have
been isolated and described: Buwchfawromyces
(Callaghan et al. 2015), Feramyces (Hanafy et al.
2018), Liebetanzomyces (Joshi et al. 2018),
Oontomyces (Dagar et al. 2015), Pecoramyces
(Hanafy et al. 2017), Agriosomyces, Aklioshbomyces, Capellomyces, Ghazallomyces, Joblinomyces, Khoyollomyces and Tahromyces
(Hanafy et al. 2020). All are monocentric and,
414
V. Flad et al.
fungi meet this challenge via the secretion of a
vast array of intricately organized and environmentally regulated enzymes (Haitjema et al.
2017; Solomon et al. 2016a, 2018; Youssef et al.
2013).
In this chapter, we briefly describe the phylogeny, unusual life style, isolation procedures,
and growth requirements of these uniquely
anaerobic fungi. We further discuss how
“omics” technologies facilitate the identification of useful proteins and metabolic pathways
and give examples of successful efforts to transfer gut fungal proteins to model microbes. Further, we provide an overview of current and
future biotechnological uses of anaerobic
fungi, including a survey of their intellectual
property landscape. We conclude that the
understudied Neocallimastigomycota are a
rich and largely untapped source of bioactivities that can be harnessed for diverse biotechnological applications, from animal nutrition to
bioprocessing technologies that seek to convert
renewable biomass into valuable fuels and chemicals.
A. Background and Taxonomy
The Neocallimastigomycota were first proposed
as fungi following Orpin’s seminal work in the
1970s analyzing the morphology of rumen “flagellates” that proliferated on fiber-rich diets in
the guts of ruminant animals (Orpin 1975).
Subsequent studies conclusively demonstrated
that these were true fungi with a chitinous cell
wall, which challenged the previously held
dogma that all fungi required oxygen to grow
(Orpin 1977). It is estimated that the anaerobic
fungi from the phylum Neocallimastigomycota
diverged from other fungi in the late Cretaceous
period (66 Æ 10 million years ago), around the
time when grasses and mammalian grazers
emerged (Emerling et al. 2018; Prasad et al.
2005; Wang et al. 2019). Similar to their relatives in the basal fungal phyla Chytridiomycota
and Blastocladiomycota, Neocallimastigomycota alternate between motile zoospores and
vegetative thalli (James et al. 2006) as part of
their life cycle (Fig. 17.1). In the animal gut,
fungal zoospores (Fig. 17.1a) are released from
mature sporangia and swim toward and attach
to ingested plant material, likely attracted by
soluble sugars and phenolic acids (Wubah and
Kim 1996). Following attachment, the zoospores encyst and germinate, penetrating the
plant surface and developing either to rhizoidal
or bulbous thalli (Fig. 17.1d). Anaerobic fungi
thrive in herbivores via enzymatic degradation
of plant carbohydrates, and they directly utilize
sugar-rich breakdown products from degradation—this leads to expansion of fungal rhizoids/holdfasts, and zoosporangia develop and
enlarge. Finally a new generation of zoospores
is produced and matured in the zoosporangium
(Fig. 17.1f), with the timing of zoospore release
likely triggered by ingestion of fresh forage,
though the mechanistic underpinnings remain
unclear (Orpin 1977). The full life cycle is
reported to take 8–32 h (Mountfort and Orpin
1994; Theodorou et al. 1996a).
Anaerobic fungal taxa have historically
been grouped according to thallus morphology
and the number of flagella per zoospore, as
exemplified in Fig. 17.2 (Wang et al. 2017).
For example, fungi with monocentric thalli
have one center of growth, while polycentric
fungi have multiple centers of growth—they
are readily distinguishable via light microscopy. Based on these morphological features,
six genera were originally identified: Neocallimastix (rhizoidal, monocentric, polyflagellate),
Piromyces (rhizoidal, monocentric, monoflagellate), Anaeromyces (rhizoidal, polycentric,
monoflagellate), Orpinomyces (rhizoidal, polycentric, polyflagellate), Caecomyces (bulbous,
monocentric, monoflagellate), and Cyllamyces
(bulbous, polycentric, monoflagellate) (Griffith
et al. 2010).
Recently multiple additional genera have
been isolated and described: Buwchfawromyces
(Callaghan et al. 2015), Feramyces (Hanafy et al.
2018), Liebetanzomyces (Joshi et al. 2018),
Oontomyces (Dagar et al. 2015), Pecoramyces
(Hanafy et al. 2017), Agriosomyces, Aklioshbomyces, Capellomyces, Ghazallomyces, Joblinomyces, Khoyollomyces and Tahromyces
(Hanafy et al. 2020). All are monocentric and,
414
V. Flad et al.