C. Progress Toward Direct Genetic
Manipulation of Anaerobic Fungi
As many anaerobic fungal enzymes are posttranslationally modified (Haitjema et al. 2017),
biotechnology would benefit from enhanced
native production enabled by a robust genome
engineering toolkit (Podolsky et al. 2019). Such
a toolkit should include robust transformation
methods for genetic DNA manipulation,
including the introduction of foreign DNA
and plasmids with appropriate promoters, terminators, and other biological parts needed for
controlled gene expression. However, genetic
engineering of anaerobic gut fungi is still in its
infancy—very few genetic regulatory elements
have been identified, versatile selection markers, and reporter genes are lacking, and chromosomal integration methods are nonexistent.
An early transformation attempt in Neocallimastix frontalis used biolistic bombardment
of mature fungal biomass, which transiently
expressed the bacterial beta-glucuronidase
gene from an Escherichia coli plasmid modified
with the putative Neocallimastix frontalis enolase promoter (Durand et al. 1997). Recently, a
method for the collection of viable, competent,
and developmentally synchronized anaerobic
fungal zoospores under strict anaerobic conditions was developed (Calkins et al. 2016). The
collected spores were shown to be naturally
competent for exogenous nucleic acids supplied to the culture medium. Subsequently, an
RNAi approach leveraged this natural competency. Chemically synthesized siRNAs targeting
D-lactate dehydrogenase (ldhD) gene were
added to germinating spores of Pecoramyces
ruminantium strain C1A, resulting in marked
gene silencing and indicating the possibility of
gene knockdown approaches in anaerobic fungi
(Calkins et al. 2018).
Finally, advances in gene editing technologies promise new opportunities to manipulate
the extremely AT-rich genomes of anaerobic
fungi (Zetsche et al. 2015). While many fungal
species incorporate exogenous DNA with high
efficiency through non-homologous endjoining pathways, site-specific knockout or
gene-integration powered by nucleases will be
necessary to avoid pleiotropic effects. The efficiency and accuracy of these gene-editing
events is enhanced in multiple fungi by removing endogeneous non-homologous end-joining
pathways (Catalano et al. 2011). Coupled with
emerging gut fungal transformation methods,
these techniques are poised to revolutionize
stable engineering of anaerobic fungi for a
range of biotechnological applications.
III. Anaerobic Fungi and Their
Applications in Agriculture and
Biotechnology
Anaerobic fungal activities may also be directly
leveraged for biotechnology to improve the
economic viability of farming operations.
Anaerobic fungi are integral to the digestion
of fiber-rich animal feed in important livestock
species including cattle, goats, and sheep (Gruninger et al. 2014). Thus, anaerobic fungi may
be manipulated in the animal gut to enhance
nutrition of forage (Gordon and Phillips 1998).
Similarly, bioprocesses may be designed to
exploit the ability of anaerobic fungi to ferment
ethanol from agricultural residues and improve
methane (biogas) formation via hydrogen syntrophy with methanogens. Below we discuss
deployment of anaerobic fungi and their
enzymes into animal feed, bioenergy production from agricultural wastes by bioethanol
production, and anaerobic digestion.
A. Anaerobic Fungi for Animal Nutrition
Beyond bio-based chemical production, anaerobic fungal CAZymes have the potential to
improve the utilization of lignocellulosic feedstocks in herbivores. Two conceivable strategies have been proposed: using fungal strains
to pretreat feedstock and including fungal
strains in animal feed as a probiotic supplement. The aim of the pretreatment process is
to promote colonization of anaerobic fungi on
the feed and thus enhance its availability to the
rumen microbial flora. Such pretreatment
could theoretically improve feedstock quality
not just for cattle, but for other animals that
17 The Biotechnological Potential of Anaerobic Gut Fungi
423
Manipulation of Anaerobic Fungi
As many anaerobic fungal enzymes are posttranslationally modified (Haitjema et al. 2017),
biotechnology would benefit from enhanced
native production enabled by a robust genome
engineering toolkit (Podolsky et al. 2019). Such
a toolkit should include robust transformation
methods for genetic DNA manipulation,
including the introduction of foreign DNA
and plasmids with appropriate promoters, terminators, and other biological parts needed for
controlled gene expression. However, genetic
engineering of anaerobic gut fungi is still in its
infancy—very few genetic regulatory elements
have been identified, versatile selection markers, and reporter genes are lacking, and chromosomal integration methods are nonexistent.
An early transformation attempt in Neocallimastix frontalis used biolistic bombardment
of mature fungal biomass, which transiently
expressed the bacterial beta-glucuronidase
gene from an Escherichia coli plasmid modified
with the putative Neocallimastix frontalis enolase promoter (Durand et al. 1997). Recently, a
method for the collection of viable, competent,
and developmentally synchronized anaerobic
fungal zoospores under strict anaerobic conditions was developed (Calkins et al. 2016). The
collected spores were shown to be naturally
competent for exogenous nucleic acids supplied to the culture medium. Subsequently, an
RNAi approach leveraged this natural competency. Chemically synthesized siRNAs targeting
D-lactate dehydrogenase (ldhD) gene were
added to germinating spores of Pecoramyces
ruminantium strain C1A, resulting in marked
gene silencing and indicating the possibility of
gene knockdown approaches in anaerobic fungi
(Calkins et al. 2018).
Finally, advances in gene editing technologies promise new opportunities to manipulate
the extremely AT-rich genomes of anaerobic
fungi (Zetsche et al. 2015). While many fungal
species incorporate exogenous DNA with high
efficiency through non-homologous endjoining pathways, site-specific knockout or
gene-integration powered by nucleases will be
necessary to avoid pleiotropic effects. The efficiency and accuracy of these gene-editing
events is enhanced in multiple fungi by removing endogeneous non-homologous end-joining
pathways (Catalano et al. 2011). Coupled with
emerging gut fungal transformation methods,
these techniques are poised to revolutionize
stable engineering of anaerobic fungi for a
range of biotechnological applications.
III. Anaerobic Fungi and Their
Applications in Agriculture and
Biotechnology
Anaerobic fungal activities may also be directly
leveraged for biotechnology to improve the
economic viability of farming operations.
Anaerobic fungi are integral to the digestion
of fiber-rich animal feed in important livestock
species including cattle, goats, and sheep (Gruninger et al. 2014). Thus, anaerobic fungi may
be manipulated in the animal gut to enhance
nutrition of forage (Gordon and Phillips 1998).
Similarly, bioprocesses may be designed to
exploit the ability of anaerobic fungi to ferment
ethanol from agricultural residues and improve
methane (biogas) formation via hydrogen syntrophy with methanogens. Below we discuss
deployment of anaerobic fungi and their
enzymes into animal feed, bioenergy production from agricultural wastes by bioethanol
production, and anaerobic digestion.
A. Anaerobic Fungi for Animal Nutrition
Beyond bio-based chemical production, anaerobic fungal CAZymes have the potential to
improve the utilization of lignocellulosic feedstocks in herbivores. Two conceivable strategies have been proposed: using fungal strains
to pretreat feedstock and including fungal
strains in animal feed as a probiotic supplement. The aim of the pretreatment process is
to promote colonization of anaerobic fungi on
the feed and thus enhance its availability to the
rumen microbial flora. Such pretreatment
could theoretically improve feedstock quality
not just for cattle, but for other animals that
17 The Biotechnological Potential of Anaerobic Gut Fungi
423