anaerobic fungi, translation of these enzymes
to heterologous production, and progress
towards direct genetic manipulation of the
fungi.
A. “Omics” Analyses Identify Enzymes and
Their Regulation Patterns
While extensive work has shown that anaerobic
fungi possess significant hydrolytic potential,
the detailed function and regulation of many
of the individual genes at the transcriptional
level has only recently been understood.
Advances in next-generation sequencing
(Wilken et al. 2019) enabled the first genomes
and transcriptomes for a small collection of
isolated anaerobic fungi. The first draft
genome of the Neocallimastigomycota was published in 2013. The genome of Orpinomyces sp.
C1A (later re-classified as Pecaromyces sp. C1A)
revealed a large abundance of CAZymes (>350
glycoside hydrolase enzymes, >90 carbohydrate esterases and an array of dockerin
domain-containing proteins associated with
cellulosome assembly) (Youssef et al. 2013).
Later, high-resolution genomes of Anaeromyces robustus, Neocallimastix californiae,
and Piromyces finnis revealed that these organisms have the largest array and diversity of
biomass-degrading enzymes in the fungal kingdom (Haitjema et al. 2017; Solomon et al.
2016a). Indeed, horizontal gene transfer has
recently been shown to play a crucial role in
expanding the substrate utilization capabilities
and electron disposal pathways in the Neocallimastigomycota (Murphy et al. 2019). Anaerobic
fungi readily adapt gene expression in response
to substrate complexity and may provide a
source of enzymes tailored to degrade feedstocks based upon their composition (Couger
et al. 2015; Henske et al. 2018a; Solomon et al.
2016a; Youssef et al. 2013). Carbon catabolite
repression (Montenecourt and Eveleigh 1977;
Strauss et al. 1995) is responsible for regulation
of diverse CAZymes in anaerobic fungi (Henske
et al. 2018a; Solomon et al. 2016a). Low concentrations (0.5 mg/ml) of various sugars (sucrose,
galactose, xylose, fructose and arabinose) were
sufficient to hinder enzyme production
(Henske et al. 2018a). It has however also been
shown that several of the secreted enzymes are
highly transcribed even when cultured on glucose or other soluble sugars (Couger et al. 2015;
Wang et al. 2011). The presence of complementary natural antisense transcripts for several
CAZymes even in the presence of glucose suggests that some enzymes are also posttranscriptionally regulated (Solomon et al.
2018). Moreover, epigenetic regulation may
also play a role as adenine methylation appears
especially prevalent near transcriptional start
sites in anaerobic fungi. However, no clear
link between DNA methylation and gene
expression has been identified (Mondo et al.
2017). Nonetheless, determining and manipulating many of the regulatory strategies of
anaerobic fungi will be essential to develop
these organisms to overexpress CAZymes for
bioenergy purposes.
B. Systems for Heterologous Enzyme
Expression
In spite of the small number of sequenced
anaerobic
fungal
genomes,
numerous
CAZymes, and proteins have been successfully
cloned and produced in other organisms,
including Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris (Table 17.2). Due to
the limited number of physical isolates, only
five genera in the Neocallimastigomycota have
been sourced for enzymes: Anaeromyces, Neocallimastix, Orpinomyces, Piromyces, and
Pecoramyces. The majority of these studies
relied on functional screening of cDNA
libraries to source enzymes from anaerobic
fungi but the recent availability of anaerobic
fungal genomes and transcriptomes has
enabled “omics”-guided approaches for the
identification of highly expressed key enzymes.
The identified genes sometimes undergo codon optimization prior to being cloned into an appropriate vector
(Morrison et al. 2016b; Wang et al. 2011). Protein products are examined for validation of predicted activities
(Liu et al. 1997), and their biochemical and kinetic
properties are characterized (Morrison et al. 2016a;
Morrison et al. 2016b; Tsai et al. 2003). Determining
these properties is important for efficient consolidated
17 The Biotechnological Potential of Anaerobic Gut Fungi
421
to heterologous production, and progress
towards direct genetic manipulation of the
fungi.
A. “Omics” Analyses Identify Enzymes and
Their Regulation Patterns
While extensive work has shown that anaerobic
fungi possess significant hydrolytic potential,
the detailed function and regulation of many
of the individual genes at the transcriptional
level has only recently been understood.
Advances in next-generation sequencing
(Wilken et al. 2019) enabled the first genomes
and transcriptomes for a small collection of
isolated anaerobic fungi. The first draft
genome of the Neocallimastigomycota was published in 2013. The genome of Orpinomyces sp.
C1A (later re-classified as Pecaromyces sp. C1A)
revealed a large abundance of CAZymes (>350
glycoside hydrolase enzymes, >90 carbohydrate esterases and an array of dockerin
domain-containing proteins associated with
cellulosome assembly) (Youssef et al. 2013).
Later, high-resolution genomes of Anaeromyces robustus, Neocallimastix californiae,
and Piromyces finnis revealed that these organisms have the largest array and diversity of
biomass-degrading enzymes in the fungal kingdom (Haitjema et al. 2017; Solomon et al.
2016a). Indeed, horizontal gene transfer has
recently been shown to play a crucial role in
expanding the substrate utilization capabilities
and electron disposal pathways in the Neocallimastigomycota (Murphy et al. 2019). Anaerobic
fungi readily adapt gene expression in response
to substrate complexity and may provide a
source of enzymes tailored to degrade feedstocks based upon their composition (Couger
et al. 2015; Henske et al. 2018a; Solomon et al.
2016a; Youssef et al. 2013). Carbon catabolite
repression (Montenecourt and Eveleigh 1977;
Strauss et al. 1995) is responsible for regulation
of diverse CAZymes in anaerobic fungi (Henske
et al. 2018a; Solomon et al. 2016a). Low concentrations (0.5 mg/ml) of various sugars (sucrose,
galactose, xylose, fructose and arabinose) were
sufficient to hinder enzyme production
(Henske et al. 2018a). It has however also been
shown that several of the secreted enzymes are
highly transcribed even when cultured on glucose or other soluble sugars (Couger et al. 2015;
Wang et al. 2011). The presence of complementary natural antisense transcripts for several
CAZymes even in the presence of glucose suggests that some enzymes are also posttranscriptionally regulated (Solomon et al.
2018). Moreover, epigenetic regulation may
also play a role as adenine methylation appears
especially prevalent near transcriptional start
sites in anaerobic fungi. However, no clear
link between DNA methylation and gene
expression has been identified (Mondo et al.
2017). Nonetheless, determining and manipulating many of the regulatory strategies of
anaerobic fungi will be essential to develop
these organisms to overexpress CAZymes for
bioenergy purposes.
B. Systems for Heterologous Enzyme
Expression
In spite of the small number of sequenced
anaerobic
fungal
genomes,
numerous
CAZymes, and proteins have been successfully
cloned and produced in other organisms,
including Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris (Table 17.2). Due to
the limited number of physical isolates, only
five genera in the Neocallimastigomycota have
been sourced for enzymes: Anaeromyces, Neocallimastix, Orpinomyces, Piromyces, and
Pecoramyces. The majority of these studies
relied on functional screening of cDNA
libraries to source enzymes from anaerobic
fungi but the recent availability of anaerobic
fungal genomes and transcriptomes has
enabled “omics”-guided approaches for the
identification of highly expressed key enzymes.
The identified genes sometimes undergo codon optimization prior to being cloned into an appropriate vector
(Morrison et al. 2016b; Wang et al. 2011). Protein products are examined for validation of predicted activities
(Liu et al. 1997), and their biochemical and kinetic
properties are characterized (Morrison et al. 2016a;
Morrison et al. 2016b; Tsai et al. 2003). Determining
these properties is important for efficient consolidated
17 The Biotechnological Potential of Anaerobic Gut Fungi
421