anaerobic fungi harbor a multitude of unique
enzymes and degradation strategies, which can
be harnessed to readily degrade specific feedstocks or for biotechnological purposes, as further discussed in Sect. II.
2. Anaerobic Fungi Have Hydrogenosomes and
Live in Close Relationship with Methanogens
Physiological and genomic investigations suggest that energy generation in anaerobic fungi
is mediated by fungal hydrogenosomes,
although this hypothesis is yet to be confirmed
by molecular studies. Hydrogenosomes are
ATP-producing organelles found across a
range of unrelated anaerobic eukaryotes that
use protons rather than oxygen as the terminal
electron acceptor. They are believed to have
evolved from degenerate mitochondria
(Boxma et al. 2004; Hackstein et al. 2019; Mu ¨ller
et al. 2012), and recent genomic studies have
confirmed the fungal mitochondrial origin of
some hydrogenosomal import proteins
(Haitjema et al. 2017; Youssef et al. 2013).
Hydrogenosomes perform three major metabolic tasks:
(i) Pyruvate metabolism: pyruvate (from
cytosolic plant sugar metabolism) is converted to formate and acetyl-CoA using
pyruvate/formate lyase (PFL) (Boxma
et al. 2004). Marvin-Sikkema et al. (1993)
demonstrated the presence of a hydrogenosome membrane-associated pyruvate/
ferredoxin oxidoreductase (PFO), similar
to that found in hydrogenosomes of Trichomonas spp., and suggested that acetylCoA from cytosolic PFL activity was transported to the hydrogenosome.
(ii) ATP production: Acetyl-CoA is converted
to acetate via succinyl-CoA synthetase,
initiating ATP production exclusively via
substrate level phosphorylation.
(iii) Regeneration of reduced electron carriers: NAD(P)H is re-oxidized by an
NAD(P)H dehydrogenase, which transfers electrons to an iron-only hydrogenase
producing hydrogen (Haitjema et al. 2017;
Kameshwar and Qin 2018; Youssef et al.
2013).
Thus, the main fermentation products of
anaerobic fungi are acetate, formate, and
hydrogen which are produced in hydrogenosomes and lactate, formate, ethanol, and succinate which are produced in the cytosol.
A syntrophic partnership with archaeal
methanogens allows fungi to thrive by siphoning hydrogen to methane (Conrad 1999; Peng
et al. 2016; Ushida et al. 1997), alleviating endproduct inhibition caused by accumulation
of hydrogen. It is well-known that there is a
close relationship between anaerobic fungi
and methanogens, with interspecies hydrogen
transfer resulting in methane production
and more efficient re-generation of oxidized
nucleotides (NAD
+ , NADP
+ ) (Bauchop and
Mountfort 1981; Cheng et al. 2009). Acetate
and formate are also substrates for methanogenesis, and Li et al. (2019) have recently shown
that, when Methanobrevibacter thaueri and
Pecoramyces sp. F1 are grown together in
batch culture, the majority of formate produced
by the fungus is used for methanogenesis. More
details on such microbial interactions and their
biotechnological potential are reported in Sect.
III.C.
II. Identification of Useful Enzymes
Anaerobic fungi offer a wealth of novel proteins and enzymes for diverse biotechnology
applications. Chief among these are potentially
superior
lignocellulose-degrading
enzymes, which offer opportunities to overcome a significant bottleneck during the production of second generation biofuels and
chemicals (Seppa ¨la ¨ et al. 2017). Anaerobic fungal genomes acquired by next-generation
sequencing techniques continue to refine our
understanding of the biosynthetic potential of
anaerobic fungi and suggest new enzymes for
platform biochemical production pathways
such as isoprenoid and secondary metabolism,
and fatty acid production (Grigoriev et al.
2011; Grigoriev et al. 2014; Hillman et al.
2017). Here, we briefly discuss recent progress
on the adoption of emerging “omics” technologies to characterize the enzyme systems of
420
V. Flad et al.
enzymes and degradation strategies, which can
be harnessed to readily degrade specific feedstocks or for biotechnological purposes, as further discussed in Sect. II.
2. Anaerobic Fungi Have Hydrogenosomes and
Live in Close Relationship with Methanogens
Physiological and genomic investigations suggest that energy generation in anaerobic fungi
is mediated by fungal hydrogenosomes,
although this hypothesis is yet to be confirmed
by molecular studies. Hydrogenosomes are
ATP-producing organelles found across a
range of unrelated anaerobic eukaryotes that
use protons rather than oxygen as the terminal
electron acceptor. They are believed to have
evolved from degenerate mitochondria
(Boxma et al. 2004; Hackstein et al. 2019; Mu ¨ller
et al. 2012), and recent genomic studies have
confirmed the fungal mitochondrial origin of
some hydrogenosomal import proteins
(Haitjema et al. 2017; Youssef et al. 2013).
Hydrogenosomes perform three major metabolic tasks:
(i) Pyruvate metabolism: pyruvate (from
cytosolic plant sugar metabolism) is converted to formate and acetyl-CoA using
pyruvate/formate lyase (PFL) (Boxma
et al. 2004). Marvin-Sikkema et al. (1993)
demonstrated the presence of a hydrogenosome membrane-associated pyruvate/
ferredoxin oxidoreductase (PFO), similar
to that found in hydrogenosomes of Trichomonas spp., and suggested that acetylCoA from cytosolic PFL activity was transported to the hydrogenosome.
(ii) ATP production: Acetyl-CoA is converted
to acetate via succinyl-CoA synthetase,
initiating ATP production exclusively via
substrate level phosphorylation.
(iii) Regeneration of reduced electron carriers: NAD(P)H is re-oxidized by an
NAD(P)H dehydrogenase, which transfers electrons to an iron-only hydrogenase
producing hydrogen (Haitjema et al. 2017;
Kameshwar and Qin 2018; Youssef et al.
2013).
Thus, the main fermentation products of
anaerobic fungi are acetate, formate, and
hydrogen which are produced in hydrogenosomes and lactate, formate, ethanol, and succinate which are produced in the cytosol.
A syntrophic partnership with archaeal
methanogens allows fungi to thrive by siphoning hydrogen to methane (Conrad 1999; Peng
et al. 2016; Ushida et al. 1997), alleviating endproduct inhibition caused by accumulation
of hydrogen. It is well-known that there is a
close relationship between anaerobic fungi
and methanogens, with interspecies hydrogen
transfer resulting in methane production
and more efficient re-generation of oxidized
nucleotides (NAD
+ , NADP
+ ) (Bauchop and
Mountfort 1981; Cheng et al. 2009). Acetate
and formate are also substrates for methanogenesis, and Li et al. (2019) have recently shown
that, when Methanobrevibacter thaueri and
Pecoramyces sp. F1 are grown together in
batch culture, the majority of formate produced
by the fungus is used for methanogenesis. More
details on such microbial interactions and their
biotechnological potential are reported in Sect.
III.C.
II. Identification of Useful Enzymes
Anaerobic fungi offer a wealth of novel proteins and enzymes for diverse biotechnology
applications. Chief among these are potentially
superior
lignocellulose-degrading
enzymes, which offer opportunities to overcome a significant bottleneck during the production of second generation biofuels and
chemicals (Seppa ¨la ¨ et al. 2017). Anaerobic fungal genomes acquired by next-generation
sequencing techniques continue to refine our
understanding of the biosynthetic potential of
anaerobic fungi and suggest new enzymes for
platform biochemical production pathways
such as isoprenoid and secondary metabolism,
and fatty acid production (Grigoriev et al.
2011; Grigoriev et al. 2014; Hillman et al.
2017). Here, we briefly discuss recent progress
on the adoption of emerging “omics” technologies to characterize the enzyme systems of
420
V. Flad et al.