enzymes, anaerobic fungi harbor a wide variety
of membrane-embedded receptors and transporters that the fungi likely use to sense and
sequester carbohydrates from the environment
(Seppa ¨la ¨ et al. 2016). For example, the study
found that anaerobic fungi appear to have Gprotein coupled receptors with putative
carbohydrate-binding domains. Furthermore,
the study found that all major eukaryotic carbohydrate transporter classes are represented
in anaerobic fungi: Major Facilitator Superfamily (MFS), the Sodium Solute Symporter family,
and the Sugars Will Eventually be Exported
Transporter family (SWEET). The occurrence
of SWEETs is frequent in plant and bacterial
systems but is rare in fungi and so far exclusive
to lower lineages, such as Chytridiomycota.
Unexpectedly, the vast majority of identified
transporter components were found to be
homologous to substrate-binding proteins
(SBPs) that are typically associated with prokaryotic ATP-binding cassette (ABC) transporters (Ter Beek et al. 2014). In prokaryotes, the
extracellular SBPs deliver small molecules to
ATP-driven uptake systems, and whereas it is
likely that the fungi acquired these proteins by
horizontal gene transfer, their function in the
eukaryotic host remains unknown (Haitjema
et al. 2017). Further experimental characterization is necessary, as very few membrane proteins from anaerobic fungi have been
heterologously produced to date (Haferkamp
et al. 2002; van der Giezen et al. 1998). Recently,
it was shown that fluoride transporters from
anaerobic fungi bolster solvent tolerance of
yeast (Seppa ¨la ¨ et al. 2019). In conclusion,
anaerobic fungi are a promising source of
receptors and transporters to expand metabolite uptake modulate tolerance in model
microbes, and these proteins should be
assessed for their use in bioprocessing applications.
C. Opportunities Identified from Published
Patents
The Espacenet tool from the European Patent Office
was used to search the worldwide collection of published applications for (a) the number of anaerobic
fungal patents filed, (b) their date of publication, (c)
the location of the inventors, and (d) the exploitation
area of the published patent. The search terms used
were directed at patent titles and abstract only. All
meaningful combinations of the following search
terms were used to search the database: anaerobic,
gut, fungi, fungus, rumen, and all named fungal genera.
With these search terms, 384 published patents were
identified. On removal of duplicates and erroneously
listed applications, 51 patents were found that directly
involved the anaerobic fungi or where anaerobic fungi
were used as a “catch all” term alongside other microbial groupings. To place this number of patents into
context, replacing the search terms with Trichoderma
or Aspergillus resulted in patent lists of 3968 and >
10,000, respectively. The majority of anaerobic fungal
patents were filed by China (28%), the USA (23%),
Taiwan (14%), the UK (9%), Australia (7%), Canada
(5%), the Netherlands (4%), and Turkey (4%). Japan,
South Africa, South Korea, and Russia accounted for
the remaining patents. Chinese and Taiwanese inventors were responsible for the majority of recent (2001–
2018) patents.
The first peer review article on the anaerobic fungi
was published in 1975 (Orpin 1975). The first patent to
mention anaerobic fungi appeared 14 years later in
1989 (JPH01289488 (A)). In this patent, a Japanese
inventor listed anaerobic fungi alongside other fungi
in a “catch-all” application for water cleanup. The UK
inventors Hazelwood and Gilbert (1993) published the
first patent directly attributable to the anaerobic fungi
(WO9325693 (A1)). This patent was concerned with the
expression of truncated recombinant xylanases from
Neocallimastix patriciarum and their potential use in
the pulp and paper industries.
Approximately 70% of patents are
concerned with molecular biology-based applications, broadly focused on gene identification,
gene characterization, cloning, and expression
of recombinant enzymes and their derivatives
in heterologous hosts. Xylanase, esterase, and
cellulase genes from Neocallimastix and Piromyces are described in many patents alongside
the enhancement of transformed enzyme activities, for example, to produce xylanases with
improved thermal stability and pH tolerance.
Expression hosts are mostly Escherichia coli,
Saccharomyces cerevisiae, and Pichia pastoris.
The latter species is the methylotrophic yeast
widely employed for protein/enzyme production using recombinant DNA techniques. In
one patent (US5948667 (A)), plant cells of
Canola were transformed with a xylanase gene
from Neocallimastix patriciarum with the
intention of improving the digestion of crop
residues by ruminant livestock. Another patent
428
V. Flad et al.
of membrane-embedded receptors and transporters that the fungi likely use to sense and
sequester carbohydrates from the environment
(Seppa ¨la ¨ et al. 2016). For example, the study
found that anaerobic fungi appear to have Gprotein coupled receptors with putative
carbohydrate-binding domains. Furthermore,
the study found that all major eukaryotic carbohydrate transporter classes are represented
in anaerobic fungi: Major Facilitator Superfamily (MFS), the Sodium Solute Symporter family,
and the Sugars Will Eventually be Exported
Transporter family (SWEET). The occurrence
of SWEETs is frequent in plant and bacterial
systems but is rare in fungi and so far exclusive
to lower lineages, such as Chytridiomycota.
Unexpectedly, the vast majority of identified
transporter components were found to be
homologous to substrate-binding proteins
(SBPs) that are typically associated with prokaryotic ATP-binding cassette (ABC) transporters (Ter Beek et al. 2014). In prokaryotes, the
extracellular SBPs deliver small molecules to
ATP-driven uptake systems, and whereas it is
likely that the fungi acquired these proteins by
horizontal gene transfer, their function in the
eukaryotic host remains unknown (Haitjema
et al. 2017). Further experimental characterization is necessary, as very few membrane proteins from anaerobic fungi have been
heterologously produced to date (Haferkamp
et al. 2002; van der Giezen et al. 1998). Recently,
it was shown that fluoride transporters from
anaerobic fungi bolster solvent tolerance of
yeast (Seppa ¨la ¨ et al. 2019). In conclusion,
anaerobic fungi are a promising source of
receptors and transporters to expand metabolite uptake modulate tolerance in model
microbes, and these proteins should be
assessed for their use in bioprocessing applications.
C. Opportunities Identified from Published
Patents
The Espacenet tool from the European Patent Office
was used to search the worldwide collection of published applications for (a) the number of anaerobic
fungal patents filed, (b) their date of publication, (c)
the location of the inventors, and (d) the exploitation
area of the published patent. The search terms used
were directed at patent titles and abstract only. All
meaningful combinations of the following search
terms were used to search the database: anaerobic,
gut, fungi, fungus, rumen, and all named fungal genera.
With these search terms, 384 published patents were
identified. On removal of duplicates and erroneously
listed applications, 51 patents were found that directly
involved the anaerobic fungi or where anaerobic fungi
were used as a “catch all” term alongside other microbial groupings. To place this number of patents into
context, replacing the search terms with Trichoderma
or Aspergillus resulted in patent lists of 3968 and >
10,000, respectively. The majority of anaerobic fungal
patents were filed by China (28%), the USA (23%),
Taiwan (14%), the UK (9%), Australia (7%), Canada
(5%), the Netherlands (4%), and Turkey (4%). Japan,
South Africa, South Korea, and Russia accounted for
the remaining patents. Chinese and Taiwanese inventors were responsible for the majority of recent (2001–
2018) patents.
The first peer review article on the anaerobic fungi
was published in 1975 (Orpin 1975). The first patent to
mention anaerobic fungi appeared 14 years later in
1989 (JPH01289488 (A)). In this patent, a Japanese
inventor listed anaerobic fungi alongside other fungi
in a “catch-all” application for water cleanup. The UK
inventors Hazelwood and Gilbert (1993) published the
first patent directly attributable to the anaerobic fungi
(WO9325693 (A1)). This patent was concerned with the
expression of truncated recombinant xylanases from
Neocallimastix patriciarum and their potential use in
the pulp and paper industries.
Approximately 70% of patents are
concerned with molecular biology-based applications, broadly focused on gene identification,
gene characterization, cloning, and expression
of recombinant enzymes and their derivatives
in heterologous hosts. Xylanase, esterase, and
cellulase genes from Neocallimastix and Piromyces are described in many patents alongside
the enhancement of transformed enzyme activities, for example, to produce xylanases with
improved thermal stability and pH tolerance.
Expression hosts are mostly Escherichia coli,
Saccharomyces cerevisiae, and Pichia pastoris.
The latter species is the methylotrophic yeast
widely employed for protein/enzyme production using recombinant DNA techniques. In
one patent (US5948667 (A)), plant cells of
Canola were transformed with a xylanase gene
from Neocallimastix patriciarum with the
intention of improving the digestion of crop
residues by ruminant livestock. Another patent
428
V. Flad et al.