Biotechnology Advances xxx (xxxx) xxxx

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Biotechnology Advances
journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Mosses: Versatile plants for biotechnological applications

Marcelo Lattarulo Camposa,1, Guilherme Souza Pradob,d,1, Vanessa Olinto dos Santosb,
Lara Camelo Nascimentoc, Stephan Machado Dohmsd, Nicolau Brito da Cunhac,d,
⁎ ⁎
Marcelo Henrique Soller Ramadac,d, Maria Fatima Grossi-de-Sab,d, , Simoni Campos Diasc,d,e,
a
Integrative Plant Research Laboratory, Departamento de Botânica e Ecologia, Instituto de Biociências, Universidade Federal de Mato Grosso, Cuiabá, MT, Brazil
b
Laboratório de Interação Molecular Planta-Praga, Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil
c
Centro de Análises Bioquímicas e Proteômicas, Universidade Católica de Brasília, Brasilia, DF, Brazil
d
Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil
e
Programa de Pós-Graduação em Biologia Animal, Universidade de Brasília, Campus Darcy Ribeiro, Brasília, DF, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Mosses have long been recognized as powerful experimental tools for the elucidation of complex processes in
Biotechnology plant biology. Recent increases in the availability of sequenced genomes and mutant collections, the estab-
Bioproducts lishment of novel technologies for targeted mutagenesis, and the development of viable protocols for large-scale
Bryophyte production in bioreactors are now transforming mosses into one of the most versatile tools for biotechnological
Model organisms
applications. In the present review, we highlight the astonishing biotechnological potential of mosses and how
Physcomitrella patens
these plants are being exploited for industrial, pharmaceutical, and environmental applications. We focus on the
biological features that support their use as model organisms for basic and applied research, and how these are
being leveraged to explore the biotechnological potential in an increasing number of species. Finally, we also
provide an overview of the available moss cultivation protocols from an industrial perspective, offering insights
into batch operations that are not yet well established or do not even exist in the literature. Our goal is to bolster
the use of mosses as factories for the biosynthesis of molecules of interest and to show how these species can be
harnessed for the generation of novel and commercially useful bioproducts.

1. Introduction endeavors such as mutant development, precise gene modification and

For almost a century, mosses have been recognized as powerful
experimental tools for research in plant science (Wettstein, 1924; Wood
et al., 2000; Chang et al., 2016; Reski et al., 2018). As members of the
Bryophyta clade (Sousa et al., 2019), mosses are positioned as an
evolutionary grade between green algae and vascular plants, making
them central to the study of land plant evolution (Rensing et al., 2008;
Shaw et al., 2011). Their capacity to colonize an extensive spectrum of
habitats (ranging from the Antarctic tundra to deep forests and the
Mojave desert) has allowed scientists to identify novel strategies and
molecular circuits involved in UV, salt, drought and cold stress toler-
ance (Oliver et al., 2000; Barker et al., 2005; Khraiwesh et al., 2015;
Waterman et al., 2018). These studies also imply the existence of many
as-yet-unknown moss-specific survival mechanisms. The haploid-
dominant life cycle (gametophyte) of mosses and their high rate of
homologous recombination (HR) in mitotic cells facilitate genetic
the analysis of gene function (Strepp et al., 1998; Kamisugi et al., 2006;
Ikram and Simonsen, 2017). Combined with their ease of growth, small
stature, and fast generation time, these characteristics have led mosses
to become outstanding model species for experiments in a laboratory
environment (Wood et al., 2000).
With the increased availability of sequenced genomes and large-
scale mutant collections (Reski and Frank, 2005; Rensing et al., 2008;
Oliver et al., 2010; Cheng et al., 2018; Goruynov et al., 2018), the es-
tablishment of state-of-the-art genome-editing technologies for targeted
mutagenesis (Nomura et al., 2016; Collonnier et al., 2017) and the
development of economically viable protocols for tissue growing in
photobioreactors (PBRs) (Wood et al., 2000; Reski et al., 2018), mosses
have achieved a new status in modern biology as one of the most
powerful chassis for biotechnological applications. Indeed, mosses are
now being exhaustively explored for a plethora of biotechnological
purposes, such as use in bioindicators for environmental changes and

⁎
Corresponding authors at: Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil.
E-mail addresses: marcelolattarulo@ufmt.br (M.L. Campos), stephan@doh.ms (S.M. Dohms), marcelo.ramada@ucb.br (M.H.S. Ramada),
fatima.grossi@embrapa.br (M.F. Grossi-de-Sa), si.camposdias@gmail.com (S.C. Dias).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.biotechadv.2020.107533
Received 2 September 2019; Received in revised form 11 February 2020; Accepted 12 February 2020
0734-9750/ © 2020 Elsevier Inc. All rights reserved.

Please cite this article as: Marcelo Lattarulo Campos, et al., Biotechnology Advances, https://doi.org/10.1016/j.biotechadv.2020.107533
M.L. Campos, et al.
pollution (Suchara et al., 2011; Gonzalez and Pokrovsky, 2014), as
substrates for horticultural applications (Gaudig et al., 2017), as tissue
factories for large-scale molecular farming (Schillberg et al., 2013), as
sources of raw materials in the cosmetic industry and to improve the
nutritional value of crop plants (Reski et al., 2018). For this reason, it is
not surprising that large-scale moss production facilities are becoming
increasingly common (Reski et al., 2015; Reski et al., 2018).
In the present review, we highlight the features that led to the
transformation of mosses into versatile plant toolkits for biotechnology
applications. Our aim is to provide a concise view of key biological
features that bolster the use of mosses as model organisms for basic and
applied research. Although we concentrate on the many features that
led the moss Physcomitrella patens to become a well-established biolo-
gical system that is widely utilized for biotechnological purposes, we
also emphasize how the vast biodiversity of mosses can be explored for
the development of novel biological products. In this scenario, we
further discuss the available technologies that are now allowing the use
of mosses as green factories for commercial purposes. Readers are also
referred to a wealth of excellent reviews focusing on the general aspects
of moss biology as well as the biotechnological features of the other
lineages of bryophytes (liverworts and hornworts) (Rensing et al., 2002;
Duckett et al., 2004; Cornelissen et al., 2007; Proctor et al., 2007;
Asakawa, 2011; Chen et al., 2018; Mishler and Oliver, 2018; Goffinet
and Buck, 2018; Hyyrylainen et al., 2018; Reski, 2018; Decker and
Reski, 2020).
2. Mosses as plant models for genetic engineering
Mosses excel as toolkits for genetic engineering due to the wealth of
available genetic resources, from classic gene targeting (GT) to the
cutting-edge (and now famous) technique of clustered regularly inter-
spaced short palindromic repeats – CRISPR/Cas9 (Schaefer, 2001;
Collonnier et al., 2017). GT is a technique for introducing site-specific
genome modifications at an endogenous locus via HR with exogenous
DNA (Hohe and Reski, 2003). It is a frequently occurring molecular
mechanism in bacteria and unicellular eukaryotes, whereas in multi-
cellular eukaryotes, nonhomologous processes (e.g., nonhomologous
end joining – NHEJ) are predominant. A nonhomologous process con-
sists of ligating two double-stranded DNA segments based on targets
without homology. It often results in illegitimate recombination (IR)
and an unpredictable number of copy insertions (Mengiste and
Paszkowski, 1999; Kumar and Fladung, 2001; Britt and May, 2003;
Hohe and Reski, 2003). Surprisingly, it is well established that in
mosses such as P. patens, HR often results in site-specific transgene in-
tegration, thus allowing GT (Kamisugi et al., 2006). Transforming DNA
exhibiting homology to the P. patens genome integrates into cognate
genetic loci with frequencies of up to 90% (Girke et al., 1998), whereas
in higher plants, HR occurs with very low frequencies (10−4 to 10−5)
(Reski, 1998; Schaefer, 2001; Britt and May, 2003; Puchta, 2003;
Kamisugi et al., 2005; Kamisugi et al., 2006).
Recently, it was demonstrated that GT efficiency could be sig-
nificantly improved in mosses using the CRISPR/Cas9 system (Lopez-
Obando et al., 2016; Collonnier et al., 2017). In this system, a Cas9
endonuclease protein from Streptococcus pyogenes guided by a custo-
mizable noncoding RNA introduces site-specific double-strand breaks
(DSB) in the genome. Repair of these DSBs can lead to gene disruption if
the break is repaired by a deleterious event resulting from NHEJ. Al-
ternatively, in the presence of a homologous donor DNA template, these
DBSs can be repaired via a homology-directed repair pathway, leading
to accurate gene replacement (Ceccaldi et al., 2016). In plants, the
CRISPR/Cas9 system has been efficiently applied for the induction of
illegitimate recombination-mediated (IR) targeted mutagenesis (or
knockout) of endogenous loci (Schaeffer and Nakata, 2015; Liu et al.,
2017). In P. patens, the efficiency of the CRISPR/Cas9 system was tested
by the induction of targeted mutagenesis in an endogenous adenine
phosphoribosyl transferase gene, PpAPT. Molecular analyses confirmed
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Biotechnology Advances xxx (xxxx) xxxx
that targeted deletions, insertions and/or substitutions disrupted the
sequence of PpAPT, thus confirming the efficiency of the CRISPR/Cas9
system for gene knockout/editing in P. patens (Nomura et al., 2016;
Collonnier et al., 2017).
New genetic engineering methodologies for target integration in the
nuclear genome have been demonstrated in P. patens, including epi-
somal recombination events guided by as few as 12 bases of homo-
logous sequences (Múren et al., 2009) and HR employing short over-
lapping sequence (King et al., 2016). This approach allows for targeted
integration in the genome, similar to the typical transformation with
linearized vector. Moreover, it provides an easy way to assemble DNA
fragments in a plant system, allows for flexible genome editing and
combinatorial assembly of DNA parts. The use of in vivo DNA assembly
methodologies can improve the engineering of complex metabolic
pathways further biotechnological potential of P. patens.
A crucial question that remains to be answered is why HR takes
place so efficiently in mosses. Based on this, many possible explanations
can be proposed. First, the high efficiency of GT in mosses may be
correlated with the dominance of the gametophytic haploid phase in
the life cycle of bryophytes (Schaefer, 2001; Schaefer and Zryd, 1997).
Haploid cells have a more relaxed control of sequence homology to
prevent deleterious recombination events when compared to diploids.
As DSBs in a haploid genome may also be repaired by HR to maintain
the genome's integrity, the presence of homologous sequences of the
exogenous (transforming) DNA would increase the probability of in-
tegration through HR in haploids (Schaefer and Zryd, 1997). Interest-
ingly, transformation experiments with haploid tissue do not result in
higher GT frequencies in higher plants (Mengiste and Paszkowski,
1999; Schaefer, 2001). Another important hypothesis is based on the
fact that the moss tissue preferentially used for transformation (proto-
nema) divides synchronously every 24 h and is arrested for most of the
day at the G2 → M transition of the cell cycle (Reski, 1998). It has
already been demonstrated that, for eukaryotes, HR is more efficient at
this phase of the cell cycle, possibly due to the presence of putative cell-
cycle phase-specific factors that act as HR mediators (Hohe and Reski,
2003).
Genes encoding many of the essential components of the DNA repair
machinery are highly conserved among all living organisms, particu-
larly for protein complexes that recognize broken DNA ends (Aravind
et al., 1999). In eukaryotes, the MRN/MRX complex carries out this task
and is composed of three conserved proteins: Meiotic Recombination 11
(MRE11), Radiation-Sensitive 50 (RAD50), and Nijmegen Breakage
Syndrome 1 (NBS1). Together, these proteins form a complex that acts
as a DSB sensor, a coactivator of DSB-induced cell cycle checkpoint
signaling, and a DSB repair effector in NHEJ and HR pathways (Falck
et al., 2005). Kamisugi et al. (2011) showed that PpMRE11 and
PpRAD50, but not PpNBS1, are required to preserve genome integrity
and to achieve GT in P. patens. This observation suggests that the re-
duced number of recruited proteins for GT may explain why the process
is more likely to occur in P. patens. Recently, two RecQ genes (PpRecQ4
and PpRecQ6) were identified on the basis of their roles in the devel-
opment and maintenance of DNA integrity in P. patens (Lang et al.,
2018; Wiedemann et al., 2018). The RecQ helicases are an important
family of genome surveillance proteins that play critical roles in
genome maintenance and stability in all kingdoms of life (Croteau et al.,
2014). Wiedemann et al. (2018) showed that the PpRecQ4 gene acts as a
repressor of recombination, while PpRecQ6 functions as a potent en-
hancer of GT. A moss mutant generated for this gene (ΔPpRecQ6)
showed a change in the ratio of targeted gene replacement via HR by
two-end invasion toward NHEJ or similar modes of untargeted trans-
gene integration that are reported to occur frequently but not pre-
dominantly in P. patens (Wendeler et al., 2015). This finding is re-
miniscent of observations made in the absence of PpRAD51, a key
factor for HR, where GT is abolished, and random integration is in-
creased (Markmann-Mulisch et al., 2007; Schaefer et al., 2010).
Discovery of the highly efficient integration of foreign DNA by HR in
M.L. Campos, et al.
P. patens enabled the use of GT as a precise tool for genetically ma-
nipulating any chosen genomic sequence in this species long before
genome editing technologies (such as CRISPR/Cas9) became accessible
(Gessel et al., 2017). Surprisingly, P. patens not only exhibits a high rate
of HR, but the simultaneous targeting of different genes can also be
achieved in this species by cotransformation and the use of independent
targeting constructs. In these cases, each locus is targeted at the same
frequency as when using a single construct. However, the possibility of
achieving a mutation is dramatically increased (Kamisugi et al., 2006).
After the development of feasible and reliable genetic transformation
methods as well as the successful genome sequencing and phylogenetic
studies of P. patens, this species became a focus of plant biology re-
search, paving the way for the genetic engineering of mosses (Rensing
et al., 2008; Reski et al., 2015). In this way, P. patens has been used as a
versatile model moss plant species, especially for studies involving
functional genomics and the colonization of terrestrial ecosystems by
plants (Bierfreund et al., 2004; Sakakibara et al., 2008; Lang et al.,
2008; Rensing et al., 2008; Mosquna et al., 2009; Decker et al., 2014;
Ikram et al., 2017; Koshimizu et al., 2018). For instance, it is possible to
study development in mosses through GT, which forms the basis of
studies involving the development of vascular plants. P. patens also
appears as a promising system of choice for the stable heterologous
expression of therapeutic proteins and small natural products with
commercial and market potential (see below) (Anterola et al., 2009;
Decker et al., 2014; Reski et al., 2015; Ikram and Simonsen, 2017;
Banerjee et al., 2018).
Elucidation of novel biosynthetic pathways in P. patens, as well as
the establishment of novel heterologous systems based on mosses, could
enable the sustainable production of individual molecules (Reski et al.,
2018). Thus, the development of new genetic engineering methodolo-
gies could help to increase the biotechnological utilization of this host.
Accordingly, P. patens was engineered to produce strains with altered N-
glycosylation patterns for recombinant biopharmaceutical proteins,
making them more “human-like” and, consequently, safer for human
use, providing an advantage of P. patens as a therapeutic-producing
heterologous system (Huether et al., 2005; Decker et al., 2014; Reski
et al., 2015).
3. A chassis for synthetic biology: mosses as systems for
generating recombinant products
Mosses are now becoming a popular chassis for synthetic biology
purposes due to their fast growth, the availability of straightforward
protocols for genetic transformation, and the relatively low cost of
maintenance even compared to prokaryotic systems (Kamisugi et al.,
2006; Ikram and Simonsen, 2017). In addition, when compared to eu-
karyotic expression systems, mosses show several advantages, such as
standardization for cultivation in bioreactors (see below), higher bio-
safety, and amenable genetics for the development of mutants with
desired post-translational modifications (e.g., glycosylation) (Rosales-
Mendoza and Salazar-Gonzales, 2014). They also excel in their capacity
to grow as differentiated tissue in plain inorganic liquid media,
avoiding somaclonal variations that can occasionally affect product
formation. Thus, it is not surprising that several recombinant molecules
have already been biosynthesized in mosses (Table 1), with multiple
companies already releasing moss-based products on the market (e.g.,
Greenovation and Mosspiration Biotech). The exploration of these or-
ganisms as a system for the synthesis of recombinant molecules began
by using mosses for the medium- to large-scale synthesis of hetero-
logous proteins of interest. The first reports on this topic explored the
use of 2 L moss bioreactors for the production of beta-glucuronidase
(GUS) (Reutter and Reski, 1996), bacterial alpha-amylase, human pla-
centally secreted alkaline phosphatase (Gitzinger et al., 2009), and the
F-actin marker GFP-talin (Saidi et al., 2005) in P. patens. It is relevant to
mention that the in vivo production of a protein is a complex process,
often involving posttranslational modifications that are required for
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Biotechnology Advances xxx (xxxx) xxxx
protein folding, stability, and proper biological activity. Prokaryotic
systems are incapable of performing such modifications, and moss cells
can be employed to overcome this limitation (Desai et al., 2010).
The second major wave of the use of mosses for the recombinant
production of molecules explored the biosynthesis of secondary meta-
bolites of commercial interest, mostly those that may be used as ther-
apeutics and sesquiterpenoids for use in the cosmetic and fragrance
industries (Zhan et al., 2014; Pan et al., 2015). The genetic network
involved in the production of secondary metabolites in mosses is more
diverse than those present in conventional, seed-propagated plants
(Beike et al., 2014). This situation indicates great potential for the use
of mosses as platforms for the biosynthesis of secondary metabolites
whose functions have already been well characterized. One example is
taxadiene, a precursor of an anticancer drug (paclitaxel) that is widely
used in chemotherapy treatments (Anterola et al., 2009). In this work,
stable P. patens transformants were obtained that expressed significant
levels of taxadiene without affecting moss growth or the quantity of
endogenous diterpenoids, suggesting that this moss species is a pro-
mising host for the production of paclitaxel and its precursors. In a
more surprising example, the whole biosynthesis route of the anti-
malarial drug artemisinin was incorporated in P. patens (Ikram et al.,
2017). Five different genes responsible for the biosynthesis of artemi-
sinin were introduced into P. patens, and the transgenic lines produced
high levels of artemisinin within only 3 days of cultivation without
metabolic optimization.
The P. patens has been metabolically engineered to produce patch-
oulol and sclareol, two terpenoids isolated, respectively, from patchouli
(Pogostemon spp.) and clary sage (Salvia sclarea), which show valuable
use in the fragrance industry (Zhan et al., 2014; Pan et al., 2015). The
production of sclareol was achieved by the expression of two active
sclareol synthase genes in P. patens. This genetic modification allowed
the upregulation of a terpenoid pathway that leads to a 2- to 5-fold
higher production of sclareol in moss (Pan et al., 2015). For patchoulol,
it was observed that rerouting biosynthesis from the cytosol to the
plastids increased its yield (Zhan et al., 2014). New approaches for
maximizing the yield of specialized metabolites have been adopted to
direct the metabolic flow to specific biosynthetic pathways by using a
transit or signal peptide or a membrane-anchor domain.
Therapeutic proteins are the most attractive molecules for re-
combinant biosynthesis in P. patens due to their high commercial value
and the genetic and molecular characteristics intrinsic to recombinant
production in moss platforms. The human protein vascular endothelial
growth factor was the first recombinant drug to be biosynthesized in P.
patens. This molecule presents high therapeutic value since it plays
numerous roles in the molecular mechanisms associated with angio-
genesis and cancer (Roskoski, 2007; Goel and Mercurio, 2013). Re-
cently, a recombinant human glycoprotein, complement factor H (FH),
was expressed in P. patens devoid of potentially immunogenic plant-
specific sugar residues on protein N-glycans (Büttner-Mainik et al.,
2011; Michelfelder et al., 2017). Recombinant FH is a promising
pharmaceutical product for therapeutic intervention in patients suf-
fering from complement dysregulation. FH is the key regulator of the
alternative pathway of complement activation and a potential drug
candidate for the treatment of atypical hemolytic uremic syndrome
(aHUS) (Weismann et al., 2011; Michelfelder et al., 2017). Currently,
aHUS treatment relies on eculizumab, a monoclonal antibody that is
infamously known as one of the most expensive biopharmaceuticals
worldwide, with an estimated cost of 500,000 USD per patient per year
(Schmidtko et al., 2013). In this scenario, moss-produced FH may re-
present a straightforward alternative for reducing the costs associated
with aHUS treatment. Furthermore, FH is also efficient in treating C3
glomerulopathies and age-related macular degeneration, increasing its
therapeutic value (Bradley et al., 2011; Weismann et al., 2011).
FH has been a key molecule in the development of mosses as re-
actors with potential for the commercial production of therapeutics. Its
biosynthesis requires robustness of the expression system since the
M.L. Campos, et al. Biotechnology Advances xxx (xxxx) xxxx

Table 1
Recombinant proteins and compounds produced in P. patens cells.
Name Native source Function References

Reporter proteins and lab reagents

Beta-glucuronidase (GUS) Escherichia coli Catalyst of formation of colored products Reutter and Reski, 1996
Green fluorescent protein-talin (GFP-talin) Aequorea victoria / mouse F-actin filament labeler Saidi et al., 2005
Placental secreted alkaline phosphatase (SEAP) Human Catalysis of molecular dephosphorylation reactions Gitzinger et al., 2009
Human serum albumin (HSA) Human Stabilizer of protein drugs Baur et al., 2005a
Alpha-amylase (AMY) Bacillus stearothermophilus Catalyses the hydrolysis of glycoside bonds in starch Gitzinger et al., 2009
and glycogen

Enzymes/Pathways involved in secondary metabolite biosynthesis

Taxadiene synthase Taxus brevifolia Catalyses the biosynthesis of the precursor of the Anterola et al., 2009
anticancer agent paclitaxel
Patchoulol synthase (PTS) Pogostemon cablin Catalyses the synthesis of the fragrance patchoulol Zhan et al., 2014
Alpha/beta-santalene synthase (STS) Santalum album Catalyses the synthesis of the alpha / beta-santalo Zhan et al., 2014
fragrance
Sclareol (full biosynthetic pathway) Salvia sclarea Precursors for the perfume industry Pan et al., 2015
Artemisin (full biosynthetic pathway) Artemisia annua Antimalarial metabolite Ikram et al., 2017

Growth factors
Vascular endothelial growth fator (VEGF) Human Angiogenesis Inductor Gorr et al., 2001
Koprivova et al., 2004;
Keratinocyte growth factor (FGF7/KGF) Human Stimulates the proliferation of keratinocytes Niederkrüger et al., 2014
Epidermal growth factor (EGF) Human Stimulates the proliferation of epithelial cells Niederkrüger et al., 2014
Hepatocyte growth factor (HGF) Human Promotes tissue regeneration Niederkrüger et al., 2014

Therapeutic proteins
Asialo-erythropoietin (AEPO) Human Stimulates apoptosis Weise et al., 2007
Complement factor H (FH) Human Complement activator Büttner-Mainik et al., 2011;
Michelfelder et al., 2017
Glyco-optimized antibody IGN311 (IgG1 IGN314) Human Tumor- recognizing antibody Schuster et al., 2007;
Kircheis et al., 2012
Beta-glucocerebrosidase (GBA) Human Enzyme replacement therapy against Gaucher disease Niederkrüger et al., 2014
Alpha-galactosidase (AGal) Human Enzyme replacement therapy against Fabri disease Niederkrüger et al., 2014

Vaccine
Fusion protein from the human immunodeficiency Artificial/HIV Vaccine candidate against HIV Orellana-Escobedo et al., 2015
virus

protein has a large and complex structure, with a mass of 155 kDa and
40 disulphide bonds. To date, P. patens is the only platform available for
the recombinant synthesis of bioactive FH molecules. Moss-derived FH
not only presented the complete structure but was also functional in
vivo (Reski et al., 2018). Recently, a recombinant glycoengineered
MFHR1 was produced in the moss bioreactor (Top et al., 2019). MFHR1
is a synthetic regulator that combines the activities of FH and FH-re-
lated protein 1 (FHR1) (Michelfelder et al., 2018). Recombinant
MFHR1 was genome engineered via homologous recombination, pro-
duced in photobioreactors with high reproducibility and product sta-
bility (Top et al., 2019).
Vaccines are another group of drug candidates produced by mosses
with the potential to be commercially approved. Mosses have been
suggested as interesting platforms for the recombinant biosynthesis of
vaccines for a variety of reasons. First, no adverse effects resulting from
the consumption of mosses by cattle or humans have been reported to
date (Rosales-Mendoza and Salazar-Gonzales, 2014). The absence of
common pathogens to humans, as well as the absence of elements that
can be immunogenic, boosts the exploitation of mosses as vaccine re-
actors (Reski et al., 2018). Furthermore, transgenic explants of moss
that accumulate vaccine antigens could be consumed in natura, which
would increase the cost-effectiveness of production by avoiding ex-
pensive purification procedures for the molecules (Reski et al., 2018).
Despite their innate characteristics as vaccine reactors, there are few
reports of antigens produced on a large scale in mosses. The most no-
table example of a vaccine candidate synthesized in moss is the chi-
meric Env-derived HIV multiepitope protein, which was synthesized in
P. patens (Orellana-Escobedo et al., 2015). The expression strategy was
to utilize P. patens to fully synthesize a multiepitope protein containing
C4 and V3 epitopes from the viral glycoprotein gp120 fused to 4 var-
iants of the ELDKWA epitope from gp41. The chimeric antigen was
demonstrated to be a potent immunogenic elicitor against HIV
ELDKWA epitopes when subcutaneously administered in mice
(Orellana-Escobedo et al., 2015).
Heterologous proteins produced in plant systems with superior
properties to their counterparts synthesized in mammalian cells are
often called “biobetters” (Beck, 2011). Two examples of moss-synthe-
sized biobetters are IgG1 IGN314 and human erythropoietin (EPO).
IgG1 IGN314 is a monoclonal antibody with high medicinal interest due
to its capacity to specifically recognize glycosylation patterns in tumor
cells. Interestingly, compared with a version secreted by Chinese
hamster ovary cells (CHO cells), the glyco-optimized moss-synthesized
IgG1 IGN314 was 40 times more efficient in lysing tumor cell lines
(Schuster et al., 2007). EPO is a human hormone that stimulates red
blood cell maturation in bone marrow and is a relevant agent for the
improvement of the properties of the human circulatory system, as well
as an elicitor of angiogenesis and neurogenesis and a protective agent
against apoptosis (Weise et al., 2007; Lombardero et al., 2011). Parsons
et al. (2012) managed to use P. patens as a system to produce an EPO
version that is devoid of undesirable structures that function as markers
of certain types of cancer, hindering the development of a product at
the commercial level. It was then shown that this moss-produced new
version of EPO did not exhibit any undesirable glycan epitopes and
presented satisfactory antiapoptotic and neuron-protective activities
(Sir'en et al., 2009; Kaneko et al., 2013; Parsons et al., 2013).
In the context of therapeutic products, the glyco-engineering of
recombinant enzymes synthesized in mosses has gained considerable
importance in the last decade. The most well-known cases of enzymes
whose glycan structure has been optimized in P. patens for therapeutic
purposes are human alpha-galactosidase (aGal) and beta-glucocer-
ebrosidase, for the treatment of Fabry and Gaucher diseases, respec-
tively (Niederkrüger et al., 2014). Human aGal and beta-glucocer-
ebrosidase produced by P. patens show a high degree of glycan
homogeneity and increased batch-to-batch stability compared to

4
M.L. Campos, et al.
Fig. 1. Moss photobioreactors (PBRs). PBRs used for moss in vitro cultivation
according to their agitation type: (A) mechanical fermenters such as a stirred-
tank bioreactor (STBR) and (B) pneumatic fermenters such as an air-lift bior-
eactor (ALBR). Both bioreactors, with a capacity of 5 L and 18 L, respectively,
are shown containing suspension cultures of differentiated gametophytic tissue
from P. patens. Source: Embrapa Genetic Resources and Biotechnology.
commercially available versions synthesized by mammalian cells in
suspension (Niederkrüger et al., 2014). Treatment of Fabry-affected
mice with recombinant aGal synthesized in moss and with aGal pro-
duced in mammalian suspension cells showed that the moss-derived
enzyme not only presented no cytotoxicity but also showed improved
pharmacokinetics, which makes it a promising drug candidate for the
treatment of orphan lysosomal storage disease. Hennermann and col-
leagues have recently conducted human trials to demonstrate the safety
of moss aGal. They have shown that a single dose of moss-produced
aGal was safe and tolerable to all administered patients, further re-
sulting in a prolonged reduction in urinary dysfunctions (Hennermann
et al., 2019).
4. Moss bioreactors and scalability
Since the advent of in vitro moss culture, efforts have been made to
optimize culture conditions for the host and/or to maximize the yields
in terms of biomass or the protein of interest when moss culture is
applied for heterologous expression. In this context, reports of the use
of moss bioreactors (Fig. 1) started to appear in 1988, when Boyd et al.
set up a pneumatic fermenter for the liquid culture of P. patens tissues
on larger scales through batch-based approaches. Rudolph and
Rasmussen (1993) adapted this cultivation method for studying the
secondary metabolism of peat moss (Sphagnum spp.), and Reutter and
Reski (1996) subsequently established a reactor platform for cultivating
P. patens using mechanically stirred tanks as a novel approach for agi-
tation. Since then, this strategy has been improved to encompass ap-
plications that vary from obtaining protoplasts for functional genomics
studies (Hohe et al., 2001; Hohe and Reski, 2002) to the production of
heterologous molecules with pharmaceutical applications (see reviews
from Decker and Reski, 2004; Decker and Reski, 2008; Decker and
Reski, 2012; Decker et al., 2014).
For in vitro culture within fermenters (especially for mosses) it is
important to provide an appropriate light source for the system to
achieve effective cell or tissue growth, which can be accomplished with
the use of so-called photobioreactors (PBRs) by the coupling of external
lamps and light panels to standard bioreactors (Schillberg et al., 2013).
Regarding the volume scale, PBRs can work well at both bench (up to
approximately 10 L) (Hohe et al., 2002; Baur et al., 2005a; Baur et al.,
2005b; Hohe and Reski, 2005; Jost et al., 2005; Schaaf et al., 2005;
Weise et al., 2007; Richardt et al., 2010; Büttner-Mainik et al., 2011;
5
Biotechnology Advances xxx (xxxx) xxxx
Cerff and Posten, 2012; Beike et al., 2015; Top et al., 2019) and pilot
(Lucumi and Posten, 2006; Perner-Nochta et al., 2007) scales. The
bench scale is suitable for determining optimal parameters for the
production process, and the pilot-scale is an intermediate step for
adapting the bioprocess before entering the industrial pathway, al-
lowing researchers to gradually make changes in the system through
scale-up or scale-down assays. Although mosses can grow in very
simple culture media containing only inorganic salts, an obstacle to the
scaling up of PBRs to the industrial scale (more than 5000 L) is related
to light obstruction caused by mutual cell shading at medium-to-high
cell densities. This limitation hinders the scalability of PBRs when
compared to other bioreactors and represents a great challenge to the
large-scale production of moss-derived molecules (Huang and
McDonald, 2012). Besides its limitations, the cultivation of mosses in
bioreactors has been shown to be more robust, rapid and stable in terms
of growth than that of several other plant hosts, especially compared to
most higher plants (Wagner, 2003). Additionally, P. patens is a versatile
moss species for bioreactor cultivation, since it is tolerant to many
agitation systems without the need to use specialized bioreactors to
reduce mechanical stresses (Hohe et al., 2002).
For these agitation systems, the best-known PBRs can, therefore, be
classified into the following configurations within two large groups
(reusable and disposable/single-use bioreactors) (Huang and
McDonald, 2012). Mechanically stirred bioreactors are the most pop-
ular, with stirred-tank bioreactors (STBRs) being the most widely used
for mosses, which consist of a glass tank containing a liquid tissue
culture mixed by impellers that can be adapted to various setups, with
baffles and foam deflectors supporting the mixing process by preventing
vortex and foam formation, respectively. Several studies involving moss
bioreactors in the literature report the use of STBRs, especially for
producing biopharmaceutical proteins (Baur et al., 2005a; Baur et al.,
2005b; Schaaf et al., 2005; Weise et al., 2007; Büttner-Mainik et al.,
2011; Cerff and Posten, 2012; Top et al., 2019). On the other hand,
pneumatically agitated bioreactors consist of tanks containing a liquid-
gas dispersion in which air or a specific gas mixture is injected to
promote agitation and aeration simultaneously. Three PBRs of this class
are predominantly used: (i) bubble-column bioreactors (BCBRs), in
which liquid agitation is based on bottom-top gas displacement inside a
glass cylinder with typical height:diameter dimensions between 6:1 and
12:1; (ii) air-lift bioreactors (ALBRs), in which mixing is promoted by
air circulation between sections inside the PBR through cyclical flow
(Huang and McDonald, 2012); and (iii) tubular bioreactors (TPBRs), in
which cultures are mixed within long glass tubes via aeration moving
along connected cylinders. Although they are widely used for plant and
algae cultures in general, only ALBRs and TPBRs are reported as being
used for mosses (Boyd et al., 1988; Rudolph and Rasmussen, 1993).
Compared to mechanically stirred PBRs, pneumatic ones offer the
advantages of being more straightforward, with lower cost, low energy
consumption, and less shear stress for plants. Although these PBRs are
widely used for plant cells in general, due to the low level of shear
stress, there are very few reports of their use for mosses, probably be-
cause moss cells are usually more shear-resistant than other plant cells
and, thus, can be successfully cultivated within STBRs, according to a
large number of reports in the literature. In contrast, mechanical PBRs
are more efficient for achieving good mixing and better oxygen transfer
and are also more flexible regarding scalability (Valdiani et al., 2018).
All of these PBRs are commonly constructed as reusable systems.
Nevertheless, disposable systems for plant hosts can also be built and
are commercially available, and these systems are attractive due to
their cost-saving and contaminant-free design, besides their ease of
compliance with cGMP requirements. In the case of mosses, the most
commonly reported and used type of system is a wave bioreactor (WBR)
(Niederkrüger et al., 2014; Dabrowska-Schlepp et al., 2016; Hoernstein
et al., 2018); WBRs are mostly provided by GE Healthcare and Sartorius
Stedim and consist of a sterile plastic bag with specific capacities, inside
which the liquid culture is mixed by constant rocking in a wave-like
M.L. Campos, et al. Biotechnology Advances xxx (xxxx) xxxx

movement. WBR guarantees good mixing and low cell damage due to

Dabrowska-Schlepp et al., 2016; Hoernstein et al.,

et al., 2005a; Beike et al., 2015; Top et al., 2019.
Reutter and Reski, 1996; Hohe et al., 2002; Baur

Lucumi and Posten, 2006; Perner-Nochta et al.,

shear stress. Moreover, troubles concerning foam formation and diffi-
culty of aeration are not common because of the type of agitation; in
addition, a WBR is easy to handle, since it does not require the ster-
ilization-in-place (SIP) or clean-in-place (CIP) procedures necessary for
reusable systems, although its scalability is very limited - usually up to a

2018; Niederkrüger et al., 2014

Rudolph and Rasmussen, 1993
Not yet published for mosses

1000 L total volume (Weathers et al., 2010). A summary of each of the

described PBRs that are most commonly used for plants and can be used
for mosses, including their advantages and disadvantages, is presented
Boyd et al., 1988;

in Table 2.
Another approach is related to the operation mode used for culti-
References

vation. The main operation modes used in bioprocesses involving

mosses are as follows: (i) single-batch: some amount of biomass/in-
2007

oculum is cultured with a specific volume of nutrients, and the final

product is collected; (ii) fed-batch: addition of one or more nutrients to
Poor mixing, low oxygen transfer, not easy scalability, not

Limited and complex scalability, requires new vessels for

transfer, not easily scalable, not adaptable parts, requires
Complex, high operation cost, high energy consumption,

the medium in a given period after starting the culture; (iii) semi-
requires SIP/CIP, shear stress and cell damage-causing

Complex, poor axial mass transfer, limited scalability,

Poor mixing compared to STBR, low-medium oxygen

continuous batch: removal of partial medium and replenishment of the

bioreactor with the same volume of fresh medium; (iv) continuous
high operation cost, high energy consumption

batch: continuous replenishment of nutrients replacing the collected

fermented medium volume by equal volume of fresh medium; and (v)
perfusion: similar to the continuous batch mode, but with cells/tissues
being retained for biomass enrichment, leading to higher product yields
adaptable parts, requires SIP/CIP

(Georgiev et al., 2013).

To sum up, the selection of the reactor design as well as the agita-
tion system and the batch/operation mode should be performed con-
sidering each particular case and taking into account all parameters
involved in the success of the cell proliferation process (e.g., shear
Disadvantages

new batches

stress, effective mixing, and oxygen transfer), bioprocess variants, the

required degree of scalability and the final application of the bioprocess
SIP/CIP

to ensure suitable culture conditions and provide a supply of substrates

to confer the desired biomass and product yield (Georgiev et al., 2013).
minimal shear stress and low cell damage, no foaming, easy handling, does not
Simple, low operation cost, low energy consumption, minimal shear stress and

Simple, low operation cost, low energy consumption, minimal shear stress and

Cost-effective, sterile ready-to-use vessels, good mixing, low operation cost,

5. Beyond P. patens: exploring moss biodiversity for novel

BCBR: bubble-column bioreactor; STBR: stirred-tank bioreactor; SIP: sterilization-in-place; CIP: clean-in-place.
Good mixing, good oxygen transfer, maximized capture of light, reduced
potential for contamination, minimal shear stress and low cell damage,
Main types of photobioreactors (PBRs) used for in vitro plant culture that are capable of moss cultivation.

biotechnological applications
Good mixing, good oxygen transfer, easy scalability, adaptable parts

With a plethora of genetic and molecular tools ready to use, nu-

low cell damage, better mixing and productivity than BCBR

merous mutant genotypes that are widely available, draft chromosomes

and a fully sequenced genome at hand, it is not surprising that P. patens
became the “moss of choice” for biotechnological applications and
biological research (Rensing et al., 2008; Khraiwesh et al., 2015; Shen
et al., 2016; Ako et al., 2017; Lang et al., 2018). However, it is now
becoming clear that the vast moss biodiversity can be explored as a
source of novel genes, molecules, and traits of interest for biotechno-
logical applications (Cheng et al., 2018). One interesting example in
this regard is Funaria hygrometrica (common cord moss), a widely dis-
tributed moss species that thrives in a diverse environment. Protonemal
tissues of this moss species can tolerate and accumulate high levels of
multiple geometries

lead (Pb), predominantly in their cell walls but also in their chloroplasts
low cell damage

require SIP/CIP

and endoplasmic reticulum (ER) (Itouga et al., 2017). Although the

Advantages

mechanisms involved in Pb accumulation in F. hygrometrica have not

been elucidated to date, the species is already demonstrating the en-
ormous potential to serve as a biomaterial for the bioremediation of Pb-
contaminated areas and the recovery of these compounds. In such a
Disposable

way, heavy metal tolerance appears to be a recurrent topic among moss

Reusable

Reusable

Reusable

Reusable

research. Several studies have indicated copper tolerance in Scopelo-

Usage

phila cataractae, a moss species whose habitat is mostly restricted to Cu-

rich environments (Kobayashi et al., 2006; Konno et al., 2010). Inter-
Agitation type

estingly, the optimal growth of S. cataractae occurs when the species is

Mechanical

Pneumatic

Pneumatic

Pneumatic

exposed to high concentrations of Cu. In this scenario, S. cataractae can

Rocking

also be seen as a suitable research model for studying the mechanisms

of heavy metal tolerance. Targeted genome editing by the CRISPR/Cas9
system was demonstrated to be applicable in this moss (Nomura et al.,
Bubble-column
Stirred-tank

2016). This state-of-the-art gene targeting approach is now allowing

Bioreactor

Tubular

scientists to understand the molecular mechanisms underlying Cu tol-

Table 2

Air-lift

Wave

erance in S. cataractae. The combination of this technology with whole-

genome sequencing and transcriptome analysis may provide a useful

6
M.L. Campos, et al.
strategy for understanding the molecular basis of metal tolerance in
plants (Nomura et al., 2016).
Ceratodon purpureus, also known as fire moss, is a model plant used
for genetic and physiological studies (Thornton et al., 2005; Cove and
Quatrano, 2006; McDaniel et al., 2007; McDaniel et al., 2013). This
bryophyte is widely distributed in a variety of ecosystems and can also
be found in urban environments and nearby degraded areas, such as
mining sites, where the levels of heavy metals are notably high (Jules
and Shaw, 1994). Studies indicate that C. purpureus is also resistant to
high levels of UV radiation by enhancing the synthesis of cell wall UV-
B-absorbing compounds in exposed populations (Waterman et al.,
2018). Moreover, C. purpureus thrives under cold-freezing tempera-
tures, as observed in Antarctica (Lenné et al., 2010). The occurrence of
this moss under such conditions suggests the presence of physiological
mechanisms to deal with environmental stress. Thus, it will not be
surprising if novel stress-related genes are identified by C. purpureus
genome analysis. Additionally, C. purpureus carries a full set of im-
portant molecular and genetic features required for plant transforma-
tion, such as efficient GT by homologous recombination. Although
Trouiller et al. (2007) have shown that GT efficiency in C. purpureus is
considerably lower compared to that in P. patens (1.05% and 20.8%,
respectively), this mechanism is still two orders of magnitude higher
than in general vascular plants. In this context, Trouiller et al. (2007)
suggest that efficient GT could be a conserved characteristic among
bryophytes. Currently, the Joint Genome Institute (JGI) is in the process
of conducting the C. purpureus genome sequencing project. The data
acquired will provide more insights into genetic/molecular tool appli-
cations and plant evolution and will identify genes involved in toler-
ance to metal contamination (see https://jgi.doe.gov/why-sequence-
ceratodon-purpureus-moss/). Previous studies demonstrated that a C.
purpureus population from a metal-rich site grew significantly more in
contaminated soil rather than non-contaminated soils. In contrast,
plants from non-contaminated areas demonstrated no or severely re-
duced growth in metal-contaminated soil treatment (Jules and Shaw,
1994). Additionally, other populations were grown on agar media
supplemented with various concentrations of metal ions (copper, zinc,
lead, and cadmium). Plants originating from metal-rich sites showed
less sensitivity to metal concentration than other C. purpureus popula-
tions (Jules and Shaw, 1994).
In the context of whole-genome sequencing, JGI has already over-
seen a community science project (The Sphagnome Project) for
Sphagnum fallax and Sphagnum magellanicum (Weston et al., 2018). A
draft version of the S. fallax genome is available (Sphagnum fallax v0.5,
DOE-JGI), while that of S. magellanicum is currently being assembled
(Shaw et al., 2016). The project still aims to sequence 31 individuals
from 15 species that comprise the five major clades of this genus
(Weston et al., 2018). This genomic information will provide critical
insights into the genetic basis of the evolution of important Sphagum
functional traits such as its ability to modify its habitat, the associations
with methane-oxidizing bacteria and the role of Sphagnum genetics and
physiology in biogeochemistry and hydrology at ecosystem to global
scales (Weston et al., 2018).
Sphagnum spp. (peat mosses) are the primary components of peat-
land ecosystems, which have a remarkable influence on global cycling
dynamics due to high rates of CO2 uptake (carbon sequestration)
(Bengtsson et al., 2016). Unfortunately, constant increase in the de-
mand for peat, which is used as growing substrates and potting material
for gardening, is leading to severe degradation of peatlands (Caron and
Rochefort, 2013). This practice, along with peat oxidation, significantly
contributes to greenhouse-effect gas emissions (Joosten et al., 2016).
Paludiculture with Sphagnum in degraded peatlands (Sphagnum
farming, as reviewed by Gaudig et al., 2017), is an interesting and
emerging approach to obtain Sphagnum biomass as an alternative to
peat extraction from natural sources and, consequently, restore de-
graded peatland ecosystems (Pouliot et al., 2015). The development of
this technology is already ongoing, but a lot remains to be done to
7
Biotechnology Advances xxx (xxxx) xxxx
increase productivity and reduce costs of Sphagnum farming (Gaudig
et al., 2017). Mass propagation of Sphagnum in PBRs is currently under
development by the MOOSzucht project (Germany) (Gaudig et al.,
2017). Genomic information carried out by the Sphagnome project will
be important to correlate genes to trait and to make Sphagnum spp.
clone selection more productive.
Another application of Sphagnum moss was carried out by the FP7
European project “MOSSclone” (www.mossclone.eu). This research
consortium aimed to use Sphagnum palustre as a novel, precise, in-
expensive biotechnological tool to monitor air quality, especially for the
presence of heavy metals in the atmosphere. The applied method was a
standardized version of the “moss bag technique” developed in the
1970s by Goodman and Roberts (1971) and involved a spherical device
(moss weight/bag surface ratio of 10 mg cm2, 2 mm net mesh size, ø
~11 cm) containing 3 g of devitalized moss material, referred to as the
“Mossphere”. In this project, S. palustre was chosen from among four
other mosses used for pollution biomonitoring because it exhibited the
highest adsorption rates of different toxic compounds and presented
important physicochemical properties that fulfilled the criteria for use
(Gonzalez and Pokrovsky, 2014). Axenically, in vitro cultures, were
performed and optimized in PBRs to obtain large quantities of biomass.
After manipulation in different media compositions, along with manual
tissue disruption, inoculum density, and pH control, S. palustre biomass
increased by around 10- to 30-fold after 4 weeks in PBRs (Beike et al.,
2015). Moreover, chemical and molecular traits of the selected clones
of S. palustre were characterized (Di Palma et al., 2016). The results of
Mossphere apparatus trials after six weeks of exposure at 10 different
sites with diverse air quality conditions in Spain and Italy showed a
more efficient uptake ability of S. palustre compared to the native moss
Pseudoscleropodium purum (Capozzi et al., 2017).
6. Conclusions and prospects
Research on mosses has contributed to significant advances in cel-
lular biology and fundamental biology. As rather lower plants, most
moss tissues are just a one-cell layer, making this group of plants re-
latively easy to manipulate for biotechnology purposes. To fully exploit
the technological potential of mosses, additional species should con-
tinue to be investigated. Efforts to increase the knowledge of systema-
tics, herbal characteristics and lists of species are of major scientific
importance, as they are sources of valuable information for many re-
searchers who investigate physiological and genetic aspects of mosses.
Thus, increases in the numbers of institutions and/or groups of re-
searchers who study mosses can address gaps and improve knowledge
of the biodiversity and biotechnological uses of these plants. Because
mosses can be easily maintained under laboratory conditions and whole
genomes of some moss species have been sequenced, we believe that
they can be among the most suitable plants for gene targeting strate-
gies. This is corroborated by the fact that a moss species, P. patens, has
become one of the most attractive model systems for plant biology and
also a marvelous tool for plant biotechnology purposes.
Several studies have reported the moss-mediated production of
compounds with high commercial value, such as those that can be used
as cosmetics or biopharmaceuticals and terpenoids. However, the po-
tential of mosses for applied research with implications for agriculture
and human health has not been fully appreciated or explored. The
characterization of new biosynthetic routes and the establishment of
new approaches for maximizing the yield of specialized metabolites
should be continued to direct metabolic flows to specific biosynthetic
pathways, enabling sustainable production of individual molecules.
Compared to other systems used for the production of recombinant
molecules such as bacteria, yeasts, animals and vascular plants, mosses
present great advantages and have proved to be a versatile chassis in
the synthetic biology context. The main factors responsible for this
success stem from their biological properties, such as their ability to
grow as differentiated tissues in simple inorganic liquid media,
M.L. Campos, et al.
avoiding somaclonal variations that may occasionally affect product
formation. Additionally, fast growth and high capacity for tissue re-
generation, as well as well-defined transformation protocols and rela-
tively low maintenance costs in liquid culture, make mosses a cost-ef-
fective platform for heterologous expression.
Finally, the discovery of the highly efficient integration of foreign
DNA by HR in P. patens and the ability to achieve multiplex targeting of
genes allowed mosses to stand out as toolkits for genetic engineering,
including GT and genome engineering approaches through synthetic
biology tools such as the CRISPR/Cas system. These new technologies
give rise to a wide range of possibilities for the genetic manipulation of
any genomic sequence chosen in this species and, consequently, for
crop improvement and the generation of biofactories for economically
important pharmaceuticals. However, as for other plants, much work
still needs to be done to optimize and perform CRISPR/Cas gene
knockin with high efficiency and lower rates of off-target effects in the
genome editing of mosses.
Acknowledgements
This work was supported by fellowships from UCB, Brazil;
EMBRAPA, Brazil; CNPq, Brazil; INCT PlantStress Biotech, CAPES
Brazil; and FAPDF, Brazil. Authors declare no conflict of interest.
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