ArticlePDF AvailableLiterature Review

Abstract and Figures

Spider silk threads have exceptional mechanical properties such as toughness, elasticity and low density, which reach maximum values compared to other fibre materials. They are superior even compared to Kevlar and steel. These extraordinary properties stem from long length and specific protein structures. Spider silk proteins can consist of more than 20,000 amino acids. Polypeptide stretches account for more than 90% of the whole protein, and these domains can be repeated more than a hundred times. Each repeat unit has a specific function resulting in the final properties of the silk. These properties make them attractive for innovative material development for medical or technical products as well as cosmetics. However, with livestock breeding of spiders it is not possible to reach high volumes of silk due to the cannibalistic behaviour of these animals. In order to obtain spider silk proteins (spidroins) on a large scale, recombinant production is attempted in various expression systems such as plants, bacteria, yeasts, insects, silkworms, mammalian cells and animals. For viable large-scale production, cost-effective and efficient production systems are needed. This review describes the different types of spider silk, their proteins and structures and discusses the production of these difficult-to-express proteins in different host organisms with an emphasis on plant systems.
Content may be subject to copyright.
Recombinant Spider Silk: Promises
and Bottlenecks
Maryam Ramezaniaghdam
1
,
2
, Nadia D. Nahdi
1
and Ralf Reski
1
,
2
*
1
Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany,
2
Cluster of Excellence livMatS at FIT
Freiburg Center for Interactive Materials and Bioinspired Technologies, University of Freiburg, Freiburg, Germany
Spider silk threads have exceptional mechanical properties such as toughness, elasticity
and low density, which reach maximum values compared to other bre materials. They are
superior even compared to Kevlar and steel. These extraordinary properties stem from
long length and specic protein structures. Spider silk proteins can consist of more than
20,000 amino acids. Polypeptide stretches account for more than 90% of the whole
protein, and these domains can be repeated more than a hundred times. Each repeat unit
has a specic function resulting in the nal properties of the silk. These properties make
them attractive for innovative material development for medical or technical products as
well as cosmetics. However, with livestock breeding of spiders it is not possible to reach
high volumes of silk due to the cannibalistic behaviour of these animals. In order to obtain
spider silk proteins (spidroins) on a large scale, recombinant production is attempted in
various expression systems such as plants, bacteria, yeasts, insects, silkworms,
mammalian cells and animals. For viable large-scale production, cost-effective and
efcient production systems are needed. This review describes the different types of
spider silk, their proteins and structures and discusses the production of these difcult-to-
express proteins in different host organisms with an emphasis on plant systems.
Keywords: bre, moss, recombinant production, expression systems, biomaterial, smart material, bioproduction
INTRODUCTION
Spider silks have fascinated scientists for decades due to their outstanding mechanical
properties. A combination of high tensile strength and large extensibility makes them
remarkably tough; they are ve times stronger than steel and possess toughness threefold
than that of Kevlar (Gosline et al., 1999;Vollrath and Knight, 2001). In addition to mechanical
properties, spider silks have bio-properties, such as biocompatibility and slow degradability.
They have been used as sutures for wound healing for centuries (Altman et al., 2003). Because of
these properties, spider silks are regarded as a promising material for medical applications such
as selective microbial-resistant coatings (Kumari et al., 2020), organic and degradable
biosensors for biomonitoring of analytes in the body (Xu et al., 2019), wound healing
(Öksüz et al., 2021), creating lenses useful for biological imaging (Tian et al., 2020), tissue
engineering (Salehi et al., 2020)suchasarticial blood vessels (Dastagir et al., 2020), nerve
regeneration (Kornfeld et al., 2021;Millesi et al., 2021) and scaffolds creation (Gellynck et al.,
2008). In addition, these silks have potential for use as smart materials. It was reported that they
can be used in lithium-ion batteries to retain the capacity and decrease the volume expansion of
silicon (Choi and Choy, 2020), for aerospace application (Mayank et al., 2021), as vision-based
vibration sensors (Liu et al., 2020a), silk-based humidity sensors (Liu et al., 2020b), or protein-
based adhesives for transparent substrates (Roberts et al., 2020). In addition, application of
Edited by:
Johannes Felix Buyel,
Fraunhofer Society (FHG), Germany
Reviewed by:
Gefei Chen,
Karolinska Institutet (KI), Sweden
Sanyuan Ma,
Southwest University, China
*Correspondence:
Ralf Reski
ralf.reski@biologie.uni-freiburg.de
ORCID:
Maryam Ramezaniaghdam
orcid.org/0000-0003-3182-7521
Ralf Reski
orcid.org/0000-0002-5496-6711
Specialty section:
This article was submitted to
Industrial Biotechnology,
a section of the journal
Frontiers in Bioengineering and
Biotechnology
Received: 14 December 2021
Accepted: 01 February 2022
Published: 08 March 2022
Citation:
Ramezaniaghdam M, Nahdi ND and
Reski R (2022) Recombinant Spider
Silk: Promises and Bottlenecks.
Front. Bioeng. Biotechnol. 10:835637.
doi: 10.3389/fbioe.2022.835637
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356371
SYSTEMATIC REVIEW
published: 08 March 2022
doi: 10.3389/fbioe.2022.835637
spider silk proteins in the cosmetic industry has been explored
recently, for example as a new technology for face lifting
(Qing et al., 2021).
Spider Silks and Their Properties
More than 41,000 species of spiders produce silks (Vierra et al.,
2011). Seven different silks are produced (Figure 1), each of them
having different properties. Each silk is produced by a specic
gland and extruded from spinnerets located on the posterior end
of the spiders abdomen (Vollrath and Knight, 2001).
Dragline silk (major ampullate silk) is the most studied spider
silk and makes the framework of the web (Figure 1). This silk has
been at the center of attention for several years, since it is the
strongest silk analyzed so far. Its estimated strength is 1,290 MPa
(megapascal) in Argiope trifasciata (Hayashi et al., 2004). A
variation is seen in the measured mechanical properties of silk
bres collected from nature (Table 1), which may be a result of
the difculties in collecting a particular type of silk for reliable
mechanical testing. Another reason can be the regulation of
spinning rate by the spider to make the property of the silk
suitable for the specic task (Yarger et al., 2018). Moreover, it was
suggested that various nutrition conditions (prey variation) may
affect silk composition and mechanics. However, this assumption
was rejected by Kono et al. (2019), who investigated mechanical
properties after starvation or directly after feeding and found no
effect of nutrition conditions on protein components and
mechanical properties.
This silk is composed of two proteins, major ampullate
spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2),
with estimated molecular masses of over 250 kDa (Table 2).
MaSp1 and MaSp2 proteins in Latrodectus hesperus consist of
a non-repetitive N-terminal domain (NR-NTD), a large and
highly repetitive core region and a non-repetitive C-terminal
domain (NR-CTD) (Ayoub et al., 2007)(Figure 2). The
N-terminal region contains non-repetitive amino acids and
may play a crucial role in the transport of the spidroin into
the glandular lumen (Ayoub et al., 2007;Zhang et al., 2013).
N-terminal domains are formed as antiparallel dimers due to
surface charges and control protein interaction and elongation
(Hagn et al., 2011). Dimerization of the N-terminal domain is
dependent on pH. This region responds to ionic and mechanical
changes and can promote the solubility of spidroins at neutral pH
(Askarieh et al., 2010;Bauer and Scheibel., 2017;Chakraborty
et al., 2020). Three conserved residues of the N-terminal domain
of dragline silk in Euprosthenops australis, Glu79, Glu84 and
Glu119, are protonated to form a homodimer from a monomer
when the pH changes from 7 to lower pH values (Jiang et al.,
2019). N- and C-terminal domains lead to an increase in Youngs
modulus, stress, and toughness of recombinant proteins (Zhu
et al., 2020). Sequence alignments of N- and C-terminal domains
of spidroins show that these domains are highly conserved
(Ayoub et al., 2007). The core repetitive region in MaSp1 is
composed of four types of ensemble repeat units (ERUs) which
are tandemly arrayed in a consistent pattern. This pattern is
iterated 20 times with near-perfect delity. Each ensemble
consists of a glycine-rich region followed by a poly-A region.
Amino acid motifs of each ensemble are GGX (X = A, Q, or Y),
GX (X = Q, A, or R) and poly-A. Similar to MaSp1, the core
repetitive region of MaSp2 is organized into four types of ERUs,
which are more variable than those of MaSp1. Moreover, they are
not always tandemly arrayed in the same order. Core region
motifs of MaSp2 comprise GPX (X = G or S), GGX (X is usually
A), GSG, QQ and poly-A (Ayoub et al., 2007). Techniques such as
X-ray diffraction, NMR measurements and transmission electron
microscopy (TEM) revealed aligned nanocrystalline β-sheets in
the predominant crystalline component (Van Beek et al., 2002;
Trancik et al., 2006). Further studies showed that mechanical
properties of spider dragline silk correlate with their molecular
structure (Yarger et al., 2018;Htut et al., 2021). Poly-A motifs
form β-sheet structures (Trancik et al., 2006;Van Beek et al.,
2002), in which large numbers of hydrogen bonds are created
between the backbone amine and carbonyl groups (Zhang et al.,
2014). The strength of dragline silk directly correlates with this β-
sheet structure (Yarger et al., 2018). On the contrary, GGX and
FIGURE 1 | Schematic representation of different types of spider silk and their role in spider webs (compiled according to Vollrath and Knight 2001).
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356372
Ramezaniaghdam et al. Recombinant Spider Silk
GPX peptide motifs, which are less orientated and amorphous,
have αhelical and type II β-turns structures, respectively. These
are not as constrained as β-sheet structures (with high density of
hydrogen bonds) so that they grant extensibility to dragline bres
(Jenkins et al., 2010;Gray et al., 2016;Yarger et al., 2018)
(Table 3). Cysteine residues in the C-terminal domain are
involved in intermolecular disulde formation. The pH value,
salt concentration, and shear-force-induced partial unfolding of
the disulde-bridged dimeric C-terminal domain control the
correct alignment of polyA/polyGA sequences to form
microcrystalline structures facilitating the assembly of bres
(Hagn et al., 2011). The properties of this silk can change
upon exposure to water (vapour or liquid), leading to an
increase in diameter and a decrease in length. This behaviour
is called supercontraction, during which a loss of molecular
structural order is induced and bre stiffness is decreased. It is
proposed that this property is related to the content of proline,
making type II β-turns structures (Liu et al., 2005;Blackledge
et al., 2009;Lang et al., 2017).
A short type of dragline silk protein called MaSp1s, consisting
of 439 aa and a molecular mass of 40 kDa, has been identied in
Cyrtophora moluccensis (Table 2). It contains a non-repetitive
N-terminal domain (149 aa), core region (192 aa) and a
C-terminal domain (98 aa) (Figure 2). Two terminals are
homologous to that of other dragline spidroins. There is an
apparent signal peptide in the N-terminal region of MaSp1s
and a putative cleavage site between amino acids 24 and 25.
An obvious repetitive region is not seen in this protein. It has only
7 short repeat units which are not extremely homogenized.
However, this protein comprises all the motifs of MaSp1 such
as GGX (X = A, Q, or Y), GX (X = Q, A, or R) and polyA. MaSp1s
has less repetitive units as other dragline spidroins, so that they
may not have a key role in determining the mechanical properties
of the silk. The abundance of this small protein is lower than that
of MaSp1 and MaSp2, implying that it may not have a dominant
effect on the strength and elasticity of the silk (Han et al., 2013).
With presenting the rst genomes in the genus Araneus, the
full spidroin gene set was obtained and conrmed with the
transcriptome of silk glands as well as the proteome of the silk
(Kono et al., 2021). As a result, the full length (7854 bp) of a new
paralog in the MaSp gene family, MaSp3, was isolated. This gene
is found in Araneus ventricosus and Argiope argentata (Kono
et al., 2019) and was previously reported partially (Collin et al.,
2018). MaSp3 is highly expressed in the major ampullate gland
and the MaSp3 protein is the most abundant in dragline silk. A
Principal Component Analysis (PCA) revealed that MaSp3 does
TABLE 1 | Mechanical properties of seven types of spider silks.
Silk Young´s modulus
(GPa)
Strength (MPa) Extensibility (%) Toughness (MJ/m
3
) References
Species of
spider
Dragline silk 906.9± 19.55 ± 5.02 84.28 ± 31.91 Kono et al. (2019)
Araneus vetricosus
Dragline silk 8.3 ± 0.54 ——141.2 ± 0.77 Swanson et al. (2006a)
Araneus diadematus
Dragline silk 9.3 1,290 ± 29 22 145 Hayashi et al. (2004)
Argiope trifasciata
Dragline silk 3.411.5 1,030 ± 176 2535 149 ± 25 Kerr et al. (2018)
Nephila pilipes
Dragline silk 1,030 ± 206 73.22 ± 7.60 Kerr et al. (2018)
Nephila plumipes
Dragline silk 13.8 ± 3.6 1,215 ± 233 11.2 ± 30 Swanson et al. (2006b)
Naphila clavipes
Dragline silk 10.2 ± 0.75 ——180.9 ± 11.19 Swanson et al. (2006a)
Latrodectus hesperus
Minor ampullate silk Argiope trifasciata 8.5 342 54 148 Hayashi et al. (2004)
Minor ampullate silk Latrodectus hesperus 3.9 ± 2.9 245.4 ± 120.5 0.57 ± 0.02 66.7 ± 46.4 Vienneau-Hathaway et al. (2017)
Minor ampullate silk 2.6 ± 1.3 174.4 ± 17.6 0.54 ± 0.02 43.6 ± 20.1 Vienneau-Hathaway et al. (2017)
Latrodectus geometricus
Minor ampullate silk steatoda grossa 2.1 ± 0.7 251.3 ± 106.2 0.74 ± 0.02 57.3 ± 20.7 Vienneau-Hathaway et al. (2017)
Flageliform 0.003 500 270 150 Gosline et al. (1999)
Araneus diadematus
Flagelliform 0.0120.08 800 ± 100 200 Perea et al. (2013)
Argiope trifasciata
Pyriform 0.2 ± 0.1 100 ± 40 5080 61 ± 47 Greco et al. (2020)
Cupiennius salei
Aciniform 9.6 687 86 367 Hayashi et al. (2004)
Argiope trifasciata
Clyndriform 9.1 390 40 128 Zhao et al. (2006)
Argiope bruennichi
Cylindriform 8.7 ± 0.9 400 ± 50 520 Nimmen et al. (2005)
Araneus diadematus
GPa, gigapascal; MPa, megapascal; MJ, megajoule; m
3
, cubic meter.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356373
Ramezaniaghdam et al. Recombinant Spider Silk
not have a direct contribution to the mechanical properties of this
silk. Only a limited homology is seen between terminal domains
of MaSp3 and MaSp1 and MaSp2 in A. vetricosus (Kono et al.,
2019). The existence of some additional proteins was conrmed
within dragline silk, such as alpha-2-macroglobulin 2 and
peroxidasin, which are found in the peripheral layer and the
core region of the caddisy silk bre (Kono et al., 2019).
Peroxidasin contributes to the silk post-draw dityrosine
crosslinking (Wang et al., 2014).
With a multiomics approach, genomes of four closely related
Nephilinae, gland transcriptomes, and proteomes of dragline silk
are available (Kono et al., 2021), which will contribute to future
research. With this high-quality genome analyses, a conserved
MaSp3B was reported in the genera Trichonephila and Nephila.
The important residues, such as Aspartic acid40, Lysine65, Glu79,
and Glu119, which have impact on the N-terminal domain
dimerization, are conserved in this protein (Kono et al., 2021).
Non-canonical silk constituents termed SpiCEs were found in the
spider silk. Four SpiCEs proteins are highly expressed exclusively
in the MA gland. They are not homologuous to described
spidroins (Kono et al., 2019). A new SpiCE-NMa1 (SpiCE
Nephilinae Major Ampullate) was reported in Nephilinae
spiders, which are nonhomologous to those of A. ventricosus.
Composite lms of recombinant MaSp proteins and SpiCE-
NMa1 were produced to investigate the role of these new low-
molecular-weight components. It was reported that these
elements can increase the tensile strength of the composite
lm 2-fold. SpiCE contributes to the interaction between
TABLE 2 | Different types of spider silk proteins and their properties.
Silk type Role of silk in
web
Protein Molecular
mass
Number of
amino acid
Spider species Motifs References
Dragline Framework of
the web
MaSp1 >250 3130 Latrodectus
hesperus
-GGX (X = A, Q, or Y) Ayoub et al. (2007)
-GX (X = Q, A, or R)
-poly-A
Dragline Framework of
the web
MaSp1s 40 439 Cyrtophora
moluccensis
-GGX (X = A, Q, or Y) Han et al. (2013)
-GX (X = Q, A, or R)
-poly-A
Dragline Framework of
the web
MaSp2 >250 3780 Latrodectus
hesperus
-GPX (X = G or S) Ayoub et al. (2007)
-QQ
-GGX (X is usually A)
-GSG
-poly-A
Minor
ampullate (MI)
Auxiliary spiral to
stabilize the scaffold
MI >250 1766 Araneus
ventricosus
-GGX Chen et al. (2012)
-GGGX
-GX
-PolyA
-spacer
Flagelliform Capture spiral of the
orb web
Flag >250 2451 Nephila clavipes -GPGGX dos Santos-Pinto et al. (2018);
Hayashi and Lewis (1998);
Hayashi and Lewis (2000)
-GGX
-Spacer motif
Pyriform Attachment of silk
bres to surface
PySp1 400 3977 Araneus
ventricosus
-QQ containing motif Wang et al. (2019)
-Proline-rich regions
-N-linker
Pyriform Attachment of silk
bres to surfaces
PySp2 212 2155 Araneus
ventricosus
-QQ containing motif Wang et al. (2020)
-Proline-rich regions
Aciniform Prey wrapping and
inner silk of egg sac
AcSp1 330 3445 Araneus
ventricosus
Motifs are long and complex.
poly-A, GGX, GPX and poly-
GA are not present
Wen et al. (2018)
Aciniform Prey wrapping and
inner silk of egg sac
AcSp2 476 4746 Araneus
ventricosus
Motifs are long and complex.
poly-A, GGX, GPX and poly-
GA are not present
Wen et al. (2020)
Tubuliform Egg sac TuSp1 180 1921 Araneus
ventricosus
-A
n
,S
n,
SA
n
, AX, (SG)
n
Wen et al. (2017)
-linker
Aggregate Glue of capture
spiral
AgSp1 4501,400 14090 Argiope
trifasciata
-GPXG at the beginning of
subgroups
Stellwagen and Renberg (2019)
-The tail regions contain GGQ,
PGG, GPG and QGP motifs
-QQ motifs
Aggregate Glue of capture
spiral
AgSp2 20774 Mastophora
phrynosoma
-Stellwagen and Burns (2021)
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356374
Ramezaniaghdam et al. Recombinant Spider Silk
repeat regions, preferentially in the amorphous region rather than
in crystalline structure. These intermolecular interactions lead to
a decrease in the molecular weight between crosslinking points,
and thus the strength and modulus of the articial lm are
enhanced (Kono et al., 2021). New elements found in spider
silk can conrm it as a multicomponent material, which is more
complex than expected previously (Kono et al., 2019;Kono et al.,
2021).
FIGURE 2 | Schematic modular structure of different spidroins. aa, amino acid; G, glycine; C, cysteine; NR-NTD, non-repetitive N-terminal domain; NR-CTD, non
repetitive C-terminal domain; SP, signal peptide; RR, repetitive region; ERU, ensemble repeat unit; SG, subgroup.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356375
Ramezaniaghdam et al. Recombinant Spider Silk
Minor ampullate silk (MI) is used by spiders to make the
auxiliary spiral of the orb-web to stabilize the scaffold (Figure 1).
MI silk plays a similar role in the spider web as MA silk but does
not achieve its high biophysical properties (Chen et al., 2012). The
analysis of the MiSp gene shows the presence of an unusually
large intron of 5628 bp. Generally, intron length and expression
level are negatively correlated (Castillo-Davis et al., 2002;Urrutia
and Hurst, 2003;Marais et al., 2005). However, MiSp genes are
likely highly expressed. Proteins of this silk (MiSp) have
molecular masses of over 250 kDa (Table 2). Minor ampullate
protein sequences in A. ventricosus comprise a non-repetitive
N-terminal domain, one N-linker, three repetitive regions, two
non-repetitive spacer regions, one C-linker and a C-terminal
domain (Chen et al., 2012)(Figure 2). The N-terminal region of
MiSp in solution at pH 7.2 contains ve helices. Cys25 and Cys96
form an intramolecular disulde bridge. Key residues for pH-
dependent dimerization in the N-terminal domain of MiSp were
considered as Glu76, Glu115, and Glu73, which are different from
those of MaSp1. However, the monomer-to-dimer conversion
has the same mechanism in MaSp and MiSp spidroins. An anti-
parallel homodimer structure is seen in the ve-helix of this
region at pH 5.5 (Otikovs et al., 2015). There is no cysteine in the
C-terminal domain of A. ventricosus MiSp. The C-terminal area
can dimerize via hydrophobic interactions. MiSp has three
repetitive regions, which are interrupted by two non-repetitive
spacer regions. Each repetitive region can be categorized into four
types of ERUs [GGX-GGX-GX, (GX)
n
oligoA-(GX)
n
, GGX-
GGX-GGGX and (GX)
n
] iterating in a non-regular manner.
Reoccurring overall patterns are seen in the repetitive region.
However, this protein, in comparison with L. hesperus MaSp1,
lacks higher order organization. Repetitive regions are dominated
by polyA, GGX, GGGX and GX motifs. There is no proline in this
structure, which is why this silk does not supercontract in water.
There are two spacer regions having 100% identity even at the
nucleotide level. These spacers have no identity to any proteins
except other spacer regions in the proteins of MiSp or Flag. The
spacer region is not repetitive but only has a single tandem repeat
(AAASS). Spacer regions are predicted to contain α-helices (Chen
et al., 2012). The roles of these regions are not well characterized
but it has been hypothesized that they help to form bres
(Vienneau-Hathaway et al., 2017)(Table 3).
Flagelliform silk has the highest extensibility among all silks
produced by orb-weaver spiders and is used as the capture spiral
of the web (Figure 1). This silk is not as strong as dragline silk, but
it is multiple times more extensible. This bre can be stretched to
250% (Gosline et al., 1999;Rising and Johansson, 2015)(Table 1)
and dissipates the impact energy of prey. As an example, a honey
bee with ight velocity of 3.1 m/s and 120 mg body weight crashes
into the web with a 0.55 mJ kinetic energy. Flag silk (with 1m
diameter) can withstand that massive impact. This outstanding
resilience helps the spider to catch a prey even bigger than itself
(Römer and Scheibel, 2008). Therefore, this silk could have an
application for dampening vibration in material development
(Hauptmann et al., 2013b).
The Flag gene is one of the longest spidroin genes, it
comprises a total exonic region of 22.5 kb in the A.
ventricosus genome (Kono et al., 2019). The corresponding
protein of this species has not been characterized yet. In
TABLE 3 | Different motifs in spider silk proteins and their roles in spider silk.
Motif Secondary structure Structural
role of motif
Mechanical properties References
Poly-A β-sheet Crystalline Tensile strength Van Beek et al. (2002);Yarger et al. (2018)
GGX GX α-helix Amorphous Elasticity Gray et al. (2016);Yarger et al. (2018)
GPX GPGGX Type II β-turns Elastic Elasticity, toughness and
supercontraction
Hayashi and Lewis (1998);Ohgo et al.
(2006);Jenkins et al. (2010)
QQ-containing
motif
α-helix or β-sheet Self-aggregation of proteins into
bres
Wang et al. (2019)
(PX)
n
Random coil —— Wang et al. (2019);Zhu et al. (2020)
Spacer α-helix Helps to form bres or contribute to
the strength of bres
Vienneau-Hathaway et al. (2017)
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356376
Ramezaniaghdam et al. Recombinant Spider Silk
contrast, with mass spectrometry, the sequence and domains
of the Flag protein of Nephila clavipes were identied. It
contains an N-terminal domain, three modules of repetitive
regions, two spacers in each repetitive region and a C-terminal
domain (Figure 2). N-terminal and C-terminal regions have
three α-helices and one small helical section, respectively (dos
Santos-Pinto et al., 2018). Motifs of repetitive regions are
GPGGX and GGX. GPGGX likely forms type II β-turns,
which may form βspirals. It is hypothesized that this
spring-like helix is the basis for the elasticity of spider Flag
silk (Hayashi and Lewis, 1998)(Table 3). Some studies show
amorphous shapes without crystalline structure in the
repetitive regions (Römer and Scheibel, 2008). It is
suggested that abundance of proline residues prevents
forming crystalline β-sheet (Ohgo et al., 2006). However,
the presence of polyglycine II nanocrystals was
demonstrated in the agelliform silk of A. trifasciata (Perea
et al., 2013). This silk does not contain a polyA motif providing
the strength of dragline silk. Each repetitive region contains
two spacers with 100% similarity in their amino acid
composition. Spacers have charged amino acids which are
assumed to contribute to the strength of bres by crosslinks
between Flag proteins (Hayashi and Lewis, 1998;Adrianos
et al., 2013;dos Santos-Pinto et al., 2018).
Previously, gene and mRNA length of Flag in N. clavipes were
estimated at 30 and 15.5 kb, respectively. The ag gene has 13
exons. The corresponding protein was reported to have three
motifs: GPGG(X)
n,
GGX and 28 spacers. Iterations of these three
motifs are organized into complex ensemble repeats. Each
ensemble repeat is encoded by a single exon. The exons have
similar lengths (~1,320 bp) (Hayashi and Lewis, 1998;Hayashi
and Lewis, 2000).
The rst genome of N. clavipes was sequenced to investigate
spidroin genes. The authors catalogued a collection of 28
spidroins and new repetitive elements. This collection of data
was complemented by expression proling of the silk gland and
shows a diversity of spidroin genes (Kono et al., 2021). A new
agelliform gene called FLAG-b was found in the N. clavipes
genome. Transcripts of FLAG-b are highly abundant in venom
glands. Previously, two spidroin-like proteins, SmSp1 and
SmSp2b, were found in the venom gland of the velvet spider.
These ndings suggest that FLAG-b can be a new type of venom
gland-expressed spidroin (VeSp) evolving roles beyond silk-
related functions. Proteomic studies are needed to investigate
whether this protein is in the venom gland. Characterization and
functional identication of this protein may open a new venue for
using spidroins in human medical applications (Babb et al., 2017).
Pyriform silk forms attachment disks used as cement by
spiders (Figure 1). The components of this silk are dry bres
and wet glue, both produced by the same gland. The glue dries
immediately and forms a hardened disc. This glue silk is used by
spiders to join different bres or attach dragline to surfaces
(Geurts et al., 2010;Wolff et al., 2015). This silk consists of
two proteins, PySp1 and PySp2. PySp1 has a molecular mass of
400 kDa. In A. ventricosus it comprises ve regions, a non-
repetitive N-terminal domain, a long N-terminal linker, a
central largely repetitive region, a short C-terminal linker, and
a non-repetitive C-terminal domain (Figure 2). The presence of a
signal peptide cleavage site in the N-terminal region was
predicted. All silk proteins need to go through the ER and the
secretory pathway and the signal peptide paves this way. In the
N-terminal region 5 α-helices were predicted. In contrast, the
C-terminal region contains 4 α-helices. Two N-terminal cysteines
were detected between helix 1 and 2, and in helix 4. PySp1
exhibits two linkers, a long N-linker and a short C-linker. The
N-terminal linker consists of two types of repeats,
QQQYEXSQASIA and QQQYXXSQQQASIX. This linker
contains 5 α-helices and is hypothesized to control the protein
to form bre or glue through self-assembly. The core repetitive
region of PySp1 spidroin is made up of sixteen remarkably
homogeneous units. Two motifs are seen in this region,
proline-rich motifs (PXPXP) and QQ-containing motifs. The
(PX)
n
motifs seem to form random coils and QQ-containing
regions form α-helix or β-sheet conformations. Glutamin
segments seem to have a role in spidroin self-assembly (Wang
et al., 2019;Zhu et al., 2020)(Table 2 and 3).
The A. ventricosus PySp2 has a molecular mass of 212 kDa and
lacks the long linker regions. PySp2 has a more complex core
repetitive region than PySp1. This protein has seven repetitive
regions which can be classied into four types: The repetitive
regions 2, 4, and 6 have the same repeats (containing QQ, QX
and A
n
). The repetitive regions 3 and 5 are also similar in repeat
sequences (Figure 2). The repetitive region 7 is the shortest one
lacking glutamine and may perform specic functions. The
three regions 2, 4, and 6 may contribute to PySp2
aggregation or self assembly due their QQ-containing regions
(Wang et al., 2020).
Aciniform silk acts as prey wrapping and forms the inner silk
of the egg sac (Figure 1). This silk is one of the toughest silks,
367 MJ/m
3
(megajoule per cubic meter), among seven silks
(Table 1). Aciniform silk protein (AcSp1) has a calculated
mass of ~330 kDa with 3445 aa (Table 2). Similar to Pyriform,
this protein is composed of ve regions, a non-repetitive
N-terminal domain, a N-terminal linker, a central largely
repetitive region, a short C-terminal linker, and a non-
repetitive C-terminal domain (Figure 2). The N-terminal
region has a signal peptide with 23 aa. The analyses show ve
α-helices and two cysteines in the locations corresponding to
helix 1 and 4. The non-repetitive C-terminal domain has four α-
helices. Two terminals of AcSp1 from A. ventricosus are
homologous to that of other spidroins and species. The
repetitive region contains 15 iterated repeats units. The rst 14
repeats have the same length (230 aa) and the last one is 197 aa
long. There are ve α-helices in each repeat. Repeat units are
highly conserved, and several repeats are 100% identical to each
other. In the alignment of 15 repeats, only 59 sites are variable.
Most of the variation belongs to the rst repeat unit. Amino acid
motifs such as poly-A, GGX, GPX and poly-GA, which are
abundant in dragline, Flag and MiSp proteins, are not present
in the AcSp1 protein (Wen et al., 2018).
Recently, a second type of protein (AcSp2) was identied in A.
ventricosus with a molecular mass of 476 kDa. This protein has
4746 aa composed of three regions: an N-terminal region with a
predicted signal peptide, a core repetitive region comprising 25
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356377
Ramezaniaghdam et al. Recombinant Spider Silk
repeat units with extreme intragenic homogenization and a
C-terminal region (Wen et al., 2020).
Tubuliform (cylindriform)silk forms the outer shell of the
egg case (Figure 1). This is the only silk produced during a
specic period of the female spiders life, the reproductive season
(Tian and Lewis, 2006). This silk is robust and able to protect
offspring from predators, temperature uctuation and parasitoid
invasion. Tubuliform silk protein (TuSp1) is predicted to have a
molecular mass of 180 kDa (Table 2). TuSp1 in A. ventricosus,
like PySp1, has ve regions, a non-repetitive N-terminal domain,
a short N-terminal linker, a central repetitive region, a short
C-terminal linker, and a non-repetitive C-terminal domain
(Figure 2). The N-terminal domain has ve α-helices, while
there are ve α-helices and three βstrands in the C-terminal
region (Wen et al., 2017). In the N-terminal domain two
conserved cystein residues were identied in helix 1 and helix
4. The core region of this protein is dominated by nine tandem
repeats. These repeats are highly conserved, >90% identical at
amino acid level. Typical motifs such as polyA, GGX, GX and
QQ, which were identied in other spidroins, are not seen in the
TuSp spidroins. Instead, common sequence motifs in the TuSp
repetitive sequence are A
n
,S
n
,SA
n
, AX and (SQ). Pyriform,
aciniform and tubuliform spidroins have long and complex
repeats. Analysis of repeat regions across species demonstrates
extreme homogeneity of intragenic repeats in the proteins of
these three silks (Ayoub et al., 2013;Wen et al., 2017).
Aggregate silk is a kind of glue to aid in prey capture
(Figure 1). This glue comprises two proteins, AgSp1 and
AgSp2, which are modied members of the spidroin family
but they are not spun into bres (Collin et al., 2016). The
predicted mass is 4501,400 kDa (Tillinghast et al., 1992)
(Table 2). In A. trifasciata, AgSp1 has 14,090 amino acids and
consists of an N-terminal region, N-terminal repeats (NRP), an
N-terminal transition (NTT, a region with degenerate, repeat-
similar structure), repetitive regions, a C-terminal transition
(CTT) and a C-terminal region (Figure 2). N- and C-terminal
regions are conserved across spidroins. Following the N-terminal
region, there is a short region (586 aa) including repetitions of
TGSYITGESGSYD. In the repetitive region two similar distinct
motifs were predicted. This region includes 43 iterations of repeat
motif 1 (129 aa) and 38 iterations of repeat motif 2 (113 aa).
Repeat motifs contain four subgroups (SGs) and a variable tail
region. Each subgroup begins with GPXG. The tail region
contains GGQ, PGG, GPG and QGP motifs and poly-
threonine stretches on both ends. The C-terminal transition
region has the same organization as the internal repeats
(Stellwagen and Renberg, 2019).
Mastophora phrynosoma, also called bolas spider, uses a
fascinating hunting technique to capture moths. Females apply
a single and large droplet of glue, which is suspended at the end of
the silk thread. The bolas spider AgSp2 is the longest spidroin
discovered so far. A massive intron of 31.5 kb was predicted
within the gene. AgSp2 spidroin from this orb weaver spider has
20,774 amino acids (encoded by nearly 62 kb of genomic DNA).
It remains a mystery what functions this long length has. This
protein is comprised of ~47 repeats and does not have glutamine-
rich regions seen in the other reported AgSp2 of classic orb
weaving species (Stellwagen and Burns, 2021). Glutamine was
hypothesized to promote self-aggregation of spidroins into bres
(Geurts et al., 2010). Previously, two short aggregate proteins
TABLE 4 | Detected/predicted PTMs on spider silk proteins.
Silk/
protein
Species Detected/predicted PTMs Detection method References
Phosphorylation Hydroxylation O-glycosylation N-glycosylation
Dragline N. clavipes Glycosylation was conrmed.
However, the pattern of glycosylation
and the type of spidroin (MaSp1 or
MaSp2) were not identied
Concanavalin A Sponner et al.
(2007)
Spidroin1 N. clavipes The major PTMs in
spidroin-1
yes N.D. N.D. Gel-based MS strategy
involving CID and ETD
fragmentation
dos Santos-Pinto
et al. (2014)
N. edulis 8 sites on S, Y
N.
madagascariensis
2 sites on S, Y
4 sites on S, Y
Spidroin2 N. clavipes 36 sites on S, Y, T N.D. N.D. Gel-based MS strategy
involving CID and ETD
fragmentation
Santos-Pinto et al.
(2016)
Aggregate A. aurantia Yes Gas-LC Tillinghast et al.
(1992)
Aggregate N. clavipes Yes Yes Choresh et al.
(2009)
Aggregate A. trifasciata Yes Yes Stellwagen and
Renberg (2019)
Flag N. clavipes on Y 45 hydroxylated P
residues
N.D. N.D. NanoLC-ESI-CID/
ETD-MS
dos Santos-Pinto
et al. (2018)
The recognition
motif is GPGGS
PTMs, post translational modications; S, serine; Y, tyrosine; T, threonine; P, proline; LC, liquid chromatography; ESI, electrospray ionisation; CID, collision-induced dissociation; ETD,
electron-transfer dissociation; MS, mass spectrometry; N.D., no data.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356378
Ramezaniaghdam et al. Recombinant Spider Silk
were reported with 407 and 715 aa in Nephila clavipes (Choresh
et al., 2009).
Post-Translational Modications of Spider
Silk Protein
Post-translational modications (PTMs) are covalent modications
that change the properties of proteins. This chemical event ranges
from enzymatic cleavage to adding a chemical group such as glycosyl,
phosphoryl, acetyl or methyl. PTMs have important roles in the
structure and function of proteins (Ramazi and Zahiri, 2021). By
Concanavalin A, it was found that the core and shell of dragline silk in
N. clavipes is glycosylated (Sponner et al., 2007). Phosphorylation sites
within the proteins of dragline silk (MaSp1 and MaSp2) were
identied by mass spectrometry (dos Santos-Pinto et al., 2014;dos
Santos-Pinto et al., 2015;Santos-Pinto et al., 2016)(Table 4). Protein
glycosylation is responsible for the adhesive qualities of aggregate
glue. It is estimated that more than 80% of threonine residues in
aggregate proteins are O-glycosylated (Tillinghastetal.,1992). In the
rst three subgroups of the AgSp1 protein high serine/threonine
regions are seen which are likely glycosylated (Stellwagen and
Renberg, 2019). Aggregate proteins in N. clavipes are reported as
glycosylated proteins. Multiple O-glycosylation and one possible
N-glycosylation site were predicted in these proteins (Choresh
et al., 2009). Moreover, Flag proteins present PTMs such as 45
hydroxylated proline residues as well as phosphorylation and
nitrotyrosination sites (Table 4). Since these hydroxylation
residues are located in the GPGGX motifs, this may explain the
mechanoelastic property of these bres (dos Santos-Pinto et al.,
2018). These PTMs may cause changes in protein conformation
and thus inuence the properties of the proteins and interactions with
other proteins as well as storage and self-assembly of silk proteins
(Heim et al., 2009;Santos-Pinto et al., 2016). There is still no
information about the recognition motifs and glycosylation
patterns on spider silk proteins. Due to the lack of
knowledge on spidroin PTMs, the PTM system of a
production host might not t the needs of the spider protein
molecules (Peng et al., 2020). Future studies of spider silk
proteomes can provide more details concerning PTMs of
spidroins contributing towards a better understanding of
their effects on mechanical properties, bre assembly and
solubility as well as selection of appropriate expression hosts.
Limitations of Spider Silk Production
Milligram amounts of natural dragline silks can be harvested by
forcibly milking spiders, or by collecting the egg sacs to retrieve
tubuliform silks. However, this process is expensive and time-
consuming. For example, one million N. madagascariensis
spiders and more than 70 working individuals are needed to
make 3.4 m textile from natural dragline silk, with an estimated
cost of over $500,000 (Vierra et al., 2011). Moreover, the
cannibalistic nature of most spiders make them unsuitable for
livestock breeding. Consequently, recombinant production of
spider silk spidroins attracted interest in research. Different
production hosts such as bacteria, yeasts, insects, mammalian
cells, animals and plants have been used to produce
recombinant spider silk proteins. Since these proteins are
very long with a multitude of highly repetitive sequences,
and therefore difcult to express in full length, currently the
main strategy is to design and produce chimeric spidroins.
Recombinant spidroins after production are spun into bres
through different methods (reviewed by Belbeoch et al., 2021;
Koeppel and Holland, 2017). Besides other challenges, which we
discuss below, a rigid down-stream processing is necessary
TABLE 5 | The lengths of complete cds of spidroins genes with their accession numbers in the NCBI.
Accession number Spider species Silk type Gene Intron Length of cds (bp or kb)
M37137 Nephila clavipes Dragline MaSp1 No Partial cds, 2247 bp
EF595246 Latrodectus Hesperus Dragline MaSp1 No Complete cds, Single exon, 9390 bp
KF032719.1 Cyrtophora moluccensis Dragline MaSp1s No Complete cds, 1,320 bp
EF595245 Latrodectus hesperus Dragline MaSp2 No Complete cds, Single exon, 11340 bp
JX112872 Argiope bruennichi Dragline MaSp2 No Complete cds, 10083 bp
U47855.1 Araneus diadematus Dragline ADF3 No Partial cds, 1911 bp
U47856.1 Araneus diadematus Dragline ADF4 No Partial cds, 1233 bp
JX513956 Araneus ventricosus Minor ampullate MI Yes, 5628 bp Complete cds, 5440 bp (after removal of the single intron)
AF027972 AF027973 Nephila clavipes Flag Flag Yes Partial cds
AH009146
KY398016.1 Argiope argentana Pyriform PySp1 No Complete cds, 17277 bp
MH376748 Araneus ventricosus Pyriform PySp1 No Full length, 11931 bp
MN704282 Araneus ventricosus Pyriform PySp2 No Complete cds, 6468 bp
MG021196 Araneus ventricosus Aciniform AcSp1 No Complete cds, 10338 bp
MT078766 Araneus ventricosus Aciniform AcSp2 No Complete cds, 14238 bp
MF192838 Araneus ventricosus Tubuliform TuSp1 No Complete cds, 5763 bp
MK138561.1 Argiope trifasciata Aggregate AgSp1 Yes, 6690 bp Gene sequence: ~49 kb
Complete cds: 42270
Mastophora phrynosoma Aggregate AgSp2 Yes, ~ 37.5 kb Gene sequence: over 100 kb
Complete cds: ~62 kb
EU780014.1 Nephila clavipes Aggregate AGS1 Complete cds, 1221 bp, 2145 bp
Eu780015.1 AGS2
bp, base pair; kb, kilo base; cds, coding sequence.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 8356379
Ramezaniaghdam et al. Recombinant Spider Silk
whenthesilksareintendedtobeusedasimplantable
biomaterial (Decker, 2018).
Bacterial Systems
Escherichia coli is one of the most used systems for recombinant
protein production. E. coli cells have rapid growth, high
productivity, potential for scale-up production and low
production cost (Chen, 2012). Consequently, bacteria were
used to produce recombinant spider silk proteins
(Bhattacharyya et al., 2021). However, low expression level was
the most prominent issue which might be due to inefcient
transcription and translation. This problem was attributed to
the repetitive core domain of sequence resulting in the high
demand for glycyl-tRNA. The lack of tRNA or codon choices may
cause premature translation termination (Fahnestock and Irwin,
1997b;Xia et al., 2010). To overcome this issue, a metabolically
engineerd E. coli, in which the glycyl-tRNA pool was elavated, could
express the 284.9 kDa protein of N. clavipes. A repeat motif 34 bp
from MaSp1 partial cds of N. clavipes (accession number M37137)
(Table 5) was used in this research. The yield was 1.2 g/L after
purication, and the tenacity, elongation and Youngsmoduluswere
similar to those of N. clavipes dragline silk bres (Xia et al., 2010).
To produce spider silk protein with longer size in E. coli, the
split inteins mediated ligation technique was utilized. DNA part
assembly allows for repeat motifs assembly by digestion and
enzymatic ligation. A single repeat unit from MaSp1 was used to
create up to 192-mer protein. Proteins with a molecular mass of
556 kDa were created at a yield of 2 g/L or 63 mg/g cell dry weight.
Tensile strength and modulus were estimated from 1.03 ± 0.11
GPa to 13.7 ± 3.0 GPa, respectively. These recombinant proteins
have similar mechanical properties as their natural counterparts.
Fibre to bre variation likely comes from genetic instability
(Bowen et al., 2018) which affects nal mechanical properties.
These recombinant proteins do not possess N- and C-terminals,
which may be the reason for shorter bres. To dissolve spidroin
powders, hexauro-2-propanol (HFIP) was used which is a harsh
reagent and is not a long-term sustainable approach (Zhang et al.,
2019). Recombinant MaSp1s proteins with a yield of
300400 mg/L of induced culture medium were produced in
E. coli BL21 (Thamm and Scheibel, 2017). Forming inclusion
bodies (IBs) is one of the problems in the production of
recombinant spider silks in E. coli. Often, harsh conditions are
used to solve this problem, for example dissolving proteins in a
high concentration of urea or guanidine hydrochloride which
often results in the poor recovery of bioactive proteins. In order to
solve this problem, a mild solubilization strategy was used
including a one-step heating method in the presence of low
concentration of urea (Cai et al., 2020). Extensive purication
is another limitation of using this host. Ammonium sulfate
precipitation is inconvenient and time-consuming, and nickel
columns are too expensive for large scale production and
purication.
Sustainable cell factory platforms are being developed due
to the awareness of climate change, food and water crises and
depletion of fossil resources. These platforms should be able to
employ sustainable bioprocesses and depend solely on
renewable non-food bioresources as feedstocks. For the rst
time, an MaSp1 protein was produced using photosynthetic
and halophilic bacteria under sea water conditions. A purple
nonsulfur bacterium, Rhodovulum suldophilum, has been
developed as a potential alternative platform to replace
heterotrophic microbial cells. Biological contamination risk
could be decreased due to the capacity to grow under seawater.
Some challenges still need to be resolved, for example the
demand for glycine and alanine tRNAs, and genetic stability of
constructs (Foong et al., 2020).
The combination of motifs of different types of spider silk
proteins was reported recently. To expand mechanical
properties of recombinat spider silk bres, two libraries of
genes from L. hesperus were created. Library A and B consists
of masp1, masp2, tusp1, acsp1 and acsp1, pysp1, misp1, ag
respectively.Random ligation of different spidroin genes from
library A or B resulted in new proteins with new mechanical
properties. Higher elastic moduli were seen in the samples
from library A compared to those of library B. In comparison
with natural silk proteins, both libraries had higher elastic
moduli (Jaleel et al., 2020). In other research, a chimeric
protein of Flag-AcSp1 was expressed in E. coli with a
molecular mass of 36.8 kDa. The mean diameter of bres
was estimated to be 1m. These bres have a toughness
of ~33.1 MJ/m
3
and a tensile strength of ~261.4 MPa (Tian
et al., 2020)(Supplementary Table S1). However, all bacterial
systems lack the capacity for proper PTMs on the recombinant
proteins and thus may limit their use.
Yeast, Insect Cell Line and Bombyx mori Silkworm
Pichia pastoris is another organism that has been used to express
dragline silk spidroins and considered as a proper replacement to
E. coli when it comes to efcient production. Additionally, this
yeast can secrete the recombinant proteins (Heidebrecht and
Scheibel, 2013). The rst report on expressing spidroin1 in Pichia
pastoris GS115 is related to Fahnestock and Bedzyk (1997b)
(Supplementary Table S1). This host cell was also used to
produce the 2E12 protein, a 113.6 kDa analogue of MaSp2
from Nephila madagascariensis (Bogush et al., 2011).Earlier,
successful production of 1F9, an analogue of MaSp1, which
encodes a 94 kDa protein in Saccharomyces cerevisiae was
demonstrated and the tensile strength of 0.10.15 GPa and
elasticity of 515% were measured (Bogush et al., 2009).The
production level was 450 mg/L in Saccharomyces cerevisiae
(Sidoruk et al., 2015). Some challenges are identied with
using P. pastoris such as poor expression in shake asks and
the need for bioreactors, proteolysis and self-assembly in vivo
(Werten et al., 2019).
Insect cells have also been taken into consideration for
research studies of spider silk proteins production. The most
important reason is that the evolutionary distance between
spiders and insects is relatively small. Cell line Sf9, derived
from the fall armyworm Spodoptera frugiperda, was employed
to express two dragline proteins of Araneus diadematus,
ADF3 and ADF4, targeted to the cytosol. Spidroin proteins
of 60 kDa were reported in the cytosol with 5 mg/L of insect
cell culture. This research showed coiled laments forming
within the cytoplasm. These laments ranged in diameter
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 83563710
Ramezaniaghdam et al. Recombinant Spider Silk
from 200 nm to 1µm, and the length was up to 100 µm. Since
the length of laments was too short due to cell size limitation,
mechanical force measurement failed (Huemmerich et al.,
2004). Moreover, complicated cloning steps and time-
consuming regeneration were drawbacks (Heidebrecht and
Scheibel, 2013).
Silkworms are good candidates for producing recombinant
spider silk as they are able to spin spider silks into bres due to
their natural spinning apparatus. With the TALEN (transcription
activator-like effector nucleases) strategy, the silkworm broin
heavy chain gene was substituted with the MaSp1 gene (1.6 kb),
and transformed cocoon shells contained up to 35.2% MaSp1
protein (Xu et al., 2018). In addition, with CRISPR/Cas9
technology, spider silk proteins of native size were successfully
produced. The MaSp1 gene (6 kb) was incorporated into the
genome of Bombyx mori. The silkworm broin heavy or light
chain (FibH or FibL) intron (FibH) was replaced with the spider
silk gene. For insertion, the intron region was used to ensure that
any CRISPR/Cas9-induced sequence changes have no inuence
on protein production. FibH or FibL-spider silk bres generated
mechanical properties like natural silk (1.2 GPa). The transgenes
were stable through subsequent generations. This study shows the
feasibility of silkworms as a natural spinner for industrial
production (Zhang et al., 2019).
Mammalian Cell Lines and Transgenic Animals
Mammalian cells are alternative expression systems for the
production of recombinant proteins. Successful expression of
dragline silk proteins of N. clavipes (MaSp1 and MaSp2) and
A. diadematus (ADF3) was reported (Lazaris et al., 2002) in two
different cell lines: bovine mammary epithelial cells excelling at
secreting proteins outside the cell, and hamster kidney cells
adapting to produce large amounts of recombinant protein.
Both cell-lines secreted soluble proteins. The recombinant
production yield ranged from 25 to 50 mg/L. The data for
toughness, modulus and strain break was estimated as
0.640.85 gpd (gram per denier), 42.8110.6 gpd and
43.459.6% (Lazaris et al., 2002). In a further attempt, Nexia
Biotechnologies tried to produce these proteins in goat milk
(Vendrely and Scheibel, 2007). These goats produced MaSp1
and MaSp2 protein analogues with approximately 65 kDa
(Karatzas et al., 2007;Copeland et al., 2015), but the quantity
was very low (Vendrely and Scheibel, 2007)(Supplementary
Table S1). The silk protein purication process from transgenic
goat milk is long, expensive and inefcient. To increase
production, the TFF (Tangential Flow Filtration) process was
optimized, and spider silk proteins were recovered at
approximately 0.5 g/L (Yazzie et al., 2017). To increase the
purity and quantity of recombinant spider silk proteins, the
CRISPR/Cas9 system was used. The alpha-s2-casein gene
coding for the native milk protein in goat was replaced with
the MaSp1 gene (2046 bp). The average maximum stress was
2173 MPa (Decker, 2018).
Mice are other transgenic animals that produced MaSp1 and
MaSp2 proteins with a molecular mass of 40 kDa in milk,
however, the tensile strength was lower than that of natural
silk (Xu et al., 2007). Recently, the development of transgenic
sheep embryos was reported. Using a liposome-mediated method,
sheep broblasts were transfected with plasmids containing a
spidroin gene. The aim of this study was to produce
recombinant spidroins in hair follicles of sheep. The authors
could successfully develop the method, and pregnancy was
observed. However, no offspring was produced (Li et al., 2020).
Plant Systems
In tobacco leaves, recombinant MaSp1 and MaSp2 proteins were
produced with a molecular mass of 60.3 and 58.5 kDa. Both
proteins were targeted to the ER by means of PR1b (secretory
signal peptide from tobacco) and accumulated in this organelle
due to the ER retention signal KDEL. The data from Western
blotting showed the single bands for proteins in stable expression,
however, a protein ladder appeared for the transient expression of
MaSp1. Some assumptions such as premature termination,
unstable rearrangement of the T-DNA, endogenous plant
proteases, trypsin-like cleavage sites and protease activity
during leaf wounding were presented as an explanation.
Moreover, different codon usage would be another reason for
this protein ladder. The maximum yield of 0.0025 and 0.025%
total soluble protein (TSP) were estimated for MaSp1 and MaSp2
proteins, respectively. Two promoters were used to determine
their impact on protein yields. Transformed tobacco with tCUP
(tobacco cryptic constitutive promoter) produce less amounts of
MaSp1/MaSp2 than those plants with CaMV-35S (cauliower
mosaic virus) promoter (Menassa et al., 2004), and there is no
data for the mechanical properties of extracted proteins
(Supplementary Table S1).
In other research, the DPIB-8p proteins (synthetic analogue of
spidroin1 with a molecular mass of 64 kDa) were targeted to the
apoplast, ER lumen and vacuole by fusing the sporamin signal
peptide, sporamin propeptide, and KDEL peptide to investigate
the organelle potential and to enhance the accumulation of
recombinant proteins. The accumulation level in the apoplast
and ER of Arabidopsis leaves was 8.5 and 6.7% TSP, but retention
in the vacuole faced failure. In addition, spidroin proteins could
accumulate in ER and vacuole of Arabidopsis seeds to 18 and 8.2%
TSP (Yang et al., 2005). Previously, recombinant spidroins (64
and 127 kDa) were produced in seeds and leaves of Arabidopsis
and soy somatic embryos wihout taking protein targeting
approaches. The synthesis of 64 kDa DB1B monomers showed
equal molecular size, while minor by-products also occurred
during the 127 kDa DP1B synthesis. The yield in these plants
ranged from 0.03 to 1.2% TSP (Barr et al., 2004).
In order to produce larger recombinant proteins, the intein-
based multimerization technique was utilized. This resulted in the
production of Flag spidroin multimers longer than 250 kDa in the
ER of tobacco leaves. The yield was estimated at 1.8 mg/50 g leaf
material. In terms of physical properties, brillae with a diameter
ranging from approximately 12 µm and a length of up to 500 µm
were detectable (Hauptmann et al., 2013a). However, mechanical
analyses such as tensile strength and toughness were not
investigated. Post-translational multimerization in vitro is a
further way to produce spider silk multimers with a large size.
Transglutamination is a crosslinking of specic aa motifs
allowing for more or less multimerization of proteins. The
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 83563711
Ramezaniaghdam et al. Recombinant Spider Silk
K-MaSp1-100xELP (K: lysine-tagged, ELP: elastin-like
polypeptide) and Q-MaSp1-100xELP (Q: glutamine-tagged)
cassettes were engineered. After purication of monomers
Q-MaSp1-100xELP and K-MaSp1-100xELP, recombinant
microbial transglutaminase (rMTG) treatment in vitro was
performed to cross-link the monomers. This multimerization
resulted in fusion proteins of more than 250 kDa. Atomic force
microscopy (AFM) measured the elastic penetration modulus of
the samples. This value measures the stiffness of a solid material
(E value, GPa). Layers of recombinant spider silk fusion
FIGURE 3 | Schematic overview of spider silk protein production in Physcomitrella.
FIGURE 4 | Overview of the expression systems used in the production of recombinant spider silk protein.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 83563712
Ramezaniaghdam et al. Recombinant Spider Silk
monomers, such as multimers linked via Q-/K-tags, were
produced by casting (Weichert et al., 2014). Indeed, the
highest E value (3.29 ± 0.03) was measured for Q-/K-MaSp1-
100xELP cross-linked multimers compared to monomers of Q-
or K-tagged MaSp1. Thus, the stiffness increased with
multimerization (Weichert et al., 2014).
The same designed Flag gene was used to study the
dimerization of a Flag monomer via cysteines in the
C-terminus. The C-terminal domain of MaSp and Flag
contain one or two cysteines, which are thought to crosslink
the spidroins during assembly via disulde bridges. Nevertheless,
the results raised many questions. Half of the Flag monomers
were dimerized. It was uncertain whether dimerization by the
C-terminal domain affects bre formation (Hauptmann et al.,
2013b). To date, it has not been tested again in plant hosts.
The advantages of long-term storage recombinant spider silk
in tobacco seeds were studied. Here, protein multimers larger
than 450 kDa were synthesised by the intein-based
multimerization technique. The GPGGX and GGX motifs of
Flag partial cds (accession numbers AF027972 and AF027973),
LeB4 legumin signal peptide and ER retention signal KDEL were
used in the construct. The intein-mediated self-splicing and
ligation of proteins did not occur straight after translation.
Hence, high molecular Flag proteins appeared only after a
while. The study showed no decrease in the accumulation or
loss of multimerization of spider silk proteins in seeds over
8 weeks at 15°C with 49% humidity. Also, long-term storage
and expression of the synthetic Flag in seeds over two or three
generations were stable (Weichert et al., 2016). In comparison to
bacterial cells, the yield of 190 mg/kg obtained for USP-FIC
(unknown seed protein promoter-ag intein c-myc) expressed
in plants was low (Whittall et al., 2020). Distinct multimeric
bands were visible due to the protein multimerization process
with different sizes. Variable sizes of linear multimers,
multimerization of epitope tag as well as the lack of N and
C-terminal areas of Flag spidroin within the construct can be
described as limitations of that technique.
One of the important problems of spider silk protein
production in plant systems so far are low yields. For more
efcient purication, ELP (elastin-like polypeptide) repeats were
used to produce recombinant spider silk proteins in tobacco and
Solanum tuberosum. ELP consists of Val-Pro-Gly-Xaa-Gly (Xaa
is any amino acid except proline). The purication method for
ELP is named ITC (heat denaturing and Inverse Transition
Cycling) which is simple, scalable and inexpensive. ELPs are
water-soluble below a specic temperature and turn into
insoluble proteins when the temperature rises. Oligomeric
repeats of ELP were fused to the spider silk sequence and after
expression and purication, up to 400 mg spider silk proteins
could be isolated from 6 kg of tobacco leaves. This report is the
highest amount of spider silk proteins puried from plants
(Scheller et al., 2004;Hauptmann et al., 2013b;Heppner et al.,
2016).
Synthetic genes of 20006000 bp based on the sequence of
MaSp2 were expressed in Medicago sativa (alfalfa) leaves and
resulted in proteins of 80110 kDa. The yield was not determined
due to storage problems. The synthetic spidroins produced in
alfalfa did not freeze well, so the proteins were insoluble, making
extraction and purication impossible. Thus, pure recombinant
spidroins and measurable yields could not be achieved (Hugie,
2019).
Methods for the production of synthetic spider silk-like
proteins in corn endosperm or plant shoot tissue were
provided (Sylvester et al., 2015). In rice, recombinant spidroins
of 22 kDa were produced and could successfully reduce blood
glucose levels in diabetic mice. No data was reported for the
production yield in transgenic rice (Park et al., 2019)
(Supplementary Table S1). To date, except for MaSp1/MaSp2
and Flag, no other spider silk protein has been recombinantly
produced in plant systems.
Alternative Production Systems
The global demand for recombinant proteins has lead to research
on transgenic microalgae as production hosts. Some features such
as rapid growth, stable transgenic transformation, cost-effective
production, scalable production as well as the ability to produce
complex proteins with PTMs are promising. Attempts were made
to produce a chimeric protein consisting of an antimicrobial
protein from a bacteriophage and a spider silk protein in
Chlamydomonas reinhardtii, which has a GC-rich genome,
and thus may be well suited to produce spider silk proteins.
The rationale was that recombinant spider silk proteins may
act as a support for other proteins. A proposed application for
these studies is the development of articial skin for burn
victims. However, the low yield of recombinant proteins (0.2%
TSP) produced in microalgae is a big challenge and hinders
commercial scale production. Several strategies including
codon optimization, development of vectors and using
proper promoter and terminator were presented to increase
the expression level (Spechtetal.,2010;Rasala and Mayeld,
2011).
Another alternative production platform could be the moss
Physcomitrella. Mosses are used in a wide variety of biotech
applications, from carbon capture in peatlands to cosmetics
(Decker and Reski, 2020). Physcomitrella especially has a
proven track record in molecular farming with several
candidate biopharmaceuticals being produced in this host
(Reski et al., 2015), in which glyco-engineering of PTMs is
possible due to precise and efcient genome engineering
(Decker et al., 2014). Transformation of protoplasts,
subculture and production in simple inorganic media devoid
of sugars, growth factors and antibiotics in Petri dishes,
Erlenmeyer asks and photobioreactors are well established for
this platform (Reski et al., 2018;Figure 3). Surprisingly, even
human complement factor H (FH) can be produced in
Physcomitrella (Büttner-Mainik et al., 2011) and is fully active
in pre-clinical trials (Michelfelder et al., 2017). This difcult-to-
express protein has a molecular mass of 155 kDa, several repeat
units and intra-molecular disulde bonds, and is heavily
glycosylated. In contrast to other plant systems, Physcomitrella
accepts a variety of animal sequences for protein production
(Gitzinger et al., 2009) and addition of additives or co-expression
of supporting proteins can stabilize the secreted protein product
(Baur et al., 2005). Moreover, a detailed analysis of the
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 83563713
Ramezaniaghdam et al. Recombinant Spider Silk
Physcomitrella genome and of expression patterns for human cDNAs
led to a codon-optimization tool that resulted in drastically enhanced
protein yield and purity for FH and for human blood-clotting factor
IX (Top et al., 2021), also making Physcomitrella a promising
candidate for the production of spider silk proteins.
Large amounts of recombinant spidroins are required for
spinning the fabrics, and the host system able to produce
spidroins on a large scale in the bioreactor is a logical choice
(Whittall et al., 2020). A recombinant spidroin containing only
the repetitive domain might have problems with solubility or
exhibit premature fold (Peng et al., 2016). However, this problem
could be circumvented by either adding native N- and
C-terminals as anking regions to improve solubility or by
fusing ELPs and targeted spidroin for selective precipitation
(Heidebrecht and Scheibel, 2013;Peng et al., 2016).
CONCLUSION AND OUTLOOK
Spider silks have attracted interest for many years due to their
superior properties in combination with biodegradability and
biocompatibility. As reviewed in this paper, many expression
systems (Figure 4) have been used to develop suitable production
systems for the efcient production of recombinant spider silk
proteins. The long length and highly repetitive nature of spider
silk genes make these attempts challenging. To overcome these
problems, chimeric genes have been used to produce chimeric
spidroins. With extensive metabolic engineering, the production
of large spider silk proteins could be possible in a bacterial system.
The solubility of spidroins produced in E. coli remains
challenging (Whittall et al., 2020). Some spidroins such as
dragline and aggregate are glycosylated proteins, however,
bacterial systems are not capable to produce such PTMs.
Plants are already in use for the production of enzymes,
carbohydrates, lipids (Ray and Behera, 2017), biodegradable
plastic-like compounds (Moire et al., 2003)andother
proteins such as collagen (Haagdorens et al., 2021). Plant
molecular farming can be integrated with material research,
for example, to produce next-generation vaccines. Instead of
using polymeric materials as nanocarriers, recombinant-
expressed VNPs (virus nanoparticles) can be used which
are very stable and can withstand temperatures outside
cold chain requirements (Chung et al., 2021). Plants offer
several advantages over conventional eukaryotic and
prokaryotic expression platforms. In comparison to
mammalian cell cultures, plants are safe and the risk of
contamination with human pathogens is low (Hauptmann
et al., 2013b;Buyel, 2019).Intermsofcostofgoods,plant
systems are generally more competitive (Dove, 2002). The
ability to produce correctly folded complex and
posttranslationally modied proteins is another benetof
plant-based expression systems compared to bacterial
systems (Hauptmann et al., 2013b). However, plant
expression systems for spider silk production have faced a
challenge (Chung et al., 2012). Recombinant spidroins have
yielded from micrograms to about 200 mg per kilogram of
plant tissue, which is still less than the commercially
acceptable level (15 g/kg). New production hosts and
strategies such as gene optimization, metabolic engineering
and purication methods may allow spider silk protein
production on an industrial scale.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material, further inquiries can be
directed to the corresponding author.
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct, and intellectual
contribution to the work and approved it for publication.
FUNDING
Funded by the Deutsche Forschungsgemeinschaft (DFG, German
Research Foundation) under Germanys Excellence Strategy
EXC-2193/1 390951807. We acknowledge support by the Open
Access Publication Fund of the University of Freiburg.
ACKNOWLEDGMENTS
We gratefully acknowledge funding by the Deutsche
Forschungsgemeinschaft (DFG, German Research Foundation)
under Germanys Excellence Strategy EXC-2193/1 390951807
(livMatS to MR and to RR). We thank Michal Rössler for the
artwork and Anne Katrin Prowse for language editing.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at:
https://www.frontiersin.org/articles/10.3389/fbioe.2022.835637/
full#supplementary-material
REFERENCES
Adrianos,S.L.,Teulé,F.,Hinman,M.B.,Jones,J.A.,Weber,W.S.,Yarger,J.L.,etal.
(2013). Nephila clavipes Flagelliform Silk-like GGX Motifs Contribute to
Extensibility and Spacer Motifs Contribute to Strength in Synthetic Spider Silk
Fibers. Biomacromolecules 14, 17511760. doi:10.1021/bm400125w
Altman,G.H.,Diaz,F.,Jakuba,C.,Calabro,T.,Horan,R.L.,Chen,J.,etal.(2003).Silk-
based Biomaterials. Biomaterials 24, 401416. doi:10.1016/s0142-9612(02)00353-8
Askarieh, G., Hedhammar, M., Nordling, K., Saenz, A., Casals, C., Rising, A., et al.
(2010). Self-assembly of Spider Silk Proteins is Controlled by a pH-Sensitive Relay.
Nature 465, 236238. doi:10.1038/nature08962
Ayoub, N. A., Garb, J. E., Kuelbs, A., and Hayashi, C. Y. (2013). Ancient
Properties of Spider Silks Revealed by the Complete Gene Sequence of the
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org March 2022 | Volume 10 | Article 83563714
Ramezaniaghdam et al. Recombinant Spider Silk
Prey-Wrapping Silk Protein (AcSp1). Mol. Biol. Evol. 30, 589601. doi:10.
1093/molbev/mss254
Ayoub,N.A.,Garb,J.E.,Tinghitella,R.M.,Collin,M.A.,andHayashi,C.Y.(2007).