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Engineered
Living
Materials
Engineered Living Materials
Wil V. Srubar III
Editor
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland
AG 2022
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
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Contents
v
Network Formation of Engineered Proteins
and Their Bioactive Properties
Seunghyun Sim
Abstract Proteins are one of the main components of the extracellular matrix in
natural biological materials. They confer a unique advantage in creating engineered
living materials (ELM) because they can be genetically encoded and rationally
designed for constructing bioactive network structures. Advances in the design,
characterization, and engineering of protein networks have been an important
multidisciplinary endeavor and should be considered when designing ELM and
understanding their behavior. This chapter describes the network-forming behavior
of recombinant proteins, as these proteins, in principle, can be genetically
programmed and synthesized directly from living cells residing in ELM. There are
three major classes of protein network-forming mechanisms relevant to this topic:
(1) phase separation and aggregation-induced recombinant protein networks,
(2) self-assembling multi-domain artificial protein networks, and (3) chemically
cross-linked recombinant protein networks. We will begin by introducing protein
hydrogels and discuss their mechanism of network formation, which is a critical
element in designing functionalities and mechanical properties of ELM. After
introducing the network-forming mechanisms in protein hydrogels, we will discuss
examples of bioactive protein networks equipped with various functionalities before
concluding with future directions and remaining challenges in this field.
1 Introduction
Natural biological materials have been a key inspiration in the broad scientific field
due to their unique ability to self-regulate, self-organize, self-heal, and respond to
complex environmental cues. We see this in bone, wood, skin, and biofilm, where
S. Sim (*)
Department of Chemistry, University of California, Irvine, Irvine, CA, USA
e-mail: [email protected]
living cells produce, regulate, and repair their own surrounding matrix. These
emergent properties result from the living cells functioning as an active component
in these materials and have yet to be demonstrated in man-made synthetic materials.
Inspired and motivated by this striking functionality and complexity of natural
biological materials, many efforts have been made in recent years to construct
engineered living materials (ELM) where living cells are the producers and modu-
lators of their surroundings. In nature, the main components of these networks made
by living cells are self-assembling proteins and exopolysaccharides. Although
exopolysaccharides produced by living cells are often the most abundant structural
component, engineering and production of designed networks are challenging as
they involve a variety of biosynthetic machinery that is not necessarily shared across
different species. Proteins, on the other hand, can be genetically encoded, rationally
designed for specific purposes, and ported between species. In addition, proteins are
an attractive building block for networks interfacing living cells from both functional
and structural aspects. They fold into a defined three-dimensional structure, can bind
specific partners even in a complex cellular environment, and form protein-protein
interactions. As a result, they can assemble into a higher-order network responsible
for the mechanical property of the ELM. Moreover, many functional protein motifs
are amenable to use in conjunction with structural units that undergo network
formation.
Self-assembled protein networks in the context of extracellular materials can be
classified as hydrogels, as they are extensively hydrated networks housing constit-
uent living cells. Therefore, throughout this chapter, we will use the terms “network”
and “hydrogel” interchangeably. Hydrogels are physically or chemically cross-
linked polymer networks that swell in water. Their ability to hold large amounts of
water stems from a delicate balance of good water solubility and interchain cross-
linking. Self-assembling proteins and aggregation-prone proteins constitute physi-
cally cross-linked hydrogel as they rely on the physical association of particular
structural motifs for the network construction. Proteins that form a covalent bond
between two separate domains upon association can serve as a building block for
chemically cross-linked hydrogels. Protein hydrogels have a significant advantage
over synthetic or bio-derived hydrogels in that one can rationally design protein
networks with specific functions and properties in mind, as protein structures can be
precisely engineered via genetic modification of DNA. For example, a protein
domain known to confer an appealing functionality can be rationally fused to
other protein domains that form a network. In addition, protein-based hydrogels
tend to be more biocompatible and biodegradable than synthetic polymeric
hydrogels. For these reasons, protein hydrogels have been extensively studied for
their utility in injectable delivery vehicles, implantable scaffolds for soft-tissue
engineering, and matrices for in vitro cell culture. Conformational changes of pro-
teins in response to temperature, pH, light, ligands, and mechanical force further
prompted the development of stimuli-responsive protein hydrogels with sensing
capability.
The main focus of this chapter is understanding the network-forming behavior of
proteins as a structural component in engineered living materials, as these proteins,
Network Formation of Engineered Proteins and Their Bioactive Properties 3
Proteins are sequence- and length-controlled linear chains of amino acids joined by
peptide bonds. This well-defined linear chain is the most basic structure of proteins,
also known as the primary structure. Proteins are synthesized by a sequential
biological process. First, the genetic information stored in DNA is transcribed into
a messenger RNA (mRNA). The second step is the translation of mRNA by
ribosomal catalysis into a linear polypeptide chain, transferring genetic information
written in a nucleic acid sequence into a protein sequence. It is possible to produce
recombinant proteins with a varying production yield by modifying the DNA of host
organisms and harnessing their transcription and translation machinery. Innovation
in synthetic and chemical biology gave birth to many tools to manipulate this
process, such as engineered ribosomal binding sites, split RNA polymerase, and
mutant tRNA synthetase. Notably, advances in co-translational incorporation of
noncanonical amino acids have allowed the expansion of native functionalities of
proteins, for example, introducing bio-orthogonal reactive handles and photo-
reactive moieties into recombinant proteins (Link et al. 2003).
Fig. 1 (a) A schematic illustration of an α-helix. It is a right-handed helix with 3.6 amino acid
residues per helical turn. Each backbone carbonyl oxygen in an α-helix is hydrogen-bonded to the
backbone amide hydrogen four residues down from it toward C-terminus. The side chains of the
amino acids (designated as R) extend outward from the helical backbone. (b) Schematic illustration
of a coiled-coil dimer and its helical wheel representation. Dimer formation is driven by hydro-
phobic interaction of residues at a and d positions. Residues at e and f positions can stabilize coiled-
coil dimers by electrostatic attractions. (c, d) Hydrogen-bonding networks between two β-strands to
form (c) parallel and (d) antiparallel β-sheet
four residues down from it toward the C-terminus, resulting in 3.6 amino acids per
one complete turn of the helix. While the amide backbone is hydrogen-bonded to
itself almost parallel to the helical axis, the side chains of each amino acid extend
outward from the backbone. The unique ability of α-helix presenting side chains of
its constituent amino acids in a regular fashion has attracted significant interest as a
model system for protein folding studies and a design template for creating protein
assembly. In particular, coiled-coil structures are formed when two or more α-helices
self-assemble (Fig. 1b). Depending on the relative orientation of helical axes, coiled-
coil forms either parallel or antiparallel arrangement. The primary structures of a
coiled-coil domain comprise heptad repeats—repetition of seven amino acids, often
represented as (abcdefg)n. In each helix in coiled-coil, hydrophobic amino acids,
including leucine and isoleucine, occupy every three or four residues (a and d ),
Network Formation of Engineered Proteins and Their Bioactive Properties 5
Motivated by the structural role they accomplish in nature, natural proteins have
been examined as building blocks for biomaterials in the form of hydrogels, films,
and others. In particular, those derived from extracellular matrices, such as collagen
and gelatin, were extensively studied in the context of in vitro tissue culture. As our
understanding of protein structure, recognition, and self-assembly deepens with the
aid of computational frameworks, engineering recombinant proteins offers the
unique opportunity to control network structure and functionality. Protein network
formation is driven either by non-covalent interactions between domains or by
covalent linkages between specific residues. The non-covalent interaction of proteins
in a network can be further classified into molecular recognition (self-assembly) and
aggregation. Although hydrogels made with short synthetic peptides also constitute
an important class of biomaterials, this chapter will only describe recombinant
protein networks and the resulting biomaterials. Considering the relevance of protein
network in constructing ELM, three important categories of recombinant proteins
Network Formation of Engineered Proteins and Their Bioactive Properties 7
Wright et al. 2002). These sequences were selected based on their phase transition
temperature, with the LCST of B being lower than that of A. At temperatures above
its LCST, end-block B undergoes aggregation, while the hydrophilic mid-block A
remains solvated, yielding physical and thermoreversible protein hydrogels. The
critical temperature and other variables affecting sol-gel transition can be engineered
through sequence variation of the triblock proteins. In a study by Chilkoti and
co-workers (Betre et al. 2002), recombinant ELP without explicit “more hydropho-
bic” aggregation domain forms a gel-like coacervate at 37 C, above its LCST
(35 C). The ELP coacervate showed three orders of magnitude increase in the
complex shear modulus and dynamic viscosity and exhibited similar mechanical
properties of the gels that were formed with cartilage extracellular matrix compo-
nents, suggesting their potential utility for cartilage tissue engineering. As described
in Sect. 3.3, ELP hydrogels have also been formed by chemical cross-linking
methods after varying the ELP guest residue to incorporate reactive moieties.
Silk is another natural protein that features impressive mechanical strength and
extensibility. For example, spider dragline silk forms robust and elastic fibers and is
three times tougher than synthetic bulletproof material Kevlar (Rising and Johansson
2015). Silk protein contains a repetitive sequence rich in glycine and alanine, which
forms β-sheet crystalline domains responsible for high mechanical strength and
hydrophilic amorphous regions. Physically cross-linked β-sheet-rich protein
hydrogels can be produced from naturally derived silk, such as silkworm fibroin.
This process involves chemical and mechanical perturbations, including low pH
(Fini et al. 2005), high temperature (Kim et al. 2004), and sonication (Wang et al.
2008). However, harvesting native silk from natural sources faces multitudes of
challenges, such as batch-to-batch variation, impurities, and difficulties in farming
particular silk proteins, especially spider silk. Successful recombinant productions of
silk proteins have been reported using bacterial (Xia et al. 2010), plant (Scheller et al.
2001), and mammalian (Lazaris et al. 2002) hosts. Although a few studies have
demonstrated hydrogel formation of recombinant silk proteins (Rammensee et al.
2006; Schacht and Scheibel 2011), most of the efforts to create biomaterials with
recombinant silk have focused on processing them into fibers, films, and foams. An
alternative approach involves a chimeric recombinant protein containing tandem
repeats of the silklike sequence GAGAGS as well as ELP domain (Megeed et al.
2002; Nagarsekar et al. 2003). The addition of ELP reduces the degree of crystal-
lization of the silklike domain and introduces flexibility and solubility. These pro-
teins spontaneously form physically cross-linked hydrogels due to crystalline
domains comprising the silklike region and show ELP-like properties, such as
temperature responsiveness.
Network Formation of Engineered Proteins and Their Bioactive Properties 9
Fig. 2 (a) Schematic illustration of coiled-coil domains. Intermolecular loop formation occurs and
competes with network formation in ACnA proteins that form antiparallel coiled-coil aggregates.
On the other hand, loop formation is suppressed in PCnP proteins with P domains that aggregates in
parallel orientation, as it requires chain stretching of the mid-block. (b) Shear-thinning and elastic
recovery of PCnP hydrogels. The shear storage modulus of PCnP hydrogels decreases upon
oscillatory strain and recovers to its original modulus within seconds (left). PCnP forms a shear-
thinning, yet self-supporting, gel (right). Reproduced with permission (Olsen et al. 2010). Copy-
right 2010, the American Chemical Society. (c) Schematic illustration of the mixing-induced,
two-component hydrogel (MITCH) where two domains in component 1 and 2 assemble via
molecular recognition. Reproduced with permission (Foo et al. 2009). Copyright 2009, the National
Academy of Sciences
Network Formation of Engineered Proteins and Their Bioactive Properties 11
Physical gel formation often requires exposure to high or low pH, temperature
change, or high ion concentration, which are not ideal from the cell or
biomacromolecule encapsulation perspective as they might lead to loss of function
or cell death. To circumvent this issue, Heilshorn and colleagues developed a peptide
association pair to trigger gelation upon mixing the two protein components
containing separate domains (Fig. 2d) and designated this system as mixing-induced,
two-component hydrogel (MITCH) (Foo et al. 2009). Domains suitable for this
purpose need to fulfill the three criteria that (1) the association domain sequence
must be short enough to be repeated in a single recombinant protein, (2) the domains
must not interfere with extracellular signaling machinery, and (3) the domain
association should be selective and tunable, and the WW and proline-rich domains
were chosen accordingly. The WW domain, named after the conserved tryptophan
residue, folds into antiparallel β-sheet structures and associates with the proline-rich
domain. A set of two artificial proteins for MITCH comprises several repeats of
either one of the domains separated by linker regions that form mostly random coils.
Two WW domains, CC43 and Nedd 4.3, were selected because they differ by an
order of magnitude in their association constants with a proline-rich domain (PPxY).
The sol-gel transition and the viscoelastic properties of the MITCH system can be
engineered by varying the stoichiometry and binding strength of the two components
(Mulyasasmita et al. 2011). Like other physical protein gels, such as PEnP, MITCH
hydrogels are shear-thinning, injectable, and self-healing. Li and co-workers have
reported a different two-component protein hydrogel with a pair of complementary
leucine zippers, CCE and CCK, that form heterodimeric coiled-coils at neutral pH
(Lv et al. 2012). Engineered bio-recognition by splitting a native protein has also
shown to be effective in creating physically cross-linked protein hydrogels, as these
split protein fragments spontaneously reconstitute the folded conformation of the
native protein. The loop elongation variant of GB1 protein, GL5, can be split into
two fragments, GN and GC, capable of spontaneous reconstitution. Protein solutions
containing repeats of the two fragments produced physical hydrogels (Kong and Li
2015).
amine residues on proteins. The reaction of THPP with amine is fast and
cytocompatible and occurs in physiological conditions. ELP proteins were cross-
linked with THPP to yield hydrogels encapsulating various cell types (Lim et al.
2008; Chung et al. 2012a). Resilin is a highly elastomeric protein found in arthro-
pods showing superior resilience and excellent high-frequency responsiveness (Qin
et al. 2012). Kiick and colleagues used the resilin-like recombinant protein, RLP-12,
which contains 12 repeats of the resilin sequence from the D. melanogaster
CG15920 (Li et al. 2011). THPP was employed to cross-link and modulate the
mechanical properties of RLP12 hydrogels, exhibiting from 500 Pa to 10 kPa storage
moduli. Despite its effectiveness in producing chemically cross-linked protein
hydrogel, THPP is no longer commercially available due to its complicated synthetic
procedure. Heilshorn and colleagues reported tetrakis(hydroxymethyl)phosphonium
chloride (THPC) as an inexpensive and widely applicable cross-linker alternative
(Chung et al. 2012b). THPC selectively reacts with amine residues, can modulate the
mechanical properties of ELP hydrogels, and is cytocompatible. Glutaraldehyde is
another type of cross-linker that reacts to amine residues, and it was employed to
cross-link a telechelic protein containing a mid-block with 12 lysine residues
sandwiched by end-blocks forming triple helix. This process produced shape-
memory protein hydrogels dependent on the thermoreversible association of the
end-block triple helices (Skrzeszewska et al. 2011). Thiol groups on cysteine
residues can form a disulfide bridge under oxidizing conditions and be used for
protein hydrogel formation. Chilkoti and colleagues showed that an ELP with
periodic cysteine residues undergoes hydrogel formation under mildly oxidative
conditions (Asai et al. 2012). The reversibility of the covalent linkage between
two sulfur atoms upon exposure to reducing agents such as glutathione confers a
significant advantage for producing biodegradable protein hydrogels.
Inspired by the biological processes that produce tough networks via enzymatic
activities, such as wound healing, extracellular matrix reinforcement, and cell wall
synthesis, it has been shown that carefully selected enzymes can construct protein
networks. Chilkoti and co-workers chose tissue transglutaminase (tTG), which
catalyzes the calcium-dependent acyl transfer reaction between glutamate and var-
ious primary amine residues. ELP recombinant proteins containing a 1:6 ratio of
lysine (ELP[KV6-112]) or glutamine (ELP[QV6-112]) to valine were incubated
with tTG to encapsulate chondrocytes in situ (McHale et al. 2005). On the other
hand, the microbial transglutaminase (mTG) does not require proenzymes or cal-
cium ions and achieves a high level of activity over a wide temperature and pH
ranges. Using mTG, gelatin hydrogels were cross-linked and studied for their utility
in thermal stability, encapsulation of HEK 293 cells, and therapeutic protein trans-
port (Yung et al. 2007, 2010). In addition to transglutaminases, the horseradish
peroxidase (HPO) system was harnessed to create di-tyrosine covalent bridges. HPO
catalyzes conjugation reactions of phenol and aniline derivatives in the presence of
hydrogen peroxide (Teixeira et al. 2012). Cross-linking resilin proteins from Dro-
sophila melanogaster with HPO resulted in both highly elastic and adhesive bio-
materials (Qin et al. 2011).
14 S. Sim
Fig. 3 (a) Splitting of CnaB2 into the protein SpyCatcher and its peptide reaction partner SpyTag (blue). Reproduced with permission (Veggiani et al. 2014).
Copyright 2014, Elsevier. (b) Schematic illustration of covalently cross-linked SpyTag-SpyTag hydrogel formed by mixing the trifunctional protein (AAA)
15
containing SpyTag and the bifunctional protein containing SpyCatcher. Reproduced with permission (Sun et al. 2014). Copyright 2014, the National Academy
of Sciences. (c) Schematic illustration describing cellular synthesis of the 4-arm starlike protein, (SpyCatcher)4GFP. Co-expression of the two fusion proteins,
16 S. Sim
Fig. 3 (continued) B10B and B11B, and subsequent split GFP reconstitution produced intracellular
(SpyCatcher)4GFP. Upon mixing with AA containing two SpyTag peptides, (SpyCatcher)4GFP
assembles into a Spy-G network. Reproduced with permission (Yang et al. 2020). Copyright 2020,
Elsevier
Network Formation of Engineered Proteins and Their Bioactive Properties 17
Fig. 4 (a) Ca2+-dependent network formation of the triblock artificial proteins. Calmodulin undergoes a conformational change and binds to proteins containing
calmodulin-binding domains, including petunia glutamate decarboxylase (PGD) and human endothelial NO synthase (eNOS), in response to Ca2+ ions.
Reproduced with permission (Topp et al. 2006). Copyright 2006, the American Chemical Society. (b) Synthesis of photoresponsive CarHC hydrogels. CarHC
tetramer undergoes disassembly upon light exposure and releases 40 ,50 -anhydroadenosine. The two telechelic proteins, ACA containing SpyTag and BCB
containing SpyCatcher, assemble into polymers and further form a protein network via AdoB12-induced CarHC tetramerization. This network can reversibly be
S. Sim
disassembled upon light exposure. Reproduced with permission (Wang et al. 2017). Copyright 2017, the National Academy of Sciences. (c) Reproduced with
permission (Huang et al. 2019). Copyright 2019, Springer Nature
Network Formation of Engineered Proteins and Their Bioactive Properties 19
2017), and adhesive proteins (Zhong et al. 2014). Bacillus subtilis, a gram-positive
microorganism, produces biofilms containing bacterial filaments of the TasA pro-
tein. Engineering TasA by fusing it with various other protein motifs enabled
programmable ELM based on B. subtilis biofilm with functional properties, includ-
ing intrinsic fluorescence, enzymatic activity, and the templated assembly of inor-
ganic nanoparticles (Fig. 4c) (Huang et al. 2019).
Grote et al. 2018). B. subtilis is widely used for the production of industrial enzymes
due to well-defined secretion pathways and the absence of an outer membrane.
However, yields are often milligram or sub-milligram quantities per liter of culture
when it comes to the production and secretion of heterologous proteins (Harwood
and Cranenburgh 2008). Engineering bacterial strains and pathways optimized for
producing a wide range of network-forming recombinant proteins in large amounts
would open possibilities to build ELM with desired mechanical properties.
The synergistic effect of living cells and protein networks in ELM beyond their
production is often overlooked. Not only do the living cells produce the protein
network, but they are also capable of manipulating them. Extensive experimental
characterizations complemented by multi-scale modeling will allow us to understand
how protein networks in the ELM change over time and respond to environmental
perturbations. The ability to understand and engineer the cellular contribution to the
mechanical properties over time will allow predictive designs of ELM.
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Living Synthetic Polymerizations
Abstract Natural materials, such as biofilms and tissues, sense and respond to
environmental signals using genetic, metabolic, and proteomic machinery. This
machinery allows natural materials to actuate changes with unmatched spatiotem-
poral precision. However, natural materials are relatively limited in morphology and
functionality compared to synthetic materials. In an effort to enhance synthetic
materials with the capabilities of living systems, we describe recent efforts to control
synthetic polymerizations using live cells as actuators. Both microbes and eukaryotic
cells have been employed in radical and oxidative polymerizations, significantly
expanding the synthetic scope available to living systems. In addition, these mech-
anisms have enabled construction of polymer networks and hydrogels that resemble
natural materials like tissues. Future efforts in synthetic biology, combined with new
methods for reprogramming metabolism to control abiotic chemistry, will enable
more platforms that synergistically enhance synthetic materials with living
functions.
1 Introduction
Living organisms detect and respond to a variety of inputs within complex environ-
ments. Specifically, living systems exist in a state of dynamic reciprocity, wherein
bidirectional flow of chemical, mechanical, and electrical information between cells
and their environment dictates micro- and macroscopic properties. This relationship
guides the development of cellular “living materials,” including biofilm growth and
tissue formation. Inputs such as food, water, light, and oxygen are processed by
cellular building blocks, instructing living material properties through growth,
morphology changes, and differentiation. At the single cell level, these environmen-
tal signals are processed and amplified using genetic, metabolic, and proteomic
networks (e.g., biological computation).
Biological computation is a central capability of living systems that enables
autonomy, self-regulation, and non-equilibrium processing. In a simple example, a
physical input such as sunlight serves as a transcriptional activator in plants, kick-
starting metabolism through the expression of photosynthesis genes. A more complex
example would be tissue homeostasis, where multiple extra- and intracellular signals
are simultaneously processed with spatial and temporal precision to maintain proper
tissue function across many length scales. This information processing is actuated and
amplified using specific biomolecules, including proteins, nucleic acids, and small
molecules. Synthetic biology, systems biology, metabolic engineering, and related
fields seek to understand and engineer control over biological computation,
harnessing the responsiveness of living systems toward outputs ranging from phar-
maceutical production to bioremediation (Meng and Ellis 2020; Tang et al. 2020;
Voigt 2020). In platforms that capitalize on the self-regulation and sustained growth
of living cells, programming both native and new functions through synthetic biology
confers added autonomy and intelligence to the engineered application. These cells
can then be deployed in dynamic and intervention-less environments.
With respect to materials, controlling biological computation toward desired
material properties has predominately focused on natural materials already produced
by the host organism. Some leading examples in the field are biofilm- and hydrogel-
based materials formed from the extracellular amyloid protein CsgA in E. coli
(Nguyen et al. 2014). A key advantage of protein-based living materials is the ability
to tailor function using amino acid sequence, such that the same general platform can
be used to design adhesives, conductors, and bioplastics. These gels also benefit
from the flexibility to harbor constituent cells as a method of continued gelation and
propagation, or to easily filter out cells for abiotic application. Another common
microbially produced material is cellulose, which can be engineered into tissue
mimetics or enzymatically functionalized surfaces (Shaffner et al. 2017; Gilbert
et al. 2021). Designer control over polysaccharide properties presents an interesting
challenge, as genetic sequence does not directly encode properties in the same way
as proteins; nonetheless, it has proven an invaluable platform for developing diverse
living materials. Organisms have also evolved relationships with inorganic mate-
rials, including synthesizing magnetic nanoparticles or forming structural elements
like bone and teeth (Furubayashi et al. 2021). In some cases, these relationships can
be metabolically tuned to control inorganic material morphology, which informs
their function. Coupling enzyme function to inorganic materials presents a novel
opportunity to apply bioengineering techniques, such as directed evolution, toward
probing and optimizing the functional landscape of these materials. It is possible that
features of this space are inaccessible to typical chemical syntheses. Overall, the
materials outlined above are notable for their tight evolutionary relationship with the
host organism, potentially facilitating genetic control over their synthesis and
Living Synthetic Polymerizations 29
In order to program synthetic polymers using living cells, catalytic activity must be
controlled by natural biological functions. One evolutionary relationship that cou-
ples the biotic and abiotic (i.e., natural and synthetic) worlds is a form of microbial
anaerobic respiration known as extracellular electron transfer (EET). In oxygen-
limited or redox stratified environments, such as the deep soil and ocean, some
microbes have evolved efficient machinery for moving electrons in/out of the cell
and across long (cm) distances. This capability allows life to persist in challenging
and competitive resource-limited environments. For example, the purple photosyn-
thetic bacteria Rhodopseudomonas palustris TIE-1 is a photoautotrophic microbe
that couples photosynthesis and the oxidation of ferrous iron, Fe(II), to carbon
dioxide reduction (Guzman et al. 2019). In this case, extracellular metals or poised
electrodes serve as electron donors to drive carbon fixation. Alternatively, microbes
such as Geobacter spp. and Shewanella oneidensis MR-1 direct metabolic electron
flux from carbon oxidation onto inorganic extracellular substrates. In nature, these
electron acceptors include metals and metal oxides, such as iron and manganese
oxides. However, synthetic materials, including various types of electrodes,
nanocrystals, metal-organic frameworks, and soluble metals, can also act as electron
acceptors (Shi et al. 2016; Dundas et al. 2018; Springthorpe et al. 2019; Graham
et al. 2021). Overall, EET provides an electronic interface between cellular metab-
olism and redox-driven processes occurring outside of the cell.
Geobacter sulfurreducens and S. oneidensis MR-1 are two of the most well-
studied organisms for understanding and applying EET. Despite both species being
electroactive, there are several key differences between these bacteria. First,
G. sulfurreducens is an obligate anaerobe, which cannot survive under highly
oxygenated conditions (Bond and Lovley 2003). In contrast, S. oneidensis is a
facultative anaerobe that is famed for its ability to utilize a variety of electron
acceptors including oxygen, DMSO, fumarate, nitrate, and various metals. Second,
the mechanisms responsible for EET are different between the two bacteria.
G. sulfurreducens and related Geobacter species exclusively use protein-based
nanowires to connect cellular metabolism to extracellular electron acceptors. The
Living Synthetic Polymerizations 31
exact structure and mechanism of these wires is somewhat controversial, but they are
required for Geobacter EET. On the other hand, S. oneidensis MR-1 uses a combi-
nation of electron transfer cytochromes bound to the outer membrane and soluble
redox shuttles, such as flavins, to facilitate EET (Coursolle et al. 2009; Coursolle and
Gralnick 2010; Brutinel and Gralnick 2012; Xu et al. 2016). In one specific example,
S. oneidensis uses the Mtr pathway (Fig. 1) to move electrons across the outer
membrane and onto extracellular electron acceptors. A final difference between the
two bacteria concerns genetic tractability. Genetic transformations in Geobacter
species, including the creation of knockouts and the insertion of exogenous DNA,
are possible, but time consuming and laborious. Although S. oneidensis is not a
model chassis, an increasing number of sophisticated genetic engineering techniques
have been demonstrated in this species (Hu et al. 2015; Corts et al. 2019; Dundas
et al. 2020; Li et al. 2020). Overall, the oxygen tolerance, relatively high growth rate,
well-understood EET machinery, and genetic tractability of S. oneidensis MR-1
make it an ideal organism for applying and manipulating EET. However, it is
important to note that Geobacter or other EET active microbes could replace
S. oneidensis in many of the EET-driven applications described below.
Our understanding of the specific EET pathways in S. oneidensis has facilitated
numerous applications using a diversity of metal electron acceptors. The most
apparent and well-studied is current generation on electrodes. S. oneidensis colo-
nizes different metal oxide surfaces to respire, which can be used to design a
microbial fuel cell (Shi et al. 2016). The diverse and facultative respiratory capabil-
ities of S. oneidensis enable long-distance electron transfer to the oxide surface even
in multicellular biofilms, where redox potential is stratified moving away from the
electrode. At appropriate potentials, electron transfer can also be redirected into the
cells through specific proteins, enabling unfavorable intracellular transformations
(termed bioelectrosynthesis). Underpinning these applications is our strong under-
standing of the Mtr and related pathways and their programmability using synthetic
biology. The wide substrate scope of S. oneidensis’ EET machinery enables contin-
ued development of new cell-substrate relationships. For instance, EET can be
directed toward oxidized soluble metals, such as Pd2+, to generate nanoparticles
(Dundas et al. 2018). Manipulating EET through both genetics and metabolism
allows control over nanoparticle synthesis rate and localization (e.g., outer
membrane-bound or intracellular). In addition, this evolutionary relationship to
metals allows cells to tune novel material properties. For example, EET can be
used to regulate the optical response of inorganic nanocrystals (Graham et al. 2021).
Overall, the tight metabolic relationship between electron transfer via the Mtr
pathway and central carbon metabolism in S. oneidensis allows for living control
over traditionally abiotic processes like producing current or programming
materials.
Given even just these few examples, it is apparent that redox-driven chemical
transformations are ubiquitous in both living and synthetic systems. However, in
practice, synthetic reactions driven by electron flow are usually controlled with
chemical reductants or in an electrochemical cell using a potentiostat. Based on
early reports that S. oneidensis could affect dehalogenation reactions in the presence
32 A. J. Graham and B. K. Keitz
of soluble metals (Workman et al. 1997), we reasoned that these bacteria may be able
to power a wide variety of material-relevant transformations via EET to synthetic
catalysts. In this chapter, we describe the efforts of several research groups to
validate this and related hypotheses. Specifically, we highlight the ability of
S. oneidensis and other electroactive organisms to control polymerization catalysts
for the synthesis of polymers and polymer networks. Although EET is an efficient
mechanism through which electrogenic bacteria can influence their redox environ-
ment, we also provide examples of non-electroactive organisms that are able to
influence their extracellular redox environment and redox-driven catalytic reactions
through molecule secretion, metabolic activity, and other means. With further
development, materials synthesized via the coupling of cellular metabolism to
synthetic catalysts will capitalize on the versatility, chemical functionality, and
robustness of synthetic materials while still maintaining the autonomy, sensing,
and computational abilities of living cells.
Radical polymerizations are central to synthetic chemistry and yield a diverse array
of soft, plastic, and glassy materials. Two well-known classes of “living” radical
polymerizations are atom-transfer radical polymerization (ATRP) and reversible
addition-fragmentation chain transfer (RAFT) (Kamigaito et al. 2001;
Matyjaszewski 2012). In the context of these polymerizations, “living” refers to
the presence of an active/dormant polymer chain end that does not spontaneously
decompose or participate in undesirable radical reactions. Both ATRP and RAFT
utilize polymer chain ends that continually switch between an active (can add
additional vinyl monomers) and a dormant (unreactive) state. The relatively low
concentration of radical species allows these polymerizations to achieve high con-
versions and produce polymers with controlled molecular weights and low polydis-
persity. The presence of a living chain end also allows for the synthesis of block
copolymers, bottlebrush polymers, and other polymer microstructures. There are
several different variants of ATRP and RAFT, but critically, there are examples for
both where an appropriately poised redox environment promotes reduction of a
chemical species, leading to radical initiation. As electrogenic microbes can be
potent reducers, we and others reasoned that endogenous cell metabolism could be
coopted to kick-start these radical polymerizations.
In RAFT, a radical initiator and a chain-transfer agent control the growth of living
polymer chains. Many common radical initiators are appropriately poised within a
reduction window achieved by microbial culture. Capitalizing on this reducing
capability of bacteria, Nothling et al. (2021) employed cultures of E. coli and
S. enterica serovar Typhimurium toward activation of a common redox-active aryl
diazonium salt. This allowed synthesis of p(oligoethylene glycol methacrylate) via a
microbially mediated RAFT mechanism (Fig. 2). The metal-free catalysis employed
nontoxic substrates, such that active cell metabolism was maintained within the
Living Synthetic Polymerizations
Fig. 2 Controlled radical polymerization (BacRAFT) facilitated by the terminal electron flux of E. coli (strain MC4100) and S. Typhimurium (strain TAS2010).
The reducing potential generated by growing bacteria activates the redox-active diazonium salt (4-bromobenzenediazonium tetrafluoroborate (4-BT)) to furnish
a carbon-centered aryl radical, which subsequently initiates a controlled radical polymerization of methacrylate monomers (OEGMA) in the presence of a chain-
transfer agent (TTC-1) via RAFT (Reproduced with permission from Nothling et al. 2021)
33
34 A. J. Graham and B. K. Keitz
While the general reducing capability of microbes is powerful for controlling the
above reactions, we reasoned that a more specific mechanism for metal reduction
could directly couple synthetic reactions to active cell metabolism. As S. oneidensis
is known to reduce a variety of catalytic transition metals, we hypothesized that EET
Living Synthetic Polymerizations
Fig. 3 A schematic of the bacteria-instructed synthesis process. (a–c) Bacteria induce polymerization in monomer-catalyst suspensions (a) to generate a
synthetic extracellular matrix of polymers (b). Recovery of polymers from the suspensions leads to two fractions (c), with polymer obtained from the aqueous
35
phase suspension around the bacteria denoted conceptually as non-templated and a second fraction obtained from a wash of the cell surfaces denoted as
36 A. J. Graham and B. K. Keitz
Fig. 3 (continued) templated. (d, e) Incubation of polymers with bacteria results in low binding of
cells for which the polymer is non-templated (d), or where a polymer templated with one cell type
(shown in red) is incubated with a cell (shown in green) of another type (e). (f) Addition of a
polymer, templated by one cell type, with its own “matched” cell population results in the formation
of large polymer cell clusters, as the templated polymers sequester the bacteria that “instructed”
their formation with high affinity. (g) The same reducing environment at bacterial surfaces that aids
the polymer synthesis can also be used to label the cells in situ via pro-fluorescent markers, which
react with cell-surface bound polymers containing “clickable” residues (Reproduced with permis-
sion from Magennis et al. 2014)
Living Synthetic Polymerizations
Fig. 4 S. oneidensis-enabled ATRP and initial polymerization kinetics. (a) Electron equivalents generated from S. oneidensis MR-1 reduce a metal catalyst
from an inactive state (MOX) to an active state (MRED). The active catalyst reacts with a halogenated initiator or polymer chain to produce a radical (gray arrow)
that can polymerize olefins. The radical can also react with the now-deactivated catalyst (MOX) to form a dormant chain (black arrow, right). (b) ATRP initiator
(2-hydroxyethyl 2-bromoisobutyrate, HEBIB) and macromonomer (OEOMA500) used in this study. (c) Monomer conversion after 24 h under various
conditions with (white) and without (purple) trace metal mix added to bacterial media. (d) Kinetics of monomer conversion in MR-1 or E. coli culture using
37
Cu(II)-EDTA as catalyst (e) Extracellular Cu(II) reduction monitored with the Cu(I)-specific fluorescent dye CF4. Data show mean SD of three independent
experiments. **P < 0.01 (Reproduced with permission from Fan et al. 2018)
38 A. J. Graham and B. K. Keitz
Fig. 5 Electron transfer proteins impact polymerization kinetics. (a) Key proteins involved in
extracellular electron transport in MR-1. (b) Effect of gene knockouts on polymerization activity
using Cu(II)-TPMA. (c) Rescue of normal polymerization activity via complementation with a
plasmid encoding mtrC, using Cu(II)-TPMA as a catalyst. Data show mean SD of three
independent experiments (Reproduced with permission from Fan et al. 2018)
a b c d
100 100 + Cu(II)-TPMA – Cu(II)-TPMA 100 MR-1 E. coli 2.5
OD = 0.2
Polymerization Rate (h-1)
OD = 0.2, Polymerization
Monomer Converion
Monomer Converion
2.0
75 75 75
(% Saturation)
Dissolved O2
after 2 h (%)
after 2 h (%)
1.5
50 50 50
1.0
25 25 25
0.5
0 0 0 0.0
0 1 2 3 Monomer + + + + HK Lysed HK Lysed
05
1
2
5
0.
0.
0.
Initiator + + +
0.
Fig. 6 S. oneidensis rapidly consumes dissolved oxygen and activates radical polymerization in
cultures for which no additional steps were taken to remove oxygen. (a) Dissolved oxygen in
S. oneidensis MR-1 cultures growing in SBM and under typical polymerization conditions. Under
both conditions, oxygen consumption outcompetes oxygen diffusion. (b) Effect of different bio-
logical and polymerization components on monomer (OEOMA500) conversion under aerobic
conditions. Monomer, initiator, catalyst, and S. oneidensis MR-1 are all required to achieve
significant monomer conversion. (c) Monomer (OEOMA500) conversion using heat-killed
(HK) and lysed E. coli MG1655 or S. oneidensis MR-1 cells under anaerobic and aerobic
conditions. Lysed cells release intracellular reductants that reduce Cu(II) to Cu(I), which activates
polymerization. Under aerobic conditions, adventitious reductants and radicals are quenched by
oxygen. Heat-killed cells do not result in polymerization under either condition as they neither
consume oxygen nor generate EET flux to reduce the metal catalyst. (d) Polymerization rate
constants of OEOMA500 using S. oneidensis MR-1 and Cu(II)-TPMA at varying inoculating
OD600 under aerobic conditions. Data show the mean SD of n ¼ 3 replicates (Reproduced
with permission from Fan et al. 2020)
40
Table 1 Monomer scope under anaerobic and aerobic polymerizations that involve S. oneidensis MR-1. Yields are listed as percentages, followed by
experimental Mn and Ð. The target Mn values for the monomer/initiator of 500:1 were polyOEOMA300 (150 kDa), polyOEOMA500 (250 kDa), polyHEMA
(65.1 kDa), polyNIPAM (56.6 kDa), polyDMAEMA (78.6 kDa), polyMMA (50.1 kDa), and PS (52.1 kDa). Average and SD values were obtained from
n ¼ 3 replicates (Reproduced with permission from Fan et al. 2020)
A. J. Graham and B. K. Keitz
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