nanomaterials

Article

The Electrosome: A Surface-Displayed Enzymatic
Cascade in a Biofuel Cell’s Anode and a High-Density
Surface-Displayed Biocathodic Enzyme
Alon Szczupak 1, Dror Aizik 1, Sarah Moraïs 2, Yael Vazana 2, Yoav Barak 3, Edward A. Bayer 2

and Lital Alfonta 1,*
1 Department of Life Sciences and the Ilse Katz Institute for Nanoscale Science and Technology, P.O. Box 653,

8410501 Beer-Sheva, Israel; [email protected] (A.S.); [email protected] (D.A.)
2 Department of Biomolecular Sciences, Weizmann Institute of Science, 234 Herzl St., P.O. Box 26,

7610001 Rehovot, Israel; [email protected] (S.M.); [email protected] (Y.V.);
[email protected] (E.A.B.)

3 Department of Chemical Research Support, Weizmann Institute of Science, 234 Herzl St., P.O. Box 26,
7610001 Rehovot, Israel; [email protected]

* Correspondence: [email protected]; Tel.: +972-8-647-9066

Academic Editor: Thomas Nann
Received: 1 May 2017; Accepted: 20 June 2017; Published: 23 June 2017

Abstract: The limitation of surface-display systems in biofuel cells to a single redox enzyme
is a major drawback of hybrid biofuel cells, resulting in a low copy-number of enzymes per
yeast cell and a limitation in displaying enzymatic cascades. Here we present the electrosome,
a novel surface-display system based on the specific interaction between the cellulosomal scaffoldin
protein and a cascade of redox enzymes that allows multiple electron-release by fuel oxidation.
The electrosome is composed of two compartments: (i) a hybrid anode, which consists of
dockerin-containing enzymes attached specifically to cohesin sites in the scaffoldin to assemble
an ethanol oxidation cascade, and (ii) a hybrid cathode, which consists of a dockerin-containing
oxygen-reducing enzyme attached in multiple copies to the cohesin-bearing scaffoldin. Each of the
two compartments was designed, displayed, and tested separately. The new hybrid cell compartments
displayed enhanced performance over traditional biofuel cells; in the anode, the cascade of ethanol
oxidation demonstrated higher performance than a cell with just a single enzyme. In the cathode,
a higher copy number per yeast cell of the oxygen-reducing enzyme copper oxidase has reduced the
effect of competitive inhibition resulting from yeast oxygen consumption. This work paves the way
for the assembly of more complex cascades using different enzymes and larger scaffoldins to further
improve the performance of hybrid cells.

Keywords: hybrid biofuel cells; enzymatic cascades; scaffoldin; cohesin; dockerin; redox
enzymes; cellulosome

1. Introduction

Biofuel cells are electrochemical devices that use enzymatic reactions to catalyze the conversion of
chemical energy to electricity in a fuel cell. They can be classified as microbial fuel cells (MFCs), which
use living microorganisms [1,2], or enzymatic fuel cells, which use purified enzymes [3,4]. Hybrid
biofuel cells combine the characteristics of both classes of biofuel cells. The concept was initially
introduced by the use of redox enzymes surface-displayed on different microorganisms and in biofuel
cells [5,6]. In the first study, glucose oxidase was displayed on the surface of Saccharomyces cerevisiae
(S. cerevisiae) yeast using the a-agglutinin yeast surface-display (YSD) system [7] for use in the anode
of a biofuel cell with an enzymatic based laccase cathode. The performance of the cell was higher than

Nanomaterials 2017, 7, 153; doi:10.3390/nano7070153 www.mdpi.com/journal/nanomaterials

Nanomaterials 2017, 7, 153 2 of 17

those employing either an enzymatic glucose oxidase anode or a microbial S. cerevisiae-based anode and
similar to that utilizing an anode consisting of both the free enzyme and yeast. The phenomenon has
been termed ‘the additive effect’. In a later study, the oxygen-reducing enzymes laccase and bilirubin
oxidase were coupled to the anode, and a fully hybrid biofuel cell was developed [8]. Yet another
advantage of enzyme surface-display is that the laborious and time-consuming purification processes
of enzymes were avoided and the surface-displayed redox enzymes were site-specifically wired
to an electrode to further improve electron transfer, as described by Amir et al. [9], who used the
Escherichia coli (E. coli) autodisplay system [10] for the display of alcohol dehydrogenase (ADH) as the
anodic reaction catalyst. This system was further improved to assemble an artificial biofilm of bacteria
surface-displaying ADH [11]. In situ processing of complexed polysaccharides such as starch into fuel
was demonstrated in a hybrid cell by the coupling of yeast displaying the starch-hydrolyzing enzyme
glucoamylase with glucose oxidase-displaying yeast in a mixed culture [12]. A mixed culture, however,
cannot be efficiently controlled over time. In this context, hybrid biofuel cells of yeast displaying
different hydrogenases on the same surface were presented [13].

In the microbial fuel cells, the metabolic pathways of the organism provide full cascades of
redox enzymes, which can catalyze the full oxidation of various fuels. The microorganisms grow
and divide, and that keeps the system alive and able to generate more catalyst for long-term usage.
However, such systems suffer from poor electron transfer to the electrode and low coulombic efficiency
due to multiple losses to the environment and to competing processes in the microorganisms. This
results in a less-defined system, which renders cell potential harder to control over time. In addition,
mass transfer problems, owing to the need for the fuel to cross the cellular membrane, result in a loss
of power outputs.

The use of enzymatic cascades in enzymatic fuel cell anodes resulted in very high power outputs,
as the electron density achieved was much higher when the fuel was fully oxidized; thus all electrons
extracted from a fuel molecule could be transferred to the anode [14–17]. The different surface-display
systems presented in different microorganisms and employed in hybrid fuel cells use the fusion of
the surface-displayed enzyme to a surface-displayed protein [5,8,9,12]. One of the major drawbacks
of the hybrid systems is the limitation of only one copy of an enzyme to be surface-displayed per
each surface-displayed protein, significantly limiting the number of redox enzymes per yeast cell.
This also limits the ability to display an enzymatic cascade, as each enzyme needs to be displayed using
a different vector, which adds complexity to the system. As the number of surface proteins in the cell
is limited, the insertion of more than one YSD vector, each encoding for another enzyme, will result in
competition over a limited number of sites and an overall loss in the cascade units. A second limitation
of the hybrid cell is that the displayed redox enzyme has to be expressed by the host organism carrying
the surface-display system, which is not ideal for enzyme overexpression and may result in lower
enzymatic activity.

These two challenges require a substrate-channeling approach in order to efficiently exploit
most of the electrons of each fuel molecule. Substrate-channeling methods involve the design of
systems in which several catalysts are designed to act in proximity, which can significantly improve
the total efficiency of a cascade of reactions [18]. Different examples for such channeling methods were
reported before, where different designs and approaches were used. Van Nguyen et al. used a DNA
scaffold in order to catalyze a cascade of reactions in which invertase of S. cerevisiae was coupled to
Aspergillus niger’s glucose oxidase to present a cascade in which sucrose was hydrolyzed by the former
enzyme, followed by an oxidation by the latter [19]. A native substrate-channeling system is inherent in
the bacterial cellulosome, in which multiple cellulolytic enzymes are bound to a non-catalytic protein,
called scaffoldin, which is displayed on the bacterial cell surface [20,21]. The scaffoldin protein consists
of several modules named cohesins, which bind with high affinity to the complementary dockerin
modules borne by the cellulosomal enzymes [22]. The binding of the scaffoldin to the bacterial cell
occurs via a second type of dockerin module in the scaffoldin protein, which binds to a cohesin
module of an anchoring scaffoldin bound to the cell surface. In a defined bacterial species, different

Nanomaterials 2017, 7, 153 3 of 17

cellulose-degrading enzymes with complementary activities share similar dockerin modules of like
specificities, and the enzymes are thus bound randomly to the scaffoldin protein [23,24], generating
heterogeneous cellulosomes. However, since the cohesin-dockerin interaction is species specific [25],
scaffoldin chimeras could also be generated by fusing genes encoding for cohesins from different
microorganisms via short protein linkers, thus generating designer cellulosomes [26–29]. In designer
cellulosomes, the number of copies and the location of any enzyme in the scaffoldin can be controlled
thus an enzymatic cascade can be self-assembled [30,31].

The gene encoding for the chimeric scaffoldin can be surface-displayed in microorganisms, thus
enabling the attachment of multiple enzymes [32–34]. Here we designed a system in which the
dockerin-containing redox enzyme will be overexpressed in E. coli and the lysate will be incubated
with the yeast displaying a chimeric scaffoldin on their surface, thereby avoiding the need to purify the
redox enzymes. Such a cascade for the oxidation of methanol has been presented before [35]. However,
electrochemical activity and/or performance in a biofuel cell was not demonstrated. Here, we have
employed cellulosome machinery and terminology and generated a redox enzymatic cascade to form
what we have termed: an electrosome. The advantage in such a system is that we exploit the versatility
and robustness of the YSD system in yeast while expressing redox enzymes in E. coli, thus avoiding
post-translational modifications (e.g., glycosylation), that occur in yeast, which would interfere with
the electron-transfer processes to and from the electrode.

Unlike the additive effect in the anode compartment observed in our earlier studies [5], in the
cathode compartment, a decrease in the hybrid biofuel cell’s performance was observed due to
competition over oxygen between the surface-displayed oxygen-reducing enzyme and the aerobic
respiration of yeast [8]. Yet, when the yeast cells in the hybrid biofuel cell were subjected to conditions
of anaerobic respiration, the advantage of the hybrid biofuel cell during long-term operation was
evident since the performance of the enzymatic cell decreased with time. Yeast anaerobic respiration
can be achieved by the addition of antimycin A, which inhibits yeast oxygen respiration [36].
Adding antibiotics is not a sustainable long-term solution since it is expensive and requires the
continuous addition of temperature-sensitive antibiotics to the fuel cells. These considerations are
even more relevant when using the biofuel cell in a continuous flow cell mode. An alternative
approach to overcome competitive inhibition is to increase the enzyme copy number or the density of
surface-displayed enzymes while keeping the yeast cell growth phase in a steady state. The approach
of scaffoldin surface-display enables an increase of the density of the cathodic biocatalyst on the yeast
surface by virtue of a scaffoldin protein that contains several cohesin domains displayed using the YSD
system, to which multiple copies of a single type of a dockerin-containing oxygen-reducing enzyme
are bound. In such a system, a monovalent scaffoldin may be used as all the enzymes are identical and
binding specificity is not required.

Depicted in Figure 1 is the electrosome that was designed for use both in an anode and
a cathode compartment; in each compartment, the unique attributes of the cellulosome scaffoldin
give a different advantage. In the anode (Figure 1A), the ethanol oxidation cascade consists of two
enzymes, ADH and formaldehyde dehydrogenase (FormDH), both containing a different dockerin
module of Acetivibrio cellulolyticus and of Clostridium thermocellum) C. thermocellum( (zADH-Ac and
pFormDH-Ct), respectively, assembled on a ‘designer’-scaffoldin chimera displayed on the surface
of S. cerevisiae. At the cathode (Figure 1B), copper oxidase (CueO) was selected for surface-display.
CueO is a multi-copper oxidase enzyme expressed by E. coli that catalyzes the oxidation of Cu(I)
ions coupled to oxygen reduction to water [37]. This enzyme is promiscuous and thus can oxidize
different aromatic compounds, some of which can act as redox mediators in the cathode compartment
of a biofuel cell. The dockerin module of C. thermocellum was fused to the enzyme (CueO-Ct), while,
in parallel, we present the display of a mini-scaffoldin bearing one to four similar cohesins that can bind
one to four copies of the dockerin-containing, oxygen-reducing enzyme CueO. The different constructs
used for assembly are depicted in Figure 1C. We report the characterization of the dockerin-containing
enzymes and their electrochemical activity using a diffusing redox mediator.

Nanomaterials 2017, 7, 153 4 of 17

2. Results and Discussion

2.1. Choice of Enzymatic Cascade
The addition of a dockerin module to an enzyme may result in significant conformational

changes as this module is relatively large (about 70 amino-acids long). Furthermore, when the native
enzyme has a quaternary structure of several subunits, dockerin fusion may inhibit the formation of
the quaternary structure and lead to a loss in activity. As a result, when selecting for enzymes, one
should avoid choosing multimeric enzymes. In this work, due to the multimeric characteristics of
most oxidoreductases, the enzymes were chosen after an analysis of their quaternary structure to
prevent major loss in activity due to dockerin fusion. Hence, based on this criterion, pFormDH was
selected to catalyze acetaldehyde oxidation in the cascade, even though it is more active towards
formaldehyde. The ADH chosen for this study exhibits very high activity towards ethanol and

Nanomaterials 2017, 7, 153 4 of 17
negligible activity towards methanol. Hence, an ethanol oxidation cascade was chosen (Figure 1A).

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of the system is given in the experimental section.

2. Results and Discussion

2.1. Choice of Enzymatic Cascade

The addition of a dockerin module to an enzyme may result in significant conformational changes
as this module is relatively large (about 70 amino-acids long). Furthermore, when the native enzyme
has a quaternary structure of several subunits, dockerin fusion may inhibit the formation of the
quaternary structure and lead to a loss in activity. As a result, when selecting for enzymes, one should
avoid choosing multimeric enzymes. In this work, due to the multimeric characteristics of most
oxidoreductases, the enzymes were chosen after an analysis of their quaternary structure to prevent
major loss in activity due to dockerin fusion. Hence, based on this criterion, pFormDH was selected to
catalyze acetaldehyde oxidation in the cascade, even though it is more active towards formaldehyde.

Nanomaterials 2017, 7, 153 5 of 17

The ADH chosen for this study exhibits very high activity towards ethanol and negligible activity
towards methanol. Hence, an ethanol oxidation cascade was chosen (Figure 1A).

2.2. Expression of Enzymes and Scaffoldin

All the scaffoldins and dockerin-containing enzymes are described schematically in Figure 1C.
The YSD of the different scaffoldins was validated by flow cytometry using an anti c-myc antibody
directed against a c-myc tag, which is part of the a-agglutinin display system (Supplementary Material
Figure S1). In all of the experiments the YSD of the scaffoldins was compared to negative controls of
non-modified yeast or yeasts known to display other enzymes on their surface (positive controls).

Colorimetric assays based on measuring the reduction of nicotine adenine di-nucleotide (NAD+) to
NADH absorbance at 340 nm for zADH-Ac and pFormDH-Ct, were used to verify enzymatic activity
of the dockerin-containing redox enzymes in bacterial lysates. Both enzymes have demonstrated
activity compared to a negative control of native bacterial lysates. The activity was compared to
the activity of a wild-type (WT), unmodified enzyme expressed in the same manner. A decrease in
activity of approximately 33% and 75% was observed for zADH-Ac and pFormDH-Ct compared
to their WT versions, respectively (Supplementary Material Figure S2A,B). This decrease in activity
may have resulted from the addition of a relatively large dockerin domain to the enzyme, which
may have reduced the expression levels and can affect affinity or substrate/co-factor accessibility to
the enzyme’s active site. In addition, since the cascade is based on EtOH oxidation, the activity of
the dockerin-containing pFormDH-Ct towards acetaldehyde was validated as well (Supplementary
Material Figure S3). As both enzymes are multimers, the addition of the dockerin module may also
interfere with inter-subunit binding. Although the observed loss in activity is quite significant,
the dockerin-containing enzymes retain the ability to bind to scaffoldin proteins directly from
bacterial lysates, avoiding a loss in protein yields, which are typical to complex purification processes.
In addition, the total amount of active dockerin-containing enzyme is sufficient for binding to all
available binding sites. The specificity of the enzymes towards their substrates was tested, and no
activity was detected for zADH-Ac towards aldehydes or of pFormDH-Ct towards alcohols.

The activity of the dockerin-containing CueO-Ct was tested by measuring the change
in absorbance at 430 nm following (orhto-phenilenediamine dihydrochloride) OPD oxidation
(Supplementary Material Figure S4). The dockerin-containing CueO-Ct exhibited 10 times more activity
in the lysate compared to a negative control consisting of native bacterial lysate. Native bacterial lysates
are also expected to have OPD oxidation activity as a result of the presence of endogenous CueO, which
is expressed when bacteria are grown in the presence of copper ions. WT CueO has demonstrated
slightly higher activity compared to the dockerin-containing CueO. The smaller reduction in the
enzyme activity of CueO compared to the other modified enzymes in this study, ca. only 20% loss of
activity, is probably due to the structure of CueO; unlike the dehydrogenases, CueO is a monomer,
which does not require an external co-factor, so the addition of the dockerin domain is less likely
to interfere with its activity as long as the active site is accessible to the substrates. As with the
dehydrogenases, with CueO as well, different expression levels may result from the dockerin addition.

Following the colorimetric enzymatic activity tests, enzymes in lysates were tested for their
electrochemical and electrode communication abilities using a redox mediator by cyclic voltammetry
and chronoamperometry. Demonstrating enzymes’ abilities to transfer electrons to and from electrodes
is essential if these enzymes are to be used in a biofuel cell. Figure 2 shows an increase in the anodic
current measured after the addition of bacterial lysate containing the dockerin-containing enzymes
zADH-Ac (Figure 2A (a)) or pFormDH-Ct (Figure 2B (a)) in the presence of their respective substrates,
ethanol or formaldehyde, respectively, and the cofactor NAD+, compared to the current measured
in the presence of a native bacterial lysate (Figure 2A (b),B (b)). No catalytic current was observed
in the absence of the respective enzymatic substrates (Figure 2A (c),B (c)). The lysates of bacteria
over-expressing native enzymes were used as positive controls (Figure 2A (d),B (d)) and demonstrated
electrocatalytic currents higher than those of the dockerin-containing enzymes which is in agreement

Nanomaterials 2017, 7, 153 6 of 17

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(cDon) s0e.0c0u2ti%vev a/dvdfiotiromnas lidne ehaycdhe sttoeply osfa (tCes) o0.f5b%a cvt/evr iEatO(aH, d oarr k(Db)l u0.e0)0e2x%p rve/svs fionrgmaadldoechkyedrien t-oco lnytsaaitneisn ogf
zbAacDteHri-aA (ca,o draprkF obrlumeD) eHx-pCrte,ssriensgp eac dtiovceklye;rin(b-c, ornetda)inninatgi vzeAbDaHct-eArica ol rl ypsFaotrems.DPHo-tCetn, trieaslpsetcetpiv: e0ly.1; (Vb,
(rvesd.)A nga/tiAveg Cbal)c,tMereiathl ylylesanteesb.l uPeot2e0nµtiMal wstaespu: s0e.1d Vas (avsre. dAogx/AmgedCila)t,o Mr aenthdyNleAnDe +bl1u.0e5 2m0 MμMw awsauss eudseads
as a redox mediator and NAD+
a co-factor for both enzymes; refer e1n.c0e5 emleMctr owdaes: Augse/dA gaCs la. co-factor for both enzymes; reference
electrode: Ag/AgCl.

TThhee eelleeccttrroocchheemmicicaal la actcitvivitiytyo fotfh tehde odcokcekreinri-nco-cnotanitnaiinnginCgu CeuOe-OCt-Cwta ws vaas lvidaalitdedatuedsi nugsiCnug (CIIu) a(IsI)t haes
rtehdeo rxedmoexd imateodriaintotrh einp trhesee pnrceesoefnocex yogfe onx. yFgigeunr. eF3igsuhroew 3s sthhoewevs otlhuet ioevnooluf taiocnat hoof dai ccactuhrorednict wcuhrernenat
lwyshaetne oaf lbyascatteer ioafe bxapcrteesrsiian gexCpureeOss-iCngt wCauseaOd-dCetd wtoast haededleedct rtooc htheem eicleacltcreolclh(Feimguicrael 3ce(all) )(Fcoigmuprea r3e d(at)o)
tchoemcpuarrreendt tmo ethaseu cruedrrewnitt hmtehaesaudreddit iwonithof tnhaet iavdedbitaicotne roiaf lnlyatsiavtee b(Faicgtuerriea3l l(ybs)a) taen (dFitgoutrhee 3b a(bck))g aronudn tdo
ctuherr ebnatckwgirthoouuntda ncuyrlryesnatt ew(iFtihgouuret a3n(cy) )l.yDsautee t(oFitghuerme u3c (hc)h)i.g Dhuerec taot htohde imc cuucrhre hnitgwhhere ncaetlhecotdroicc actuarlryesnist
twoohkenp lealecect,riotciasthaalyrdsist otooobks eprlvaecet,h iet rise vhearrsdib tloe ofobrsmervoef cthuer vreev(Ferigsiubrlee 3fo(rcm)), obfu ctutrhveet y(Fpiigcuarler3e v(ce)r)s, ibbulet
vthoelt atympmicoagl rraemvesrosifbtlhee vmoletdamiatmorowgraasmosb soefr tvheed mweidthiatthoer mwiadsd olebsoefrtvheedw waviteh atth+e 0m.0i5dVdlve so. fA tgh/e AwgaCvle. Aat
W+0T.0e5n Vzy vms.e Awga/As ugsCeld. Aas Wa Tp oesnitziyvme ceo wntarso lu(sFeidg uarse a3 p(dos))i.tiTvhe ecoonntsreotlp (oFtiegnutriea l3o (fdt)h)e. Tbhioe- eolnescetrto pcoattaenlyttiiacl
of the bio-electrocatalytic current is higher than that expected for mediated electron transfer but not
as high as the potential expected from direct electron transfer of the enzyme (ca. 0.45 V vs. Ag/AgCl).

Nanomaterials 2017, 7, 153 7 of 17

cNuarnroemnatteirsialhs i2g0h17e, r7,t 1h5a3n that expected for mediated electron transfer but not as high as the poten7 toifa 1l7
expected from direct electron transfer of the enzyme (ca. 0.45 V vs. Ag/AgCl). This is probably due to
thTehicso nist rpibruotbiaobnlyo fdbuoeth tpo rtohce scsoesntroibeuletciotrno noft rabnosthfe rp, reovceensstehso utog heltehcetreonnz ytmraenshfaesr,n eovt ebne etnhowuigrehd tohre
pehnyzsyicmaell yhastt nacoht ebdeetno twhieresdu rofar cpehoyfstihcaelleyle actttraocdhee.d to the surface of the electrode.

FFigiguurere3 .3E. Elelcetcrtorochcheemmicicaal la acctitvivitityyo off tthhee ddoocckkeerriinn–ccoonnttaaiinniinngg CCuueeOO–CCtt.. CCVVss ooff tthhee eelleeccttrroorreedduuccttiioonn
ooffO O2

2 iinn 00..11 M aacceettaattee bbyy ((aa)) llyyssaatteess ooff bbaacctteerriiaa eexxpprreessssininggC CuueeOO-C-Ct;t;( b(b))n nool lyyssaatete; ;( (cc)) llyyssaatetesso off
nnaatitviveeb abcatcetreiariwa iwthiothutoeunt zeynmzeymovee rovexepr reexsspiroens;s(ido)nl;y (sdat)e lsyosfabteasc toefr ibaaocvteerr-ieax opvreesrs-ienxgpCreusesOinwg iCthuoeuOt
awdiotchkoeuritn am doodcuklee.rCinu 2m+ o(1d0uµleM. )Csuer2v+ e(d10a sμaMre)d soexrmveedd iaatso ra; rreefderoexn cme eeldeicatrtoodr;e :reAfge/reAngcCel ;eslceacntrroadtee::
1AmgV/A/sg.Cl; scan rate: 1 mV/s.

22.3.3. .E EnnzzyymmeeB Bininddininggt tooS Suurrfafaccee–DDisispplalayyeeddS Sccaafffofoldldininss

TToo tteesstt tthhee bbiinnddiinngg oof fthteh edodcokcekrienr-icno-ncotanitnaiinngin egnzeynmzyems teos tthoe sthcaeffsocldafifno lddiisnpladyisepdl aoyne dyeaosnt,
yceoalsotr,imcoeltorriicm aecttirvicityac atsivsaityys awsesarey spewrfeorrempeedr fuosrimnge dscuafsfionlgdisnc asuffrofladcien-dsiusprflaacyei-ndgi sypelaasyti cnegllsy einacsut bcaetlelsd
in bacterial lysates. Figure 4 shows the change in absorbance at 340 nm due to NAD+

incubated in bacterial lysates. Figure 4 shows the change in absorbance at 340 nm due troedNuActDio+n
rfeodlulocwtiionng ftohlel oawctiinvigtyt hoef eaaccthiv oift ythoef aneaocdhic oefnzthyemaens owdiitch tehneziyr mreesspewctiitvhe tshuebisrtrraetsepse. cYteivaest ssuubspsternadteesd.
Yseiamstusltuasnpeeonudselyd isnim thuelt alynseaotuess lyofi nbathcteerlyias aetxeps roefsbsiancgte brioathex epnrzesysminegs bdoitshpleanyzeydm aecstidviistyp ltaoywedaradcst ivbiottyh
tosuwbasrtdrastebso t(hFigsuubres t4raAt)e.s Y(Feaigstu rceel4lsA i)n.cYuebaasttecde lwlsitihn counblyat ezdADwHith-Aocn elyxpzrAesDsiHng-A lycseaxtpesr e(sFsiignugrely 4sBa)t eosr
(FpiFgourrmeD4BH)-oCrt p(FFiogrumreD H4C-C) te(xFhiigbuitreed4 Cac)teivxihtyib iotendlya ctotiwviatrydos ntlhyetior wreasrpdesctthiveeir sruebspstercattievse. sSucbasfftoraldteins.-
Sdciasfpfolaldyiinn-gd yisepalsaty iinncguybeaatsetdi nwciuthb altyesdatwesi tohf lbyascatteersiao fthbaatc tdeirdia ntohta etxdpidrensso tdeoxcpkreersins -dcoocnktearininin-cgo nentaziynminegs
ednizdy nmoet sshdoidw nanoyt sahctoiwvityan (Fyigauctriev 4itDy).( FInig ourrdee4r Dto) .exIncluodrde earnyto acetxicvliutyd ereasunlytinagct fivroitmy nreosnu-lstpinegciffircoamlly
nboonu-nspde ecnifizcyamlleys,b tohuen ydeaesntz cyemllse ws,etrhee wyaesahsetdc ealfltsewr tehree bwinadshinegd aasfstaeyr, tahse dbeisncdriibnegda isns athye, aesxpdeersicmriebnetdal
insecthtieone,x panedri ma ecnotnaltrsoelc toifo nn,atainvde yaecaostn tcreolllso, fwnhaitcihv ewyeeraes tincceullbsa, twedh iwchithw ethree idnoccukbeartiend-cownittahintihneg
deonczkyemrines-,c ownatsa ipneirnfgoremnzeydm. Neso, awcatisvpiteyr fwoarms mede.aNsuoreadct fiovrit tyhwe caosnmtreoals euxrpeedrifmoretnhte (Fcoignutrroel 4eEx)p, ewrihmicehn its
(Fa iggouorde i4nEd)i,cawtihoinch thiast anogno-ospdecinifdici cbaitnidoningth daotens onno-ts opceccuifir.c Bboitnhd cionngtrdool eexspneoritmoecnctusr .indBiocathte ctohnatt rtohle
ebxipnedriinmge inst sspiencdificica taentdh athtatth aecbtiivnidtyin dgoiess snpoetc sifitecma nfrdomth abtaasactli vyeitaystd aocetsivnitoyt. sTtheme afcrtoivmityb aosfa tlhyee tawsto
aecntizvyitmy.eTs hbeouacntdiv tioty yoeaf stth teotwwaordens zeythmaensobl wouansd notot hyiegahset rt othwaanr dthsaett hofa nzAolDwHa-sAnco ot nhliyg,h beoruthnadn toth yaetaosft,
zpArDobHa-bAlyc odnulye, btoou tnhdet oreyleaatisvt,eplyro bloawbl yindiutieatl octohnecreenlattriavteiolynlso wofi naictieatlacldonehceyndter.a tAiosn sao fraecsuetlat,l dNehAyDdeH.
Apsroadruescutilotn, N wAasD oHnlpyr doduue cttoi oznAwDHas-Aoncl aycdtiuveityto. AzAs tDhHe p-AFcoramctDivHit-yC. tA fsoltlhoewpsF tohrem saDmHe- Cretafcotliloonw, ws thheen
saacmetealrdeeahctyidone ,cwonhceenntarcaetitoanlds eihnycrdeeasceo,n mceonrter aNtiAonDsHi naccrceuamseu, lmateosr;e hNenAcDe Hthea cincucrmeauslea tiens ;abhseonrcbeanthcee.
inTchreesaes reeisnulatbs ssourgbgaenscte t.hTath, einse fureesl uceltlslss tuhgagt eosptetrhaatte, fionr fluoenlgceerl plsetrhioadt so tphearna ttehefo drulorantgioenr pofe rthioed ascttihvainty
thexepdeurirmateinotns,o tfhteh eeffaecctti voift yane xepnezryimmeantitcs ,cathsceaedfefe wctilol fbaen oebnsezryvmeda ttioc caa hscigahdeerw exiltlebnet. oTbhsee rcvheadngtoe ian
haibgshoerrbeaxntceen ti.nT bhoethc haasnsagyesi nwaabs snoorbrmanacleiziendb toot hthaes asbaysosrwbaanscneo artm OaDliz60e0d into otrhdeera btos oerxbcalundcee avtaOriDation

600 i nin
oyredaesrt topoepxculluadtieonv adrieantisoitnyi. nTyheea sstppeocpifuicliattyi oonf deeancshit ye.nTzhyemsep eicni fitchiety coafsecaadche etnozwyamrdesi nittsh esucabssctraadtee,
toowbsaerrdvsedit sins uthbes tarsastea,yos bpseerrfvoerdmiendt uhesiansgs abyasctpeerirafol lrymsaetdesu,s winags bmaactinertiaailnleyds,a etevse,nw aafstemr eaninztyaminee db,inedvienng
to the scaffoldin. As the cohesin-dockerin interaction is species specific [38], each of the enzymes
bound to its matching cohesin domain in the divalent chimeric scaffoldin. In the case of binding of
zADH-Ac only, product inhibition may occur, but this inhibition may be avoided in the presence of
the downstream enzyme. Yeast metabolism also contributes to further oxidation of acetaldehyde

Nanomaterials 2017, 7, 153 8 of 17

after enzyme binding to the scaffoldin. As the cohesin-dockerin interaction is species specific [38],
Neancohmoatfertihales 2e0n1z7,y 7m, 1e5s3 b ound to its matching cohesin domain in the divalent chimeric scaffoldin. I8n otf h1e7
case of binding of zADH-Ac only, product inhibition may occur, but this inhibition may be avoided
(iEnttOhHe p orxeisdeantcioeno pf rtohdeudcotw) bnustt rteoa am leesnsezry emxete.nYte tahsatnm thetea ybeoalisstm suarlfsaocec-odnistrpilbauyteeds etonzfyumrthe,e pr FooxridmaDtiHon-
Coft. acetaldehyde (EtOH oxidation product) but to a lesser extent than the yeast surface-displayed
enzy me, pFormDH-Ct.

FFiigguurree 44.. BBiinnddiinngg ooff ddoocckkeerriinn–ccoonnttaaiinniinngg eennzzyymmeess ttoo tthhee ssccaaffffoollddiinn–ddiissppllaayyeedd yyeeaasstt.. CChhaannggee iinn
aabbssoorrbbaannccee aatt 334400 nnmm nnoorrmmaalliizzeedd ttoo aabbssoorrbbaannccee aatt 660000 nnmm ffoolllloowwiinngg tthhee rreedduuccttiioonn ooff 22..11 mmMM
NNAADD++ iinn tthhee pprreesseennccee ooff eeiitthheerr 11%% vv//vv eeththaannooll oorr 00.0.00022%% vv//vv foformrmaaldldeehhyyddee iinn tthhee pprreesseennccee ooff
ssccaaffffoollddiinn–ddiisspplalayyininggy eyaesatsitn cinucbuatbeadtewd itwh i(tAh ) (zAA)D zHA-ADcHa-Andc paFnodr mpDFHor-mCtDiHn -bCatc tienr iablalcytseartieasl;
l(yBs)azteAsD; H(B-A) czAinDbHac-Atecri ailnl ybsaactete; r(iCal) plyFsoartme;D (HC-)C pt FinorbmacDteHri-aCl tl yisna tbe;ac(Dte)rinaal tlivyesabtea;c t(eDria) lnlyastiavtee;
b(Ea)cnteartiiavle lyyesaasttec;e (llEs)i nncautbivatee dyewaistth cbeoltlhs ziAncDuHba-Atecda nwditphF obromthD HzA-CDt Hin-bAacc tearniadl lpyFsaotrems.DAHll -aCssta iyns
bwaecrteepriearlf olyrmsaetdesa. tA3l0l ◦aCssianyTsr wis ebruef fperer5f0omrmMe,dp aHt 83.00 .°C in Tris buffer 50 mM, pH 8.0.

For CueO-Ct, the change in absorbance at 430 nm due to OPD oxidation catalyzed by the enzyme
was fFoollroCwueedO, -aCntd, tthheec dheapnegnedinenacbes oornb athnec enautm43b0ern mof davuaeitlaobOleP bDinodxiindgat isoitnesc awtaalsy zsehdowbyn.t hFeigeunrzey 5mAe
swhaoswfso ltlhowe eindc,raenadset hine dabespoernbdaennccee uopnotnh ebinnudminbge rofo foanvea tiloa bthlerebei nedniznygmsiet ecsowpiaess sthoo aw mn.oFniogvuarele5nAt,
dshivoawlesntth, eanindc trreiavsaeleinnt ambsinoir-bsacanfcfeoludpino n(Fbiginudrein 5gAo f(i–oiniie))t. oWthhreene ae nfozyumrthe cboinpdieinsgt osiatem woanso vadaldeendt,
(dteivtraalvenalte, natn md itnrii-vsaclaefnfotlmdiinn)i, -tshcea fafcotlidviinty( Fdiegcureraes5eAd c(oi–miipi)a).reWd thoe tnhea afoctuivrtihty bwinitdhi nthgreseit ebiwndaisnagd sditeeds
((Fteitgruarvea l5eAn t(imv)i)n. iT-shciasf fsoulgdgine)s,ttsh oexaycgteivni tdyedpelectrieoans edduceo tmo pthaere rdeltaotitvheelya chtiigvhit yacwtiivthityth croenecbeinntdraintigonsi toens
t(hFeig yuereas5tA su(irvfa))c.eT whihsesnu gbginedstisngo xsyitgeesn fdore pthleet ieonnzdyumeet oarteh eavrealialatibvleel.y Thhieg hspaecctiifviictiytyc oonf cbeinntdraintigo nwoans
vthaelidyaetaesdt suusirnfagc de iwffehreenntb cinondtirnogl ssiatmespfloers tohf eneantizvyem yeeaasrt einacvuabilaatbedle .wTithhe CsupeeOci-fiCcti t(yFiogfubrein 5dAin (gv)w) oars
ovfa lsicdaaftfeodlduins-indgispdliaffyeirnegn tyceoanstt rionlcsuabmatpelde swoifthn aat iWveTy eenazsyt minec uthbaatt ecdanwniotht bCiundeO y-eCatst( F(Figiguurere5 A5A( v()v)i)o)r,
wofhsiccahf fdoelmdionn-dstirsaptleasy tihnagt ytheearset iisn cnuob aactteivditwyi tthhaat sWteTmesn fzroymm enothna-stpceacninfioctalblyin bdoyuenads ten(Fziygmurees.5 IAn b(voit)h),
cwohnitcrhold eexmpoenrismtreantetss, tthhaet athcteirveitiys nwoasa cstiigvnitiyficthanattlsyt elomwsefrr,o rmefnleocnti-nsgp eocnifilyc ablalyckbgoruonudnden yzeyamste asc. tIinvibtyo.t h
control experiments, the activity was significantly lower, reflecting only background yeast activity.

Ellecttrochemiicall acttiiviitty was measured ffor tthe same samplles.. Fiigure 5B shows an iincrease iin tthe
catthodiic ppeeaakkc ucrurerrnetnwt hwenhemni nmi-sicnai-fsfocaldffionlsdwinitsh woniteht oofnoeu rtboi nfdoiunrg bsiitneds iwnger esiuteses dw, aefrter suussepde, nadfitnegr
syuesapstenindCinuge yOe-aCstt- cino nCtauienOin-gCtl-ycsoanteta. iTnhinegc ulyrsreantet.s Twheer ecunrorremntasl iwzeedret onothrme OalDiz6e0d0 itno othrde eOr Dto60n0 einu torardlizeer
ttoh eneinuctraelaisze itnhea ibnscorrebaasne cine astbesmormbaingcef rsotemmcmeilnl g rforowmth cealnl dgrcoewlltdhi avnisdio cnelfl rdoimvistihoen nfruomb tehreo nfuamctbiveer
oefn zayctmivee ceonpzyymnuem cobperys .nFurmombeFrsi.g Furoem5B F, iigtucaren 5bBe, oitb scearnv bede othbaste,rwvehde nthuasti, nwghbeent wuesienngo bneetwanedent hornee
abnindd tihnrgees ibteins,dtihnegc suitrerse,n tthien cruerarseenst ainscerxepaseecste ads beyxpceac. t1edµ Abyw ciat.h 1e μacAh wbiinthd ienagchsi tbeinadidnegd s.iHte oawddeveedr.,
Hasoiws eviedre, nast fisro emvidtheentc ofrlomrim theetr cicolaosrsiamy,eutrpico ansbsainyd, iunpgoonf bfoinudrienngz oyfm foeusrt heenrzeyims aesd tehcerreea sise ain dceucrereanset,
in current, showing a current that is similar to that observed for two binding sites. Again, we attribute
the reduction in currents with a fourth binding site to oxygen depletion at the yeast surface.

Nanomaterials 2017, 7, 153 9 of 17

showing a current that is similar to that observed for two binding sites. Again, we attribute the
reduNcatnioomnatienriaclsu 2r0r1e7,n 7t, s15w3 i th a fourth binding site to oxygen depletion at the yeast surface. 9 of 17

FiguFrieg5u.r(eA 5).B (iAoc)h eBmioicchael manidca(lB a)nedle c(tBro) cehleemctircoaclhaecmtivicitayl oafctsiuvriftayc eo-fd issuprlfaayceed-dCisupelOay-Cedt nCorumeOal-iCzet d to
OD no

600.rm(Aa)liCzhedan tgoe OiDn 6a0b0.s (oArb) aCnhcaenagte4 i3n0 anbmsornboarnmcae laizt e4d30to n6m0 0nonrmmfaolilzloewd itnog 6t0h0e nomx ifdoalltoiowninogf OthPeD in
the porxeisdeanticoeno offy OeaPsDt dinis tphlea ypirnegseansccea offfo yldeainst odfis(ip)loaynien;g(i ai) stcwaoff;o(lidiii)nt hofr e(ei); oanned; ((iivi)) tfwoou;r (biiiin) dthinrege;s ites

and (iv) four binding sites incubated with CueO-Ct expressing bacterial lysate; (v) WT yeast
incubated with CueO-Ct expressing bacterial lysate; (v) WT yeast not displaying a scaffoldin incubated

not displaying a scaffoldin incubated with CueO-Ct-expressing bacterial lysate; (vi)
withmCouneoOv-aClet-netx spcraefsfosilndginb-dacisteprliaaylilnygs ayteea; s(tv iin) cmuobnaotevda lwenitths aca WffoTl deninz-ydmisep.l a(By)in Mgayxeiamstuimnc cuabthatoeddicw ith
a WTcuernreznytm aec.h(iBev) eMda bxyim CuVms ocfa ytheoadsti cdcisuprlraeynitnagc shciaefvfeodldbinysC oVf osnoef ytoe afostudr ibsipnldayiningg sistceasf ifnoclduibnasteodf one
to fowuritbhi CnduienOg-sCitt.e sThinec cuubrarteendt wis inthorCmuaelOiz-eCdt .toT hane caubrsroernbtainscneo artm 6a00li znemd.t Coua2n+ a(1b0s oμrMba)n wceasa tu6se0d0 nm.
Cu2+as( 1a0 rµedMo)xw maesduiasetodr;a rseaferreednocxe meleecdtiraotdoer;: rAegfe/rAegnCcel; eslceacntr oradtee:: A1 mg/VA/sg.C l; scan rate: 1 mV/s.

2.4. F2.u4e. lFCueell lCAelsl sAemssbemlybalyn danCd hCahraarcatcetreirziaztaitoionn

FollFoowllionwgitnhge dtheem odnesmtroantisotrnatoiof ne leocft roeclehcetmroicchaelmacictaivl itayc,tfiuveitlyc, elflusewl ecreellass swemerbe leadssteomdbelmedo ntsot rate
the adcetmivointystorafteth theee ancztiyvmitye sofb tohue nendzytomsecsa bfofoulnddin tos sdciasfpfolladyiends doinsptlhayeesdu ornfa tchee soufryfaecaes ot.f yFeigasut.r eFi6gusrheo ws
the 6p oshlaorwizsa tthieo npoalanrdizaptioown aenrdo puotwpuert ocuutrpvuets cuorfvetsh oef ctheell sceullss iunsginge tehtahnanool l aass ffuueel lwwhehne unsiunsgi nygeasyte ast

displaying a cascade of two enzymes (Figure 6 (a)) compared to cells of just zADH-Ac (Figure 6 (b))
dispolar ypiFnogrma DcaHsc-Cadt e(Foigfutwre o6 e(nc)z).y Amse esx(pFeigctuerde, t6h(ea m))acxoimmupmar epdowtoerc eolulstpouftj uosf t2z.7A ±D 0H.1- AμWc (·cFmig−u2 wrea6s (b))
or pFormDH-Ct (Figure 6 (c)). As expected, the maximum power output of 2.7 ± 0.1 µW·cm−2

measured with a cascade-displaying yeast. The higher power output of the yeast displaying only the
wassmeceoansdu rpeFdorwmiDthHa-Ccta, s2c.a2 d±e -0d.3is pμWlay·cimng−2,y ceoamstp.aTrehde thoi gthhoesre peoxwpreerssoinugtp ountlyo fththe efiyrseta sztAdDiHsp-Alacy ing
only the second pFormDH-Ct, 2.2 ± 0.3 µW·cm−2

enzyme, 1.6 ± 0.2 μW·cm−2, can be explained by th,e ceoxmprpesasrieodn otof tthheo sneateivxep ryeesassitn AgDoHn,l ywthhicehfi rst
zADcHat-aAlyczeesn tzhyem fiers,t 1r.e6ac±tio0n. 2in µthWe ·ccamsc−a2d,e,c awnhibche iesx tphleani nfeodllobwyedt hbey ethxep rresascitoionn ocaftathlyezenda tbiyv ethyee ast
ADHsc,awffohldicihn-bcoautanldy zpeFsortmheDHfir-sCtt.r Weahcetino nonilny tthhee zcAaDscHa-dAec, iws bhoiuchndi,s ththise ennzfoymlloew coendtrbibyutehse tor etahcet ion
catalcyelzle pderbfoyrmthaensccea, fbfuotl dthine -fboolluowndingp FreoarcmtioDnH d-oCets. nWot hoecncuor.n Tlyhet hcoenztrAoDl sHam-Aplcesis obf oyeuansdt d, itshpilsayeninzgy me
contar isbcuatfefosldtoint hweitcheolul tp berofuonrdm eanzcyem, besu t(Fthigeufroe l6lo (wd)i)n ogrr neaatcitvieo nyedaoste scenllost ioncccuubra.teTdh we citohn dtrooclksearimn-ples
of yecaosnttadiinsipnlga yeninzgymaessc a(Fffigouldrein 6 w(ei)t)h doiudt nbootu dnedmeonnzstyrmatee san(Fyi geluercetr6oc(hde)m) oicranl aactitviveityye. aInst aclle lclassiensc, uthbea ted
withobdsoecrkveedri np-ecrofonrtmaiannicneg weansz ylomweesr t(hFaignu trheat6 o(bes)e)rdveidd nfoort tdhee mcaosncasdtrea. tIen aandydietlioenct, rtohceh beamckicgarlouanctdiv ity.
In alclucraresnets ,wtihtheooubt saenryv eadddpiteiorfno ormf yaenacset wwaass vleorwy elorwth. Tahnet hpoawt oerb soeurtvpeudt wfoars tahlseo cpalsoctatedde v. eIrnsuasd tdhiet ion,

current density, showing better performance of the cascade (Supplementary Material, Figure S5).
the background current without any addition of yeast was very low. The power output was also

Comparing to the previously presented hybrid cells of one enzyme [5,13], an improvement in power
plottoeudtpvuetr sius sotbhseercvuerdr.e Ant cdoenntrsoitly c,eslhl owwitihnogubt eytetearstp werafso rumseadn ctoe toefstth tehec absaccakdgero(Suunpdp cluermreennttsa aryndM haatse rial,
FigusrheoSw5n). vCeorym lpoawr icnugrrteontths e(nporte svhioowusnl)y, lporweseern ttheadn hoybbserridvecde lilns Foifgounree 6e n(dz,yem), dee[p5i,c1t3in],ga tnheim npegroatvievme ent
in pocownetrrool usatmpuptleiss. observed. A control cell without yeast was used to test the background currents

Nanomaterials 2017, 7, 153 10 of 17

Nanomaterials 2017, 7, 153 10 of 17

and has shown very low currents (not shown), lower than observed in Figure 6 (d,e), depicting the
negative control samples.

Figure 6. Performance of yeast surface-displayed enzyme cascade. Power output curves of
anodFeisguorfe s6c.a fPfoerldfoinrm-daisnpclea yoifn ygeayseta sut rtfhaacte-wdiesrpelaiynecdu beantzeydmwe icthasc(a)dea. Pmoiwxteurr eouotfpuzAt cDuHrv-Aesc -ofa nd
pForamnDodHe-sC otf- esxcpafrfeoslsdining-dbiaspctlearyiainlgl yseaatsets t;h(abt) wzeAreD iHnc-uAbca-etexdp rwesitshin (ga) baa mctiexrtiualrel yosf aztAe;D(Hc)-ApcF-o arnmdD H-
Ct-exppFroersmsinDgHb-aCctt-eerxiaplrleysssaitneg; (db)anctaetriviael balycsteartieasl; ly(sba)t ez; A(eD) nHa-tAivce-eyxepasrtesinsicnugb abteadctweriitahl bolythsazteA; D(Hc)- Ac-
and ppFFoorrmDH–Ctt–eexxpprreessssininggb baactcetreiraialyl slyasteast.eE; (tdO)H na2t%ivev /bvacatsefruiaell ,lyNsAatDe;+ (e1).0 n5amtivMe yaneads1t imncMubMatBedh ave
beenwuistehd baostthh ezeAnDzyHm-Ae cc-o faancdto rpaFnodrmreDdHox-Cmte-edxiaptroers,sriensgp ebcaticvteelryi.a lysates. EtOH 2% v/v as fuel,

NAD+ 1.05 mM and 1 mM MB have been used as the enzyme cofactor and redox mediator,
respectively.

Hybrid biocathodes, containing yeast displaying an increasing number of CueO copies per
surface-dHisypblraiyde dbiosccatfhfolddeisn, ,cwonetraeinaisnsge myebalsetd d. iTshpleasyeinmgo adni fiiendcrsecaasifnfogl dniunm-bbeearr ionf gCyueeaOs tcwopeirees upserd as
catalsyusrtfsacine-daihspylbayriedd csacathffodldeinto, wwehrei cahssaeimr bwleads. pTuhergse dmoindiofiredde srctaoffionldcrine-abseaorixnygg yeenaslte wvelrse. uFsiegdu ares 7A
showcastathlyestpso iwn ae rhoyubrtipdu ctacthuordve stoo wf thiechd aifirfe wreans tpcuerlglse.dU inn olirkdeert htoe itnrecrnedasoeb osxeyrgveend leinvetlhs.e Fbigioucrhe e7mA ical
and sehleocwtrso tchhe epmowicearl oausstpauyts ,cuthrveehs iogfh tehset dpifofewreenrt ocueltlps.u Ut nolfikcea t.h0e .t7r6en±d o0b.0s8erµveWd ·icnm th−e2 bwioacsheombiscearlv ed
withanyde aesletcdtrioscphleamyiincagl afsosuarysb, tinhed ihnigghessitte psowfoerr toeuttrpau-vt aolfe cnat. 0m.7i6n ±i- s0c.0a8f fμoWld·icnm(−2F wigausr oeb7seArve(dd) w). itYhe ast
displyaeyaisnt gditswpolaybiinngd ifnogurs bitiensd(idngi- vsiatleesn ftomr tientri-as-cvaaflfeonltd mini)nie-xschaifbfiotleddint h(Feigseucroe n7dA h(dig)h). eYsetapsot wdiesrploauytinpgu t of
0.70 t±wo0 .b0i5ndi

µWng· csmite−s 2(,dbi-uvtaltehnatt mwinasi-socbasffeorlvdeind) aetxhhiibgihteedr vthoel tsaegcoen, idn hdiigchaetisnt gpoawheirg ohuetrpiuntt eorf n0a.7l0r e±s 0i.s0t5a nce
μW·cm−2, but that was observed at higher voltage, indicating a higher internal resistance compared

compared to that of all the other cells (Figure 7A (b)). The lowest power output for scaffoldin-displaying
to that of all the other cells (Figure 7A (b)). The lowest power output for scaffoldin-displaying yeast

yeastwwasa sacahciheiveevde dwwithit hthteh eyeyaesat sdtidspislaplaying a scaffoldin with only a single binding site (mono-valent
mini-scaffoldin), 0.054 ± 0.06 µW·cm−ying a scaffoldin with only a single binding site (mono-valent

2
mini-scaffoldin), 0.054 ± 0.06 μW·cm−2 ((FFiigguurree 77AA ((aa))).) .TThhe eddififfefreernecnec beebtwetewene etnhet hfueeflu ceelllcse allnsda tnhde the

diffedreifnfet raesnsta yasssparyess epnretesednhteedre hinerceainn bceane xbpel aeixnpeldaibnyedt hbeyf athcte tfhaactt tthheaty ethaes tyceeallsst acreelles xaproe seexdptooseddif fteor ent
conddiitfifoenresnitn cothnediftuioenlsc ienl ltsh,er efulaetl icveelltso, rtehlaotsiveei nto tthheoseel einct rthoec heelemctircoaclhceemllicoarl ctehlel obri othceh ebmiocichaelmaicctailv ity
testsa. cTtivhietyb tieosctsh.e Tmheic bailoachcteimvitcyal aascstiavyityw asssacyo nwdaus ctoendduschtoedrt lsyhourptloy nupboin dbiingdinbge tbweteweneeenn eznyzmymesesa nd
the sacnadff othlde inscainffoaldfriens hin bau frfesrh, w bhuiflfert,h wehfiule lthcel lfsuealr ecemllso raerec ommorpel ecxomlivpilnexg lsiyvisntegm sysstthematsa trheaat lalorew ed
to stalbloilwizeed atfot esrtaabsisliezme abfltyera ansdsebmebfolyr eancdh abreafcotreer iczhaatriaocnte.riAzafttieorna. nAfetqeru ailnib erqautiiolinbrpateiorino dp,ertihoed,o txhyeg en
concoexnytgraetni ocnosncinentthraetimonesd iinu mthem mayedbiuemlo mwaeyr tbhea nlowtheeri rthinanit itahleciro nincietinatlr acotinocnesn.trHatioownse.v Hero,wdeivffeurs, ion
limitdaitfifounsioisnl oliwmeitraatisonw eisll sloinwceer thaes rewieslln osinimcem tehderiae teisd enmo ainmdmfeodriahtieg hdeomxyangden fcoor nhciegnht raotxiyognena s in
the CcoVncmenetarsautiroenm aes nints t.hTe hCuVs ,mtheaesuexrepmecetnetds. iTnhcurse,a tshee ienxppeocwteedr inocurtepauset ,inst pemowmerin ogutfproumt, satemhimghincgo py
numfbroemr o af hdiigshp lcaoypeyd neunmzbyemr eosf ,diisspolbasyeerdv eendz.yTmhees, pisu orgbisnergveodf .a Tirheto ptuhreginasgs oefm abirl etdo tfhuee lascseelmlsb, lwedh ich

fuel cells, which was not performed in the activity assays, further improves mass transfer and oxygen
was anvoatilpaebrilfiotyrm toe dthien etnhzeyamcetisv. iAtys aesxspaeycste, dfu, rctohnetrroilm pporpouvleastiomnas sosf tryaenasstf etrhaatn cdanonxoytg ebnindav CauileaObi-lCitty to
the e(nFizgyumree s7.AA (es))e xopr escctaefdfo,ldcoinn-tdrioslplpaoypinugl ayteioasnts tohaf ty heaavste tbheaetnc ainncnuobtatbeidn dwiCthu ethOe- Cntat(iFvieg uenrzey7mAe (e))
or sc(aFfigfoulrdei 7nA-d (ifs))p, lwahyiicnhg caynenaostt btihnadt, ehxahvibeitbedee snigninifcicuabnattlye dlowweirth potwheern oauttipvuetse onfz 0y.1m1 e± 0(F.0i6g μuWre·c7mA−2 (f)),
whicahndc a0n.0n9o ±t 0b.0i6n dμW, e·cxmh−i2b, irteesdpescitgivneilfiyc, arenptlryeselonwtinegr ypeoaswt berasoalu atcptuivtistyo, wf h0i.c1h1 is± clo0s.0e6 toµ tWhe ·ecrmro−r 2ofa nd
0.09 t±he0 m.0e6aµsuWre·mcmen−t2s., Are csopnetcrtoilv beiloyf,ureel pcerlels wenitthionugt yeastt wbas aulseadc ttiov tietyst, twheh bicahckigsroculonsde ctuorrtehnetse arnrodr of
the mhaesa suhorewmn evnetrsy. lAowc ocnutrroenl tbs io(nfuote lshcoewllnw),i tlohwouert ytheaans tthwoases oubsseedrvteodt eins tFtihguerbe a7cAk g(reo,fu) ndegcautirvree nts
and hcoanstrsohlo swamnpvlersy. low currents (not shown), lower than those observed in Figure 7A (e,f) negative
control samples.

Nanomaterials 2017, 7, 153 11 of 17
Nanomaterials 2017, 7, 153 11 of 17

FiguFriegu7.reP 7e. rPfoerfmoramncaencoef obfi obcioacthatohdoedsecs ocmomprpirsiesdedo of fy yeeaasstt cconttaiiniing iincreasiing ccooppyy nnuummbbeer r of
surfaocf es-udrifsapclea-ydeisdpClauyeeOd .CPuoewOe. rPoouwtepru otuctupruvte csufrovre(sA f)ord i(fAfe) rdeinftfesrceanftf oslcdaifnfo-dldisinp-ldaiysipnlgayyienags tyewaistth (a)
monwovitahl e(na)t ;m(bo)nboivvaalleenntt;; ((bc)) btriivvaalleenntt; ;(acn) dtri(vda)lteenttr;a avnadle (ndt)s tceatfrfaovldaliennstw scitahffColudeinOs- Cwti;th(e )CnuaetOiv-eCyt;e ast
with(eC)u neaOti-vCet ;y(efa) smt owniothv aCleunetOs-cCatf;f o(fl)d min-obneoavrainlegnyt esacsatffionlcduibna-bteedarwinitgh yneaatsitv ienCcuubeaOt;ed(B w) Pitohw neartiovuet put
curvCeus eoOf ;c e(lBls) wPoitwh earn toimutypcuitn cAuravneds wofi thce(lals) mwoitnho avnatliemnty;c(ibn) dAi vaanledn tw; i(tch) t(raiv) amleonnt;o(vda)letnettr; a(vba) lent

divalent; (c) trivalent; (d) tetravalent mini-scaffoldins. Air was purged to the cells, Cu2+
mini-scaffoldins. Air was purged to the cells, Cu2+ 25 µM as redox mediator and enzyme c o25fa μctMor .

as redox mediator and enzyme cofactor.

TheThyeb hryidbrbidio bciaotchaothdoedseps eprefroformrmeedd weellll withoutt tthhee nneeded tot oinihnihbitb iyteyaseta asetraoebrioc briecspriersaptiiorna,t ion,
thusthinuds icinadtiincagtitnhga tththaet tchoem cpometipteivtietivineh iinbhitibioitnioenf fefcftecret sruesltuinltignfgr ofrmomo xoyxgyegnenc ocnosnusummpptitoionnb byyy yeeaasstt was
overwcoams oevbeyrctohme he ibgyh tdheen hsiigtyh odfeonxsyitgye onf- roexdyugcein-greednuzcyinmge esnoznymtheesy oena stthseu yrfeaacset scuormfapcaer ceodmtopaprreedv itou sly
repoprrteevdiobuiosclya trhepoodretse,di nbiwochatihchodthese, ienf fwechticrhes tuhlet eedffeicnt aressiuglnteidfi cina nat sliogsnsifiincapnot lwosesr ionu ptopwuetr[ 8o]u.tIpnuto [r8d]e. r to
validIna toerdtheirs tofi nvdaliindga,tea nthtiism fiyncdiningA, ,awnthimicyhciins aAn, winhhiicbhi tios rano finyheiabsittoare orfo ybeicasrte asepriorbaitci ornes[p3i6ra],tiwona s[3a6d], ded
to thweads iafdfedreedn ttoc tahteh doidffeesr,enatn cdatthhoedecsa,t ahnodd tehse’ cpaethrofodrems’ apnercfeorwmaasncceo mwapsa croemdptaorethd atot tohfact aotfh coatdheosdiens the
abseinnc tehoef aabnsteinmcey coifn aAnt.imWyeceinx pAe.c Wtede ethxpatecatsedco tphyat naus mcobpeyr npuemr cbeelrl ipnecrr ecealsle isn,ctrheeasceosm, thpee tciotimvepeintihtiivbei tion

inhibition effect will decrease. Thus, the effect of antimycin A addition on performance should be the
effeclat rwgeilslt dfoerc rmeoanseo.vaTlhenuts ,sctahfefoeldffiencst. oFfigaunreti m7By schinowAs athdadti twiohnileo fnorp oenrfeo armnda ntwceo schopoiuelsd obf eCutheeOl-aCrtg est
for mboounnodv atloe netasccha fsfcoalfdfoinldsi.nF i(gFuigruer7e B7sBh o(aw,bs))t,h tahtew pheirlfeofromraonncee aonf dthtew ocecllosp iimesporofvCeude Oan-dC thbigohuenrd to
eachmsacxaifmfouldmi np(oFwigeru roeut7pBu(tsa ,obf) )0,.6th1e ±p 0e.0r5fo μrmWa·cnmce−2 oafndth 0e.7c8e l±ls 0i.m02p μroWv·ecmd −a2 n(fdorh imgohneor-mvaalexnimt aunmd dpio-wer
outpvuatlsenotf s0c.a6f1fo±ldi0n.0, 5reµspWec·ctimve−ly2) awnedre0 .m78ea±su0r.e0d2, µinW c·ocmp−a2ri(sfoonr mwiothn o0-.5v4a l±e n0t.0a3n μdWd·ic-mva−2l eanntds 0c.a7f0f o±l din,
resp0e.c0t3i vμeWly·c)mw−e2,r reesmpecatsivuerleyd, w, iinthcooumt apnatirmisyocninw Ait. hFo0r. 5th4e± hig0h.0e3r cµoWpy· cnmum−2bearnsd su0c.h7 0as± tri0-.v0a3leµnWt a·ncdm −2,
respteectrtiav-vealyle, nwt itshcaofufotladnintsim, tyhcei npAow. eFro routhtpeuht igwhaesr sciompiylanr utom bthearts sauchcihevaesdt rwi-ivthaloeunt t antdimtyectrina -vAa, lent
scaffdoelmdionnss,ttrhateinpgo wthe rsoamutep ruetsuwltass wsiimthiilna rthteo sthimaitlarc hmievaseudrewdi tehrroourt (aFnigtuimrey 7cBin (cA,d, )d).e Tmhoenses trreastuinltgs the
samesurgegsuesltt sthwaitt choinmtpheetitsiivme iilnahr imbietiaosnu craend beer roovre(rFciogmuer ew7iBth(ocu,dt )t)h.eT ahdedsietiroens uofl tasnstuimggyecisnt Ath,a wt choicmh,p feotri tive
inhiblointigo-ntecrman ubseaogev,e risc onmote wa istuhsotuaitntahbelea dapdpitriooancho finan MtimFCysc.i nInA o, rwdheri chto, ffourlllyo nogv-etrecrommeu soaxgyeg,eins not
a sucsotaminpaebtilteioanp, mproorae cchominpMlexFeCd ss.caInffooldrdine rprtootefuinlsly, wohviechrc coomnseisot xoyf gmeonrec ocomhpeseitni tmioond,umleos,r sehcooumldp blee xed
scaffsoulrdfaincep-droistpelianyse,dw. hTihceh ucosen soifs thoigfhmero rreedcooxh epsointenmtioadl ulalcecsa,ssehso aunldd bimepsruorvfaedce -edleicstprolany etrda.nTsfheer uors e of

wiring will further improve fuel cell performance. In addition, CueO may be engineered for
highiemrprreodvoexd ppoetrefonrtmiaalnlcaec cians ebsioafunedl icmelpl rdoevveicdese. leTchter opnowtrearn sofuetrpourtsw wireirneg awlsoil lpfluotrttehde ragimainpsrto vtheef uel
cell cpuerrrfeonrtm daennsciety. I(nSuapdpdleimtioennt,aCryu MeOatmeriaayl Fbieguerneg Si6n)e aenredd fufortrheimr dpermovoendstrpaeterdfo trhme aimncperoinvebmioenfut einl cell
devicceelsl .pTerhfoerpmoawnceer uopuotnp tuhtes awdderiteioanls oof CpuloetOte cdopaigeas ipnesrt ytheaestc cuerlrl eanntdd theants, iitny th(Seu hpigphleerm coenpyta nruymMbaerte, rial
FigutrheeS a6n)tiamnydcfiun rAth iesr ndoet mcoonntrsitbruattiendg tthoe thime cperlol vpeemrfoernmt ainnccee, lul npleikrfeo irnm thane cloewu pcoopnyt hneumadbderit. ion of CueO
copies per yeast cell and that, in the higher copy number, the antimycin A is not contributing to the
cell performance, unlike in the low copy number.

Nanomaterials 2017, 7, 153 12 of 17

The characterization of each compartment (anode and cathode) presented herein was performed
separately in order to avoid the influence of the other compartment on characterization. Furthermore,
the assembly of a two-compartment hybrid cell requires also the presence of a proton-exchange
membrane, which affects cell performance due to the introduction of higher resistances. Power output
losses that might occur as a result of cell architecture, which is not relevant to the systems presented
in this study, are also excluded in a semi-biofuel cell configuration. The use of a potentiostatically
controlled electrodes in a membraneless assembly may affect the system. Therefore the counter
electrode in the three-electrode system used herein had a higher surface area, as described in the
experimental section. In the case of the hybrid anode, setting the potential of the cathode to a higher
potential may result in the oxidation of the redox mediator. However, the usage of a membraneless
assembly has given us an advantage in terms of internal resistance, which is crucial in the simple
assembly used for the proof of concept. Different control cells, including a cell of background current,
were used to demonstrate that the differences in performance are a result of the different yeast
populations that were used in this study.

3. Conclusions

We have designed a novel yeast surface-display system displaying different scaffoldin proteins in
order to improve the performance of hybrid biofuel cells. This approach enabled us to display a cascade
of ethanol oxidation enzymes bound to a chimeric scaffoldin in the anode, which demonstrated
higher performance than a cell displaying only a single enzyme. The system has demonstrated
comparable performance to that of our yeast surface-displayed cellobiose dehydrogenase (CDH)
system [13]. Taking into consideration that, in the CDH system, four electrons oxidation occurs, which
is similar to the number of electrons resulting from the ethanol oxidation cascade presented herein.
This performance is significantly higher than our first example of a hybrid biofuel cell with a yeast
surface-display of glucose oxidase [5]. The main advantage of the cascade assembly is that it is not
limited to one enzyme reaction, meaning that, by further addition of enzymes, wiring and surface
coverage improvement can be achieved in follow-up studies.

In addition, this approach allowed us to use crude lysates. Thus, we were able to avoid the
purification of two different enzymes. In the cathode, we displayed the oxygen-reducing enzyme
CueO in a sequential addition of copy-numbers; from one enzyme bound to a mono-valent scaffoldin
to up to four enzymes bound to a tetra-valent mini-scaffoldin in a single cell. This approach resulted in
reduced competitive inhibition effects resulting from oxygen consumption by yeast, thereby avoiding
the need for the inhibition of yeast aerobic respiration using antibiotics or other inhibitors. It should be
noted that the power output demonstrated in the fuel cells presented, is low compared to previously
reported enzymatic biofuel cells systems. However, the system presented herein is a proof of concept,
demonstrating that, with further engineering of the enzymatic cascades and scaffoldin, as well as
biofuel cell’s architecture and engineering, much higher power outputs may be achieved while
exploiting the advantages of the hybrid fuel cell, which is a living system that does not require enzyme
purification and is more suitable for long-term operation.

This approach paves the way for the assembly of more intricate cascades using different enzymes
and larger scaffoldins such as the recently described adaptor scaffoldins [28], which will further
improve hybrid cell performance. Considering that mediated electron transfer is less suitable for
the long-term operation of the system, further studies require the engineering of systems that are
able to perform direct electron transfer. The demonstration of Amir et al. [9] of the site-specific
wiring of an enzyme can be used. In addition, the ability of CueO to perform through direct electron
transfer can further improve fuel cell performance. As the structure of the enzyme influences its
activity when fused to a dockerin module, this should be considered when choosing enzymes
for such an application. The improvement of an enzymatic cascade and substrate channeling
strategies should be performed hand in hand with improvements in electrode material as well as
enzyme/microorganism-immobilization approaches.

Nanomaterials 2017, 7, 153 13 of 17

4. Materials and Methods

4.1. Strains and Constructs

The genes encoding dockerins of Acetivibrio cellulolyticus (dockerin from ScaB scaffoldin strain
ATCC 33288) and Clostridium thermocellum (dockerin from Cel48S strain ATCC 27405) were cloned
and ligated to the C-terminus of Zymomonas mobilis alcohol dehydrogenase and to Pseudomonas putida
formaldehyde dehydrogenase (zADH-Ac and pFormDH-Ct, respectively) by standard methods [39].
The dockerin module of C. thermocellum was also ligated to the C-terminus of CueO (CueO-Ct) of E. coli
(all the sequences are listed in the supplementary material (SI) section). All the dockerin-containing
enzymes encoding genes have been cloned into the pET15b vector for expression in E. coli, yielding
the pET15b-zADH-Ac, pET15b-pFormDH-Ct, and pET15b-CueO-Ct vectors. For controls, the genes
encoding the native enzymes without an appended dockerin module were also cloned in the same
vector, yielding plasmids pET15b-zADH, pET15b-pFormDH, and pET15b-CueO. All the chemicals
used in this study are detailed in the SI section.

4.2. Protein Expression

For protein expression, a 10-mL culture of E. coli bacteria was grown overnight at 37 ◦C in
standard Luria-Bertani broth with carbenicillin (Chem-Impex International, Wood Dale, IL, USA) at
a concentration of 100 µg/mL. A volume of 1.0 mL of the culture was used to inoculate a 100-mL
culture in the same medium containing carbenicillin at the same concentration. The culture was
incubated at 37 ◦C until it reached an optical density (O.D.) of 0.5 at 600 nm. Then 1 mM Isopropyl
β-D-1-thiogalactopyranoside (IPTG, Inalco, San Luis Obispo, CA, USA) was added to induce protein
expression, followed by the overnight incubation of the cultures at 20 ◦C. The bacteria cultures were
lysed by sonication, and the lysates containing the proteins were separated by precipitation. The cells
were lysed in 50 mM Tris buffer, pH 8.0, containing 1 mM CaCl2. For cells expressing CueO, lysis was
performed using 0.1 M acetate buffer, pH 5.0, containing 1 mM CaCl2 (for proper dockerin folding)
and 800 µM CuSO4. For all the lysates preparations, the bacterial cultures were diluted to the same
optical density of 1.5 in 1:20 dilution in the binding buffer in order to ensure that the same number of
bacteria was lysed from each culture and to measure the differences resulting from enzyme expression
and activity levels.

4.3. Enzyme Activity Assays

The activity of the enzymes was tested in their respective lysates. The activity of zADH and
pFormDH was tested by adding the bacterial lysates and following the change in absorbance at 340 nm
due to the reduction of β-Nicotinamide adenine dinucleotide (NAD+, Sigma-Aldrich, Rehovot, Israel),
which is a co-factor of both enzymes. For the zADH assay, 1–5% v/v EtOH was used as a substrate, and,
for the pFormDH assay, 0.002% v/v formaldehyde or 50 mM acetaldehyde was used. A concentration
of 1.05 mM of NAD+ was added to both enzymatic assays. All assays were performed in 50 mM Tris
buffer at pH 8.0 containing 1 mM CaCl2 (for proper cohesin-dockerin interaction).

The activity of CueO was tested by following the change in absorbance at 430 nm due to
the oxidation of 3.7 mM o-phenylenediamine (OPD), catalyzed by CueO. A SigmaFast OPD kit
(Sigma-Aldrich, Rehovot, Israel) was used while bacterial lysates expressing CueO were added to the
kit reaction mixture. The assay was performed at 0.1 M acetate buffer pH 5.0, containing 1 mM CaCl2
and 800 µM CuSO4.

4.4. Construction of YSD of Chimeric Scaffoldins

Genes encoding monovalent scaffoldins (one cohesin module) from A. cellulolyticus (cohesin 3
from the ScaC scaffoldin), Bacteroides cellulosolvens (cohesin 3 from ScaB), and C. thermocellum (cohesin
3 from the CipA scaffoldin) were cloned into the pCTCON a-agglutinin YSD vector. In the process,
the cellulose binding module (CBM) of the scaffoldin was removed, yielding vector pCTL20 (-CBM).

Nanomaterials 2017, 7, 153 14 of 17

The B. cellulosolvens cohesin was inserted to enable the binding of a third enzyme in future studies.
Scaffoldins with two, three, and four cohesins of C. thermocellum (cohesins 8 and 9 from CipA or
cohesins 1, 2 and 3 or cohesins 2, 3, 4 and 5, respectively) were cloned into the same vector, yielding
vectors pCT2Ct, pCT3Ct, and pCT4Ct, respectively. The cloning was performed between the Aga2p,
which enables the binding of the YSD system to the yeast cell, and the c-myc tag, which can be used for
YSD validation [7]. The genes were transformed to the EBY100 S. cerevisiae yeast strain. The expression
of the YSD system was performed as described earlier [5,12]. YSD was validated by flow cytometry
using the c-myc tag of the YSD system, as described and shown in detail in the SI section.

4.5. Enzyme Binding to Scaffoldin

For enzyme binding, 2.0 mL of yeast cells displaying scaffoldin, for which absorbance at
a wavelength of 600 nm was 1.0 (full description of the surface-display process can be found in
the SI section), were incubated with bacterial lysates containing the expressed enzymes at room
temperature for 1 h. 1.0 mL of the bacterial lysates were used for the binding, which was performed in
a final volume of 15 mL. As a binding buffer, 50 mM Tris buffer at pH 8.0 with 1 mM CaCl2 was used.
Upon binding, the yeast cells were precipitated, and binding was repeated using fresh lysate. After the
second binding cycle, the yeast cells were washed four times in the buffer to remove non-specifically
bound enzymes. For the CueO-Ct binding, the yeast cells were suspended in 0.1 M acetate buffer pH
5.0 containing 1 mM CaCl2 after the last wash. Following binding, the yeast calls were resuspended in
2.0 mL of buffer.

4.6. Cyclic Voltammetry (CV) and Chronoamperometry (CA)

A standard three electrode electrochemical cell was used. 0.9 mm diameter graphite rods (Pilot,
Tokyo, Japan) served as both working and counter electrodes, and Ag/AgCl was used as the reference
electrode (ALS, Tokyo, Japan). The assembly was designed so the counter electrode would have
a surface area 10 times greater than that of the working electrode. All measurements were conducted
using a PalmSens potentiostat (PalmSense BV, Houten, The Netherlands). For CV measurements,
100 µL of bacterial lysates prepared as described above were added to the electrochemical cell
containing the enzyme’s substrate and cofactors. For zADH and pFormDH, 20 µM methylene blue
(MB), as a redox mediator, and 1.05 mM NAD+ were added. For the CueO, 20 µM CuSO4 was added
as the redox mediator, which also functions as the enzyme cofactor. The scan rate was 1 mV/s in all
the measurements in a potential range of −0.3–0 V versus Ag/AgCl for the zADH and pFormDH or
−0.1–+0.3 V versus Ag/AgCl for CueO. The final reaction volume was 2.0 mL. The CV measurements
with yeast were performed under the same conditions, substituting the bacteria lysate with 200 µL of
enzyme-bound yeast suspension. CA was performed to demonstrate the electrochemical activity of the
anodic cascade dockerin-containing enzymes at a potential of +0.1 V vs. Ag/AgCl. The same volume
of bacterial lysates as in the CV, 100 µL, was added to a final volume of 2.0 mL. 20 µM MB served as
a redox mediator and 1.05 mM NAD+ as the cofactor of both enzymes. 0.5% v/v of EtOH or 0.002%
v/v of formaldehyde were added. The bacterial lysates of native bacteria were used as a negative
control. All electrochemical measurements have been performed in triplicates and have given stable
and similar curves.

4.7. Biofuel-Cell Assembly and Characterization

The fuel cell assembly was performed in 50 mL glass vials. For the cascade assay, 5.0 mL of bound
yeast was precipitated and added to the cell containing 1.05 mM NAD+ and 1 mM MB. Prior to the
addition of yeast, Argon was purged to the medium for 1 h. 2% v/v EtOH was added following
the addition of yeast, and the cells were sealed using Parafilm (Bemis, Oshkosh, WI, USA). The total
volume of the cells was 10 mL. A potentiostatically controlled cathode was set to +0.5 V vs. Ag/AgCl
using a PalmSens MultiEmStat potentiostat (PalmSens BV, Houten, The Netherlands), as performed by
Xia et al. [11]. For the CueO-based fuel cells, the CueO-Ct bound yeast were added to a cell containing

Nanomaterials 2017, 7, 153 15 of 17

25 µM CuSO4 and the antibiotics antimycin A (10 µM). Air was continuously purged to the fuel-cells.
A potentiostatically controlled anode set to −0.2 V versus Ag/AgCl was used. In all experiments,
the cells were left to stabilize overnight, following fuel cell assembly, before characterization was
performed. The characterization of fuel cell performance was done by measuring the voltage of the
cells under variable external loads. A background current cell was used as a negative control for all
fuel cell experiments and did not contain any yeast. Graphite rods of 5 mm diameter served as both
anodes and cathodes. The counter electrode that served for the potentiostatically controlled electrode
was of a larger surface area, as described for the CV and CA measurements.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-4991/7/7/
153/s1, Table S1: Strains and plasmids used in this study, Table S2: scaffoldin gene sequences, Table S3:
dockerin-containing genes sequences, Figure S1: FACS analysis, Figures S2–S4: enzymatic activity assays in lysate,
Figure S5: power output vs. current density curves for the anodic cascade biofuel cells, Figure S6: power output
vs. current density curves for the biocathodes biofuel cells.
Acknowledgments: This research was supported by an Israel Science Foundation (ISF) Program (232/13 to
Lital Alfonta and 1349/13 to Edward A. Bayer), as well as by the Israeli Ministry of National Infrastructure and
Energy (Lital Alfonta and Edward A. Bayer). An Azrieli and Merage. Fellowships are greatly acknowledged
(Alon Szczupak). We thank Shirley Tzelik for technical assistance.
Author Contributions: L.A., A.S., Y.B. and E.A.B. conceived and designed the experiments; A.S., D.A. Y.B.
Y.V. performed the experiments; A.S., D.A., S.M. and L.A. analyzed the data; L.A. and E.A.B. contributed
reagents/materials/analysis tools; A.S., L.A., S.M. and E.A.B. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
The following abbreviations are used in the text:

MFC microbial fuel cell
YSD yeast surface-display
ADH alcohol dehydrogenase
AldDH aldehyde dehydrogenase
FormDH formaldehyde dehydrogenase
GOx glucose oxidase
zADH Zymomonas mobilis alcohol dehydrogenase
pFormDH Pseudomonas putida formaldehyde dehydrogenase
CueO Escherichia coli copper oxidase
BOD bilirubin oxidase
Ac Acetivibrio cellulolyticus
Ct Clostridium thermocellum
CBM Cellulose-binding module
CV cyclic voltammetry
CA chronoamperometry
MB methylene blue
NAD+ β-Nicotinamide adenine dinucleotide
OPD o-phenylenediamine dihydrochloride
DMSO dimethyl sulfoxide
IPTG Isopropyl β-D-1-thiogalactopyranoside
PEG polyethylene glycol
WT wild type
EtOH ethanol
MeOH methanol
FACS Fluorescence Activated Cell Sorting

Nanomaterials 2017, 7, 153 16 of 17

References

1. Logan, B.E.; Hamelers, B.; Rozendal, R.; Schröder, U.; Keller, J.; Freguia, S.; Aelterman, P.; Verstraete, W.;
Rabaey, K. Microbial Fuel Cells: Methodology and Technology. Environ. Sci. Technol. 2006, 40, 5181–5192.
[CrossRef] [PubMed]

2. Lovley, D.R. Bug juice: Harvesting electricity with microorganisms. Nat. Rev. Microbiol. 2006, 4, 497–508.
[CrossRef] [PubMed]

3. Cracknell, J.A.; Vincent, K.A.; Armstrong, F.A. Enzymes as working or inspirational electrocatalysts for fuel
cells and electrolysis. Chem. Rev. 2008, 108, 2439–2461. [CrossRef] [PubMed]

4. Cooney, M.J.; Svoboda, V.; Lau, C.; Martin, G.; Minteer, S.D. Enzyme catalysed biofuel cells. Energy Environ.
Sci. 2008, 1, 320–337. [CrossRef]

5. Fishilevich, S.; Amir, L.; Fridman, Y.; Aharoni, A.; Alfonta, L. Surface-display of Redox Enzymes in Microbial
Fuel Cells. J. Am. Chem. Soc. 2009, 131, 12052–12053. [CrossRef] [PubMed]

6. Szczupak, A.; Alfonta, L. The Use of Yeast Surface-display in Biofuel Cells. In Yeast Surface-Display: Methods,
Protocols, and Applications; Liu, B., Ed.; Springer: New York, NY, USA, 2015; pp. 261–268.

7. Boder, E.T.; Wittrup, K.D. Yeast surface-display for screening combinatorial polypeptide libraries.
Nat. Biotechnol. 1997, 15, 553–557. [CrossRef] [PubMed]

8. Szczupak, A.; Kol-Kalman, D.; Alfonta, L. A hybrid biocathode: Surface-display of O2-reducing enzymes for
microbial fuel cell applications. Chem. Commun. 2012, 48, 49–51. [CrossRef] [PubMed]

9. Amir, L.; Carnally, S.A.; Rayo, J.; Rosenne, S.; Melamed Yerushalmi, S.; Schlesinger, O.; Meijler, M.M.;
Alfonta, L. Surface-display of a redox enzyme and its site-specific wiring to gold electrodes. J. Am. Chem. Soc.
2013, 135, 70–73. [CrossRef] [PubMed]

10. Lattemann, C.T.; Maurer, J.; Gerland, E.; Meyer, T.F. Autodisplay: Functional Display of Active beta-Lactamase on
the Surface of Escherichia coli by the AIDA-I Autotransporter. J. Bacteriol. 2000, 182, 3726–3733. [CrossRef] [PubMed]

11. Xia, L.; Ravenna, Y.; Alfonta, L. Layer-by-layer assembly of a redox enzyme displayed on the surface of
elongated bacteria. Chem. Commun. 2015, 51, 2633–2636. [CrossRef] [PubMed]

12. Bahartan, K.; Amir, L.; Israel, A.; Lichtenstein, R.G.; Alfonta, L. In situ fuel processing in a microbial fuel cell.
ChemSusChem 2012, 5, 1820–1825. [CrossRef] [PubMed]

13. Gal, I.; Schlesinger, O.; Amir, L.; Alfonta, L. Bioelectrochemistry Yeast surface-display of dehydrogenases in
microbial fuel-cells. Bioelectrochemistry 2016, 112, 53–60. [CrossRef] [PubMed]

14. Zhu, Z.; Tam, T.K.; Sun, F.; You, C.; Zhang, Y.P. A high-energy-density sugar biobattery based on a synthetic
enzymatic pathway. Nat. Commun. 2014, 5, 1–8. [CrossRef] [PubMed]

15. Kim, Y.H.; Campbell, E.; Yu, J.; Minteer, S.D.; Banta, S. Complete oxidation of methanol in biobattery devices
using a hydrogel created from three modified dehydrogenases. Angew. Chem. Int. Ed. 2013, 52, 1437–1440.
[CrossRef] [PubMed]

16. Sokic-Lazic, D.; de Andrade, A.R.; Minteer, S.D. Utilization of enzyme cascades for complete oxidation of
lactate in an enzymatic biofuel cell. Electrochim. Acta 2011, 56, 10772–10775. [CrossRef]

17. Tasca, F.; Gorton, L.; Kujawa, M.; Patel, I.; Harreither, W.; Peterbauer, C.K.; Ludwig, R.; Nöll, G. Increasing
the coulombic efficiency of glucose biofuel cell anodes by combination of redox enzymes. Biosens. Bioelectron.
2010, 25, 1710–1716. [CrossRef] [PubMed]

18. Wheeldon, I.; Minteer, S.D.; Banta, S.; Barton, S.C.; Atanassov, P.; Sigman, M. Substrate channelling as
an approach to cascade reactions. Nat. Chem. 2016, 8, 299–309. [CrossRef] [PubMed]

19. Van Nguyen, K.; Giroud, F.; Minteer, S.D. Improved Bioelectrocatalytic Oxidation of Sucrose in a Biofuel Cell with
an Enzyme Cascade Assembled on a DNA Scaffold. J. Electrochem. Soc. 2014, 161, H930–H933. [CrossRef]

20. Bayer, E.A.; Chanzy, H.; Lamed, R.; Shoham, Y. Cellulose, cellulases and cellulosomes. Curr. Opin. Struct.
Biol. 1998, 8, 548–557. [CrossRef]

21. Bayer, E.A.; Shimon, L.J.; Shoham, Y.; Lamed, R. Cellulosomes-structure and ultrastructure. J. Struct. Biol.
1998, 124, 221–234. [CrossRef] [PubMed]

22. Carvalho, A.L.; Dias, F.M.V.; Prates, J.A.M.; Nagy, T.; Gilbert, H.J.; Davies, G.J.; Ferreira, L.M.A.; Romão, M.J.;
Fontes, C.M.G.A. Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex.
Proc. Natl. Acad. Sci. USA 2003, 100, 13809–13814. [CrossRef] [PubMed]

Nanomaterials 2017, 7, 153 17 of 17

23. Carvalho, A.L.; Dias, F.M.V.; Nagy, T.; Prates, J.A.M.; Proctor, M.R.; Smith, N.; Bayer, E.A.; Davies, G.J.;
Ferreira, L.M.A.; Romão, M.J.; et al. Evidence for a dual binding mode of dockerin modules to cohesins.
Proc. Natl. Acad. Sci. USA 2007, 104, 3089–3094. [CrossRef] [PubMed]

24. Mechaly, A.; Fierobe, H.P.; Belaich, A.; Belaich, J.P.; Lamed, R.; Shoham, Y.; Bayer, E.A. Cohesin-dockerin
interaction in cellulosome assembly: A single hydroxyl group of a dockerin domain distinguishes between
nonrecognition and high affinity recognition. J. Biol. Chem. 2001, 276, 9883–9888. [CrossRef] [PubMed]

25. Pagés, S.; Bélaïch, A.; Bélaïch, J.P.; Morag, E.; Lamed, R.; Shoham, Y.; Bayer, E.A. Species-specificity of the
cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: Prediction
of specificity determinants of the dockerin domain. Proteins Struct. Funct. Genet. 1997, 29, 517–527. [CrossRef]

26. Bayer, E.A.; Morag, E.; Lamed, R. The cellulosome—A treasure-trove for biotechnology. Trends Biotechnol.
1994, 12, 379–386. [CrossRef]

27. Fierobe, H.P.; Mingardon, F.; Mechaly, A.; Belaich, A.; Rincon, M.T.; Pagès, S.; Lamed, R.; Tardif, C.;
Belaich, J.P.; Bayer, E.A. Action of designer cellulosomes on homogeneous versus complex substrates:
Controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin. J. Biol. Chem. 2005,
280, 16325–16334. [CrossRef] [PubMed]

28. Stern, J.; Moraïs, S.; Lamed, R.; Bayer, E.A. Adaptor Scaffoldins: An Original Strategy for Extended Designer
Cellulosomes, Inspired from Nature. mBio 2016, 7, e00083-16. [CrossRef] [PubMed]

29. Moraïs, S.; Morag, E.; Barak, Y.; Goldman, D.; Hadar, Y.; Lamed, R.; Shoham, Y.; Wilson, D.B.; Bayer, E.A.
Deconstruction of lignocellulose into soluble sugars by native and designer cellulosomes. mBio 2012, 3,
e00508-12. [CrossRef] [PubMed]

30. You, C.; Myung, S.; Zhang, Y.P. Facilitated Substrate Channeling in a Self-Assembled Trifunctional Enzyme
Complex. Angew. Chem. Int. Ed. 2012, 51, 8787–8790. [CrossRef] [PubMed]

31. You, C.; Zhang, Y.-H.P. Self-assembly of synthetic metabolons through synthetic protein scaffolds: One-step
purification, co-immobilization, and substrate channeling. ACS Synth. Biol. 2013, 2, 102–110. [CrossRef] [PubMed]

32. Tsai, S.-L.; DaSilva, N.A.; Chen, W. Functional display of complex cellulosomes on the yeast surface via
adaptive assembly. ACS Synth. Biol. 2013, 2, 14–21. [CrossRef] [PubMed]

33. Wen, F.; Sun, J.; Zhao, H. Yeast surface-display of trifunctional minicellulosomes for simultaneous
saccharification and fermentation of cellulose to ethanol. Appl. Environ. Microbiol. 2010, 76, 1251–1260.
[CrossRef] [PubMed]

34. Moraïs, S.; Shterzer, N.; Lamed, R.; Bayer, E.A.; Mizrahi, I. A combined cell-consortium approach for
lignocellulose degradation by specialized Lactobacillus plantarum cells. Biotechnol. Biofuels 2014, 7, 1–15.
[CrossRef] [PubMed]

35. Liu, F.; Banta, S.; Chen, W. Functional assembly of a multi-enzyme methanol oxidation cascade on
a surface-displayed trifunctional scaffold for enhanced NADH production. Chem. Commun. 2013, 49, 3766–3768.
[CrossRef] [PubMed]

36. Boumans, H.; Grivell, L.A.; Berden, J.A. The Respiratory Chain in Yeast Behaves as a Single Functional Unit.
J. Biol. Chem. 1998, 273, 4872–4877. [CrossRef] [PubMed]

37. Outten, F.W.; Huffman, D.L.; Hale, J.A.; Halloran, T.V.O. The Independent cue and cus Systems Confer Copper
Tolerance during Aerobic and Anaerobic Growth in Escherichia coli. J. Biol. Chem. 2001, 276, 30670–30677.
[CrossRef] [PubMed]

38. Vazana, Y.; Barak, Y.; Unger, T.; Peleg, Y.; Shamshoum, M.; Ben-Yehezkel, T.; Mazor, Y.; Shapiro, E.; Lamed, R.;
Bayer, E.A. A synthetic biology approach for evaluating the functional contribution of designer cellulosome
components to deconstruction of cellulosic substrates. Biotechnol. Biofuels 2013, 6, 1–18. [CrossRef] [PubMed]

39. Vazana, Y.; Morais, S.; Barak, Y.; Lamed, R.; Bayer, E.A. Designer cellulosomes for enhanced hydrolysis of
cellulosic substrates. Methods Enzymol. 2012, 510, 429–452. [PubMed]

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