iGEM 2025 — Team greatbay-scie

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### Notebook

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{% extends "layout.html" %} {% block title %}Engineering Success{% endblock %} {% block lead
%}Demonstrate engineering success in a technical aspect of your project by going through at least
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[Abstract](#abs)
[Chitinase](#chi)
[Glucanase](#glu)
[CBM addition](#cbm)
[Geraniol](#ger)
[Conclusion](#conc)

[Reference](#reference)

### Abstract

---

Aiming to address the widespread issue of indoor mold growth, GreatBay-SCIE developed
ArMOLDgeddon, an antifungal spray designed to disrupt the fungal outer protective
layer and induce cell lysis. The synergistic formula contains three types of enzymes
(chitinases, glucanases, lysozyme), supplemented with a monoterpenoid, geraniol.
Chitinases and glucanases degrade the key structural components of fungal cell wall,
chitin and glucan, and destroy the hyphal tips and mycelia, while geraniol disrupts
fungal cell membrane, leading to membrane permeabilization
[[1]](#ref1). Additionally, human lysozyme contributes to cell
wall
hydrolysis, disrupts membrane integrity and attacks fungal cell wall through its
cationic properties
[[2]](#ref2). Chitinases and glucanases were expressed in
*Escherichia
coli*, while lysozyme was produced in *Pichia pastoris*. Geraniol was
synthesized in *Escherichia coli* through integration of a mevalonate (MVA)
pathway and a geraniol synthesis module carrying truncated synthase variants. Its
solubility and stability were improved through encapsulation with 𝛾-cyclodextrin
produced by 𝛾-CGTase, enabling controlled release and prolonged antifungal action
[[3]](#ref3).

Enzyme performance was improved through
computational redesign and additional binding domain. The wild-type enzymes
*Pr*ChiA, GlxChiB, and BglS27 were optimized using ProteinMPNN and LigandMPNN,
to improve soluble expression, stability, and catalytic efficiency. Additionally, a
carbohydrate-binding module (CBM2 from *Bacillus sp.*, *Ba*CBM2) was
fused to the enzymes to enhance binding affinity to polymeric substances and hence
realize surface immobilization, thereby prolonging their reaction time and
efficacy.

Together, these components form a synergistic and sustainable
spray that not only eradicates existing mold but also inhibits future growth. By
integrating multi-enzyme degradation with antifungal compounds, ArMOLDgeddon provides
a safe, effective, and eco-friendly alternative to chemical fungicides, with potential
applications in a variaty of scenarios including households, healthcare, and
food-related settings.

### Chitinase

Cycle1

Design

Cell wall, a critical structure for spores and mature fungal
cells, derives its mechanical strength and resilience from a dense layer of chitin
(**Fig. 1**a,b). This β-1,4-linked polymer of
*N*-acetyl-D-glucosamine units is essential for fungal growth and
morphogenesis, rendering chitin hydrolysis a critical strategy for antifungal control.
Chitinases, the natural hydrolytic enzymes of chitin, serve as ideal agents to execute
this strategy by directly dismantling the structural core of the fungal wall
[[4]](#ref4).

Based on literature screening, we
selected four
wild-type chitinases with reported antifungal potential: r*Mv*EChi,
*Pr*ChiA, GlxChiB and *Bc*ChiA1 (**Fig. 1**c). These enzymes
come from different glycoside hydrolase (GH) families with different sites of action.
For instance, r*Mv*EChi (GH18) specifically targets the germinating hyphal tips
to inhibit fungal elongation
[[5]](#ref5); GlxChiB (GH19) and *Pr*ChiA (GH18) can damage
both hyphal
tips and lateral walls
[[6]](#ref6)
[[7]](#ref7); *Bc*ChiA1 (GH18) has high chitinolytic activity
[[8]](#ref8), contributing additional chitin-degrading activity.
They act
complementarily, attacking different regions of the mold body.
![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-1.webp)

Fig. 1 | Conceptualization of our chitinase selection. (a)
schematic representation of mold hyphal cell wall with chitin labelled (chitin
polymers shown as green bricks); (b) illustration of cell wall of mold spores; (c)
Targeted area of each enzyme on fungal hyphae (adapted from
[[9]](#ref9)); (d) plasmid construct of chitinases.

Build

The plasmids carrying the codon-optimized coding sequences of
the enzymes were synthesized by gene synthesis companies, and were transformed into
*E. coli* BL21(DE3) cells. A single transformed colony was used to inoculate a
5 mL LB starter culture supplemented with either kanamycin or ampicillin. After
overnight culture, this starter culture was then used to inoculate 50 mL of LB medium,
which was incubated at 37°C with shaking at 220 rpm until the OD₆₀₀ reached 0.6–0.8.
Protein expression was induced by adding 0.3 mM isopropyl-β-D-thiogalactopyranoside
(IPTG), followed by incubation at 18°C for 12 h. The cells were then harvested by
centrifugation at 12,000 rpm for 7 min at 4°C. The pellet was resuspended in a lysis
buffer, adjusted to the enzyme's optimal pH using a 0.1 M citric acid and 0.2 M
Na₂HPO₄ system, and lysed on ice using a sonicator. The mixture was then centrifuged
again (12,000 rpm for 7 min at 4°C) and the cell supernatant is separated from the
pellet.

Test

To verify expression, SDS-PAGE was performed. Induced cultures
were normalized to the same cell density (OD₆₀₀=10.0), sonicated, and centrifuged. The
pellet was resuspended in hydroxymethylaminomethane (Tris) buffer, and, together with
the supernatant and whole-cell lysate, analyzed by gel electrophoresis. Results showed
that *Bc*ChiA1, r*Mv*EChi, and GlxChiB were successfully expressed in the
supernatant, while *Pr*ChiA was expressed as inclusion bodies (**Fig.
2**a).

Fig. 2 | Summary of testing assays on our chitinase. (a)
Expression of chitinases in E.coli BL21(DE3) chassis cell; cell fractions of BL21(DE3)
with empty vector are used as control (LB: supernatant after overnight fermentation;
wc: whole cell samples; s: supernatant after ultrasonic cell lysis; p: pellet after
ultrasonic cell lysis); (b) enzyme concentration of the supernatant of cell lysate.
*Pr*ChiA was not tested for enzyme concentration due to inclusion body
expression; (c) specific enzyme activity of each enzyme. *Pr*ChiA was not tested
due to unsuccessful expression, GlxChiB has no activity detected. \* denotes not tested
due to unsuccessful expression. Activity is calculated after subtracting the measured
absorbance with that of the control; (d) specific activity curve of *Bc*ChiA1;
(e) specific activity curve of r*Mv*EChi. Error bars represent±SD (*n* =3).

Learn

For enzymes with confirmed antifungal properties, the
literature demonstrates that the antifungal efficacy is positively correlated with the
degree of chitinolytic activity (quantified by DNS assay).
[[19]](#ref19). Among our four enzyme candidates, three
successfully achieved
supernatant expression, with *Bc*ChiA1 and r*Mv*EChi showing high levels of
hydrolyzing activity. After consultation with expert Mr. Su, we were informed that
deficiency in protein folding fidelity, solubility and enzymatic activity might
underlie the unsatisfactory results for *Pr*ChiA and GlxChiB, highlighting the
need for engineering strategies to optimize enzyme performance. Since literature
evidence clearly confirms that both *Pr*ChiA and GlxChiB can degrade fungal
hyphae and could significantly strengthen the antifungal effect of our final product,
we decided to engineer these two enzymes to improve their soluble expression and
catalytic function.

Cycle 2

Design

For the two enzymes *Pr*ChiA and GlxChiB, we applied AI
modeling tools ProteinMPNN and LigandMPNN to redesign the amino acid sequences based
on the enzymes' 3D backbone structures. For the aim of exploration, we only redesigned
the catalytic domain of each chitinase. The primary objectives were to improve soluble
expression levels and enhance catalytic activity.

Multiple variants were
generated for each enzyme, and we selected the 6 most promising sequences for each
enzyme based on their RMSD, Rosetta score, docking score and SASA scores, filtering
out poor candidates (**Fig. 3**).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-3.webp)

Fig. 3 | Representation of enzyme redesign workflow. (a)
superposition of LigandMPNN and ProteinMPNN generated de novo protein structures of
the chitinases *Pr*ChiA and GlxChiB; (b) plasmid construct of MPNN designed
sequences.

Visit our
[**Model**](%7B%7B%20url_for%28%27pages%27%2C%20page%20%3D%20%27model%27%29%7D%7D) page for more
information.

Build

The codon-optimized coding sequences were cloned into
pET-22b(+) vectors with GoldenGate assembly and transformed into *E. coli*
strain DH5α. Positive clones were identified by colony PCR (**Fig. 4**)
and verified by sequencing. Verified plasmids were then transformed into *E.
coli* BL21(DE3) for protein expression, which followed the standard induction
and fermentation procedures described in cycle 1.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-4.webp)

Fig. 4 | Colony PCR of the redesigned enzyme coding fragment for
*Pr*ChiA (a) and GlxChiB (b).

Test

The redesigned catalytic domains, together with the original
set of non-catalytic protein regions, were expressed in *E. coli* BL21(DE3).
Without modifying the fermentation protocol, five of the six redesigned *Pr*ChiA
variants were successfully expressed in the supernatant, as confirmed by the clear
bands in the SDS-PAGE analysis (**Fig. 5**a). *Pr*ChiA-4 has not
realized successful extended inoculation after several attempts and is excluded from
the following assays.

Unfortunately, all GlxChiB variants formed inclusion
bodies (**Fig. 5**b), stopping us from continuing downstream enzyme
characterization.

Soluble expression yield of the *Pr*ChiA variants is
assayed with Bradford assay (**Fig. 5**c) and chitinolytic activity is
assayed with DNS assay under the reaction conditions same as the assay for wild-type
(1 µL supernatant and 200 µL substrate for 20 min under 40°C) (**Fig.
5**d). The specific activity was obtained by subtracting the glucose
concentration of the control, which is BL21(DE3) carrying an
empty vector, from that of the enzyme sample, and divided by enzyme mass. Among the variants, *Pr*ChiA-3 and *Pr*ChiA-5 exhibited
measurable chitinolytic activity (2.407 Umg-1 and 1.967
Umg-1, respectively), confirming that successful sequence
optimization.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/fig-5-n9.webp)

Fig. 5 | SDS-PAGE, Bradford assay, and chitinolytic activity
assay results of redesigned *Pr*ChiA variants. (a) protein expression of
MPNN-redesigned sequences for *Pr*ChiA; cell fractions of BL21(DE3) with empty
vector are used as control (LB: supernatant after overnight fermentation; wc: whole
cell samples; s: supernatant after ultrasonic cell lysis; p: pellet after ultrasonic
cell lysis) (b) protein expression of MPNN-redesigned sequences of GlxChiB; cell
fractions of BL21(DE3) with empty vector are used as control (c) enzyme
concentrations for *Pr*ChiA expressed (\* denotes not tested due to inclusion
body expression); (d) specific enzyme activity assay of the redesigned
*Pr*ChiA. Error bars represent±SD (*n* =3).

Learn

The successful soluble expression of *Pr*ChiA in the
supernatant and its demonstrated enzymatic activity validate our computational
optimization strategy, while engineering of GlxChiB did not yield satisfactory
results. To address this, a second round of protein redesign is carried out on
GlxChiB using SolubleMPNN. The redesigned sequences are screened, and prospecting
candidates identified, awaiting future wet lab effort.

### Glucanase

Cycle1

Design

To target the β-1,3-glucans, β-1,3-1,4-glucans, β-1,6-glucans
and α-1,3-glucans present in the fungal and spore cell walls (**Fig.
6**a,b)
[[1]](#ref1), we selected four glucanases that have been shown in
the
literature to exhibit significant antifungal effects, BglS27, Bglu1, *Fl*Glu30
and aglEK14 (**Fig. 6**c)
[[10]](#ref10)
[[11]](#ref11)
[[12]](#ref12)
[[13]](#ref13). These enzymes were selected based on their
optimal
temperature and pH conditions, which align with the requirements of our application.
To facilitate expression in *E. coli*, the codon-optimized coding sequences of
the glucanases were cloned into suitable vectors compatible with bacterial expression
systems (**Fig. 6**d).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-6.webp)

Fig. 6 | Selection of our glucanases. (a) schematic
representation of mold hyphal cell wall with the glucan layer labelled (glucan
polymers shown as strings of green balls with different colors representing different
types of glucan molecules); (b) illustration of cell wall of mold spores with glucan
labelled; (c) illustration of the type of glycosidic bond each enzyme targets; (d)
plasmid constructs of the glucanases.

Build

The target gene sequences for Bglu1 and *Fl*Glu30 were
cloned into pET-28a(+) expression vector, while BglS27 and aglEK14 were cloned into
pET-22b(+). The plasmids were transformed into *E. coli* BL21(DE3). The proteins
were expressed under our standard fermentation procedures described in chitinase cycle 1 (37°C
for extended culture and 18°C overnight after inducing with IPTG).

Test

Following ultrasonic cell lysis and centrifugation, SDS-PAGE
analysis confirmed the successful expression of BglS27, Bglu1, and *Fl*Glu30
(**Fig. 7**a), with the majority of the proteins present in the
supernatant and smaller amounts in the precipitate. Among them, BglS27 showed the
lowest expression level, as indicated by the faintest target band. The concentration
of each protein was measured following the same procedures as chitinase, yielding the
specific concentrations of Bglu1 (0.233 ± 0.005 mg/mL), BglS27 (0.113 ± 0.003 mg/mL),
and *Fl*Glu30 (0.159 ± 0.008 mg/mL) (**Fig. 7**b). However, for
aglEK14, even after several expression trials, we were not able to observe a band with
the correct length (**Fig. 7**a).

The enzymatic activities of
the expressed proteins were assayed using the DNS assay, with each enzyme tested
against its specific substrate (**Fig. 7**c). Since the initial results
for BglS27 and *Fl*Glu30, when tested with laminarin and pustulan (carbohydrate
with β-1,3-glycosidic and β-1,6-glycosidic linkages respectively), were not
significant, they were retested with lichenan (β-1,3-1,4-glycosidic bonds), the
substrate of Bglu1 (**Fig. 7**d). The results consistently proved that
Bglu1 exhibited the highest catalytic activity of 532.08 ± 0.282 U/mg, followed by it
of BglS27, which is 89.38 ± 2.178 U/mg. Under identical reaction conditions, we
plotted a curve showing hydrolysis activity against time lapse (**Fig.
7**e). Specifically, 200 μL of 2 mg/mL lichenan solution was used as the
substrate with 1 μL of enzyme solution added to react at 37°C. The results showed that
the concentration of reducing sugars increased sharply within the first 10 min, and by
approximately 20 min, the value had reached a plateau at around 0.30 g/L. After 30
min, the limiting factor of the reaction became the depletion of substrate and
accumulation of product, rather than insufficient enzyme activity.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/fig-7-n.webp)

Fig. 7 | (a) SDS-PAGE results proving protein expression of the
four glucanases: Bglu1, BglS27, *Fl*Glu30, aglEK14; cell fractions of BL21(DE3)
with empty vector are used as control (LB: supernatant after overnight fermentation;
wc: whole cell samples; s: supernatant after ultrasonic cell lysis; p: pellet after
ultrasonic cell lysis); (b) enzyme concentration of the three glucanases; (c) specific
enzyme activity assay of glucanases, tested with their own substrate (i.e. substrate
containing the bond type targeted by the respective enzyme). Note: due to large
discrepancy in specific enzyme activity, the scale on the left is used by Bglu1, while
the scale on the right is for BglS27 and *Fl*Glu30; (d) specific activity assay
using DNS reagent of the three glucanases with lichenan as substrate; (e) specific
activity curve of glucanase Bglu1. Error bars represent±SD (*n* =3).

Learn

Upon using lichenan as substrate in DNS assay, BglS27 exhibited
a remarkable increase in specific activity, rising from 2.062 U/mg to 89.379 U/mg,
whereas *Fl*Glu30 only increased from 0.570 U/mg to 13.134 U/mg. This sharp
improvement highlights the catalytic potential of BglS27. Moreover, its natural
substrate, β-1,3-glucan, takes up 65% to 90% of the total amount of fungal glucans
[[14]](#ref14), making BglS27 particularly important for
effective
degradation. These two factors, substantial activity potential coupled with biological
relevance, underscore the importance of optimization of BglS27. Therefore, we plan to
apply ProteinMPNN and LigandMPNN to redesign its wild-type sequence, aiming to enhance
soluble expression, catalytic efficiency, and stability, ultimately improving its
effectiveness in our application.

Cycle2

Design

Based on the predicted 3D backbone structure, we employed
ProteinMPNN and LigandMPNN to generate novel sequences. From these sequences, six
candidates were selected for experimental validation (**Fig. 8**a). Our
engineering strategy specifically targeted the catalytic domain of BglS27 to mirror
the performance enhancements observed in *Pr*ChiA, with the goal of improving
both expression yield and catalytic efficiency over the wild-type. Different from
chitinase, we expressed redesigned catalytic domain with only an additional C-terminal
His tag, aiming to specifically explore only catalytic efficiency. These redesigned
BglS27 variants were then cloned into the pET-22b(+) vector for subsequent expression
testing (**Fig. 8**b).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-8.webp)

Fig. 8 | Workflow of enzyme redesign for BglS27. (a)
superposition of LigandMPNN and ProteinMPNN generated de novo protein structures of
BglS27; (b) plasmid construct of redesigned BglS27 candidates for wet lab validation.

Build

The designed plasmids were transformed into *E. coli*
BL21 (DE3), with overnight culture in LB with ampicillin. The proteins were expressed
under our standard fermentation procedures described in cycle 1 (37°C for extended
culture and 18°C overnight after inducing with isopropyl β-d-1-thiogalactopyranoside
(IPTG)). Supernatant and precipitate were collected after ultrasonic cell lysis.

Test

Protein expression was analyzed by SDS-PAGE. Among the six
variants, candidates 2, 4, and 6 showed weak expression and were mostly localized in
the insoluble fraction (precipitate) (**Fig. 9**), against our intended
outcome.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-9.webp)

Fig. 9 | Expression of the redesigned BglS27 (LB: supernatant
after overnight fermentation; wc: whole cell samples; s: supernatant after ultrasonic
cell lysis; p: pellet after ultrasonic cell lysis).

Learn

From these results, we concluded that the AI-based predictions
were not fully ideal in this round: while some variants were expressed, their
insolubility suggested that MPNN optimization did not yield stable and soluble
proteins. On the other hand, the insoluble expression could underscore the importance
of other domains in maintaining correct folding and solubility. Thus, further
iterative computational design was attempted using SolubleMPNN. Potential candidates
were identified for future wet lab validation.

Lysozyme

Cycle 1

Design

Realizing the antifungal potential of lysozymes due to
its ability to disrupt fungal cell wall
[[15]](#ref15), we decided to incorporate human lysozyme
(*h*LYZ)
to further enhance the fungicidal effect of our enzyme cocktail.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-10.webp)

Fig. 10 | Schematic representation for the flow of
lysozyme transformation.

As hLYZ is a highly folded protein with 4
disulfide bonds, expression in *E. coli* is not ideal. Thus, yeast *P.
pastoris* strain GS115 is chosen for lysozyme expression
[[16]](#ref16).

Two *h*LYZ sequences were
selected for
expression: codon optimized *h*LYZ and a previously reported AI optimized
m*h*LYZ
[[17]](#ref17)
[[18]](#ref18). The respective sequences were engineered
onto the
vector pPp-P2S0 with an α-factor secretory signal peptide fused to the
N-terminus of the lysozyme sequence for extracellular excretion. A 6×His tag is
connected to the C-terminus, which could be used for future protein purification
(**Fig. 10**).

Build

m*h*LYZ, *h*LYZ V110S, characterized with
higher activity and better soluble yield, is constructed by SOE PCR
(**Fig. 11**a)

Fig. 11 | Gel images showing successful plasmid
construction of both plasmids (a) colony PCR of DH5ɑ strain harboring
pPp-P2S0-m*h*LYZ; (b) unlinearized plasmid of pPp-P2S0-*h*LYZ and
linearized plasmid of pPp-P2S0-m*h*LYZ and pPp-P2S0-*h*LYZ. "p"
denotes unlinearized plasmid.

Learn

The linearized plasmids were electrotransformed into
*P. pastoris* GS115 competent cells and cultured on high zeocin
concentration plates to obtain high copy transformants, which are expected to
carry the two human lysozyme sequences accordingly. However, colony PCR of these
yeast transformants failed to show any positive results after multiple trials
even with continual modifications to experimental procedures. In the remaining
time during October, we will aim to further reflect upon and improve our
experimental procedures on the expression of human lysozyme in *P.
pastoris*, and hopefully proceed to the following steps of validation.

### CBM Addition

Design

Inspired by professor Desen Ke, incorporation of carbohydrate
binding domain into our project design is proposed for immobilizing our enzymes onto
wall surfaces and AC filter materials. We thus selected a family 2
carbohydrate-binding module from *Bacillus anthracis*(*Ba*CBM2), which was
identified to exhibit strong affinity for crystalline PET and insoluble
polysaccharides such as cellulose and xylan, materials commonly found in wallpapers,
paints, coatings, filters, and construction fibers. Additionally, *Ba*CBM2 (abbr.
CBM2) was originally found as part of a chitinase, suggesting its potential to be a
chitin-binding domain, making it well-suited for our application
[[19]](#ref19). A modified version of *Ba*CBM2 (dubbed
CBM2e) with an
additional peptide sequence attached was also incorporated for investigation, as the
module is theorized to have stronger binding affinity towards PET substrate
(**Fig. 12**)
[[20]](#ref20)
[[21]](#ref21). A comparative analysis was carried out for the
two
modules.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-12.webp)

Fig. 12 | Designs of carbohydrate binding modules(CBM) (a)
Plasmid construct of pET-CBM2-GFP and pET-CBM2-*Bc*ChiA1/Bglu1. (b) Illustration
of the application, in which the CBM-fused proteins are bound to the surface of an
air-conditioner filter.

Build

Binding affinity of CBM2 and CBM2e were
assayed in a simple GFP-reporter binding assay (**Fig. 13**a); CBM2e is
also fused with Bglu1 and *Bc*ChiA1, to assess its impact on enzymatic
performance (**Fig. 13**b). These constructs were subsequently
transformed into *E. coli* BL21(DE3) for protein expression.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-13.webp)

Fig. 13 | Colony PCR of DH5ɑ strain harboring
pET-28a(+)-CBM2/2e-GFP (a) and pET-28a(+)-CBM2e-*Bc*ChiA1/Bglu1 (b), showing
successful construction.

Test

SDS-PAGE analysis confirmed the successful expression of the
target proteins in the supernatant (**Fig. 14**a). To evaluate CBM
binding performance, we compared the affinity of CBM2/2e-GFP fusions proteins with
free GFP on a variety of materials including cellulose and PET film. After lysis, 1 µL
of the crude supernatant was pipetted onto a pre-cut piece of PET and cellulose
material and dried for 12 h. The surface was then washed under flowing water for 10
seconds. Under blue light, both GFP fused with CBM2 and CBM2e demonstrated stronger
retention on the materials compared to free GFP, with CBM2 showing higher affinity
than CBM2e (**Fig. 14**c, **14**d). Further testing on CBM
domains is presented on our
[**Implementations**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementations%27%29%7D%7D) page

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-14.webp)

Fig. 14 | Testing results for CBM-related experiments. (a)
expression of CBM2 and CBM2e fused with GFP indicator as well as fusion of CBM2e with
*Bc*ChiA1 and Bglu1; cell fractions of BL21(DE3) with empty vector are used as
control (s: supernatant after ultrasonic cell lysis; p: pellet after ultrasonic cell
lysis); (b) comparison of enzymatic activity between wild type *Bc*ChiA1, Bglu1
and fusion protein of each with CBM2e. Error bars represent±SD (*n* =3). The data
showed a statistically significant result in the t-test (*p* < 0.01),
indicated as \*\*, (*p* < 0.0001), indicated as \*\*\*\*; (c,d) Binding assay of
CBM2 and CBM2e on cellulose (left) and PET film (right).

At the same time, we conducted the DNS enzyme activity assays
on the fused enzymes, and compared the data with those from the original enzymes.
(**Fig. 14**b) Surprisingly, the results revealed that CBM2e enhanced
enzymatic activity. Under the same reaction conditions, the fusion proteins showcase a
specific activity of 228.8 U/mg and 662.3 U/mg for CBM2e-*Bc*ChiA1 and
CBM2e-Bglu1 respectively, compared to the original 193.8 U/mg and 531.5 U/mg. This
effect is likely due to the binding module increasing the binding affinity between the
enzyme and substrate during the reaction, thereby boosting hydrolysis efficiency.

Learn

Due to time constraints, we were unable to successfully express
the constructs with enzymes fused to CBM2 – the CBM domain with higher
binding affinity to a range of surface materials. However, with demonstrated increase
in activity for fusion proteins CBM2e-*Bc*ChiA1 and CBM2e-Bglu1, it is expected
that CBM2 would achieve the same effect. Moving forward, we plan to continue
optimizing the constructs and expression of CBM2 fusion proteins by testing different
host strains and fermentation conditions, with the goal of obtaining more
comprehensive data to share with the community.

### Geraniol

Cycle 1

Design

Considering the antifungal and fungistatic properties as well
as fragrance of monoterpenoid geraniol, we decided to add it to our enzyme cocktail to
corroborate the efficacy of ArMOLDgeddon
[[22]](#ref22).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/fig-15-n.webp)

**Fig. 15** | General workflow of our design on the
expression of monoterpenoid geraniol. (a) metabolic pathway for geraniol synthesis;
(b) plasmid construct of pW1-t86*Ag*GPPS2-*Ob*GES and
pW1-t86*Ag*GPPS2-t65*Ob*GES; (c) dual plasmid system containing pMVA plasmid
and geraniol synthesis plasmid.

In order to achieve synthesis of monoterpenoid geraniol
(**Fig. 15**a), we attempted to incorporate two fragments,
t86*Ag*GPPS2 and *Ob*GES, into our plasmid construct (**Fig.
15**b). t86*Ag*GPPS2 is a truncated geranyl diphosphate synthase (GPPS)
from *Abies grandis*, and *Ob*GES is a geraniol synthase (GES) from
*Ocimum basilicum*. According to a previous study on ObGES, truncation of the
65-amino acid plastid-targeting signal peptide on the N-terminal would greatly enhance
geraniol biosynthesis for heterologous expression in *E. coli*
[[23]](#ref23). We used pW1 (pET28a-ptac-RiboJ-BsaI) as our
cloning vector.
As it is derived from pET-28a, pW1 retains the kanamycin resistance marker and
pBR322-type origin, but the native T7 promoter was replaced with the Ptac
(IPTG-inducible, LacI-regulated) promoter, with a RiboJ insulator introduced
downstream of Ptac to standardize the 5' UTR. A pMVA plasmid constructed by
GreatBay-China 2018 was also used to improve the production of DMAPP (IPP), the
precursor of GPP and geraniol, forming a dual plasmid system (**Fig.
15**c).

Build

pW1-t86*Ag*GPPS2-t65*Ob*GES and
pW1-t86*Ag*GPPS2-*Ob*GES are constructed using GoldenGate Assembly and
transformed into *E. coli* strain DH5α competent cells. Constructs are verified
by colony PCR (**Fig. 16**) and sequencing.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-16.webp)

Fig. 16 | Colony PCR of pW1-t86*Ag*GPPS-t65*Ob*GES and
pW1-t86*Ag*GPPS-*Ob*GES in DH5ɑ.

Test

Plasmids verified by sequencing were extracted and subsequently
transformed into DH5α ΔtnaA competent cells with pMVA plasmid for: knockout of tnaA
prevents the production of foul indole, reducing background odor and allowing the
floral scent of geraniol to be more prominent. Gernaiol synthesis was induced by IPTG
with glycerol as additional carbon source, and dodecane as organic solvent. Geraniol
yield was quantified using gas chromatography-mass spectrometer (GC-MS) (**Fig.
17**a,b), suggesting t65*Ob*GES has a nearly threefold geraniol yield
comparing to *Ob*GES (0.1571 g/L vs. 0.05929 g/L) (**Fig.
17**c).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-17.webp)

Fig. 17 | Results for biosynthesis of geraniol. (a) standard
curve for geraniol concentration (b) Mass spectrum of geraniol sample (c) comparison
of geraniol yield of untruncated and truncated *Ob*GES. Error bars represent±SD
(*n* =3). The data showed a statistically significant result in the t-test
(*p* < 0.01), indicated as \*\*; (d) comparison of enzymatic activity between
enzyme-only solution and enzyme solution with 23.7 mM geraniol. The difference
displays no significance (*p* > 0.05).

To ensure that geraniol did not affect activity of chitinase
and glucanase in our product, we measured the activities of *Bc*ChiA1 and Bglu1
with 10 µl of 500mM geraniol solution added to the reaction system(200 μL of
substrate, 1 μL of supernatant sample), making a final geraniol concentration of
23.7mM – a concentration far higher than the minimum inhibition concentration (MIC)
for most mold species
[[24]](#ref24)
[[25]](#ref25). A control with 10μl of double-distilled water
added to the
enzyme solution was set up for comparison.

After 10-minute reaction under
the standard conditions (500 rpm, 37°C for Bglu1 or 40°C for *Bc*ChiA1), the
results showed that the activity of Bglu1 decreased by approximately 4.5%, while the
activity of *Bc*ChiA1 slightly increased by about 10%, showing no significant
difference and thus confirming that geraniol does not affect the catalytic performance
of either enzymes (**Fig. 17**d).

Learn

From our GC-MS assay, we observed that truncating the first 65
codons of *Ob*GES significantly improved geraniol biosynthesis efficiency. By
integrating our improved *Ob*GES with the existing production system, we achieved
a 62.8% increase in yield compared to the 96.5 mg/L reported by GreatBay\_SZ in 2018.
That is, the truncation strategy enhanced recombinant terpene synthase expression,
proving a success in optimizing heterologous metabolic pathways. We also confirmed
that geraniol solution at around 20mM does not significantly affect catalytic
performance for both chitinase and glucanase, confirming the feasibility of our
product design.

Cycle 2

Design

Further investigation revealed that integration of geraniol
into our anti-mold mixture poses challenges due to geraniol's hydrophobicity, high
volatility, and susceptibility to oxidation. After extensive screening of past
research, we discovered that covalent grafting or physical encapsulation of geraniol
with γ-cyclodextrin (γ-CD), a cyclic oligosaccharide with a hydrophobic inner cavity
and a hydrophilic outer surface (**Fig. 18**a), can significantly enhance
its thermal and chemical stability as well as solubility and biocompatibility
[[26]](#ref26).

To manufacture γ-CD, codon-optimized
coding
sequence of γ-cyclodextrin glycosyltransferase (γ-CGTase), which catalyzes the
hydrolysis and cyclization of starch to produce γ-cyclodextrin, was cloned into the
pET-22b(+) expression vector (**Fig. 18**b).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-18.webp)

Fig. 18 | (a) schematic illustration of γ-cyclodextrin molecule;
(b) plasmid construct of pET-22b(+)-γ-CGTase.

Build

The plasmids designed were transformed into *E. coli*
BL21(DE3) cells, following standard procedures with kanamycin as selection marker.
Protein expression was induced with 0.3 mM IPTG, followed by incubation at 18°C for
12-16 h. Supernatant and precipitate were collected after ultrasonic cell lysis

Test

SDS-PAGE was performed to confirm the successful expression of
γ-CGTase (**Fig. 19**a).

When 20 μl of γ-CGTase supernatant
was mixed with 1 mL of 2% starch suspension overnight, the solution became almost
transparent, indicating the enzyme's ability to convert starch into γ-cyclodextrin
through cyclization (**Fig. 19**b).

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-19.webp)

Fig. 19 | (a) expression of enzyme γ-CGTase; cell fractions of
BL21(DE3) with empty vector are used as control (s: supernatant after ultrasonic cell
lysis. p: pellet after ultrasonic cell lysis); (b) overnight reaction of γ-CGTase with
substrate starch.

Learn

The transparency observed after adding γ-CGTase to the starch
suspension suggests the enzyme successfully converted starch (insoluble) into
γ-cyclodextrin (soluble). Confirmation of enzyme expression and activity underscores
the potential of γ-CGTase as a reliable tool for scalable γ-cyclodextrin production.
Such efficiency not only validates our design but also opens opportunities for our
team to integrate this process into broader applications, such as the encapsulation of
functional compounds. Due to time constraints, encapsulation was not attempted;
however, the results strongly support its feasibility for future work, in line with
recent advances demonstrating the economical synthesis of γ-cyclodextrin via oriented
CGTase displayed on bacterial polyhydroxyalkanoate (PHA) nanogranules
[[27]](#ref27). Most importantly, this promotes the feasibility
of
incorporating γ-cyclodextrin into ArMOLDgeddon, empowering us to deliver safer, more
effective, and sustainable mold-removal solutions.

### Conclusion

This engineering journey has successfully established a robust,
multi-faceted platform for a novel enzyme-terpene-based antifungal mixture. We have
not only achieved heterologous expression of core anti-fungal ingredients —
chitinases, glucanases, and monoterpenoid geraniol — but have also systematically
enhanced their potential through model-guided protein design, we significantly
improved the catalytic efficiency of our key hydrolases, chitinase and glucanase.
Furthermore, fusion of carbohydrate-binding modules (CBMs) allows superior substrate
affinity and diversifies our application scenarios. Collectively, these efforts
culminate in a potent and rationally designed antifungal strategy, poised for further
application and scaling. Through dedicated effort and precise engineering,
ArMOLDgeddon (**Fig. 20**) is a promising and powerful solution to
address real-world mold problems, aiming to tackle one of the greatest threat to
public health.

![](https://static.igem.wiki/teams/5614/imagebase/engineering/img-20.webp)

Fig. 20 | An overview of the final "ArMOLDgeddon" antifungal
system.

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

[Cycle1-1](#Abstract)

[Cycle1-2](#background)
[Cycle1-3](#solution)
[Cycle2-1](#enzymes)

[Cycle2-2](#enzymes)
[Cycle3](#enzymes)
[Conclusion](#enzymes)
[Reference](#reference)

{% endblock %}