iGEM 2022 — Team estonia-tuit

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Notebook

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Engineering

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Design of melanin-shielded yeast was taken step-by-step, supported by research articles, modeling, and data from
our
experiments. In this part of the wiki, we reason our choices, ideas, and solutions used to create yeast strains
that are
capable of sustaining the most strenuous space danger of solar and cosmic radiation.

![](https://static.igem.wiki/teams/4440/wiki/wiki/half-arc-divider.svg)

# The aims and uncertainties

The goal of our project is to engineer yeast strains that are able to survive high radiation environments in
space.
Moreover, these yeast strains should be able to produce nutrients or other beneficial compounds for humans. To
achieve
this, we engineer yeast to synthesize a sufficient amount of melanin that will absorb space radiation to protect
the
cells while avoiding the potential cytotoxicity that melanin biosynthesis can cause. A precursor of melanin
synthesis,
L-DOPA, can auto-oxidate, leading to the formation of free radicals that are also damaging to the cells. Another
cause
of L-DOPA toxicity arises from translation errors it might cause. Due to its similarity to tyrosine, L-DOPA can
be
incorporated into newly synthesized proteins instead of tyrosine (Giannopoulos *et al*., 2019). Although
the
protective effect of melanin has been observed in several species, it is not known how the localization of
melanin
synthesis (intra-, extracellular, particle-limited) affects its protective functions and the potential toxicity
of the
synthesis pathway.

# Design: multiple approaches for melanin synthesis in yeast

We took different approaches to design a melanin production system in yeast cells. In the center of our selected
strategy is an enzyme, tyrosinase, that mediates the synthesis of melanin precursors (L-Dopa, dopaquinone) from
amino
acid tyrosine, which is synthesized in *S. cerevisiae* natively in the shikimate pathway (Fig. 1).

![](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/engenering-tyrosine-path.svg)

**Figure 1. Pathway of tyrosine synthesis in yeast.**

To find an optimal setup for melanin production, we undertook multiple strategies, including:

• considering multiple enzymes for melanin production;
• addressing cytotoxicity of melanin precursors (L-Dopa);
• comparing extracellular and intracellular melanin synthesis.

In the first design step, we looked for the most efficient enzymes for melanin production. We found two
candidates:
laccase from *Escherichia coli* and tyrosinase from *Bacillus megaterium* (Gustavsson *et al*.,
2016).
Most tyrosinase enzymes are heterodimeric complexes, but the one from *B. megaterium* functions as a
homodimer,
simplifying its use. We found that tyrosinase catalyzes all reactions in the pathway (tyrosine - L-Dopa, L-Dopa
-
Dopaquinone) (Fig. 2). Laccase, however, cannot convert tyrosine to L-Dopa. Moreover, we constructed a
computational
model describing the dependencies of these enzymes' reaction rates from substrate concentrations, which showed
tyrosinase to be more efficient than laccase (See Modeling). Also, while tyrosinase uses tyrosine as the
substrate,
laccase requires L-Dopa. So, for our project, we decided to use the tyrosinase gene from *B. megaterium*
for
heterologous expression in yeast for melanin production (Fig. 2).

![](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/melanin-pathway.svg)

**Figure 2. Melanin pathway introduced into *S. cerevisiae* by addition of tyrosinase from *B.
megaterium*.**

To address the question of the importance of localization of melanin synthesis, we developed three different
strategies.
In our first strategy, we overexpress tyrosinase by combining three biobricks: galactose inducible promoter
*GAL1*,
tyrosinase coding sequence, and *CYC1* terminator, with the aim to produce melanin in the cytoplasm (Fig.
3).

![](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/figure1-engenering.svg)

**Figure 3. Construct for cytoplasmic expression of tyrosinase in yeast.** *GAL1* - strong
galactose-inducible
*S. cerevisiae* promoter. *Tyr1* - gene encoding for *B. megaterium* tyrosinase.

Our second approach includes the formation of nanocompartments. These nanostructures aim to increase the local
concentration of enzymes, protect the enzymes from degradation, and physically separate cytotoxic intermediates
from the
cytoplasm. This approach relies on two components: *VP1* gene that encodes a major viral capsid protein,
and a
fusion of *VP2C* and *Tyr1* gene (encodes for tyrosinase fused to the nanocompartment anchor protein)
(Cheah
*et al*., 2021). Expression of these genes is controlled by a bidirectional *GAL1/GAL10* promoter
(Fig. 4).

![](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/figure2-engenering.svg)

**Figure 4. A dual expression strategy for VP1 and VP2C-Tyr1 proteins for assembly of melanin-producing
nanoparticles.** *GAL1/GAL10* - bidirectional galactose-inducible *S. cerevisiae* promoter.
*VP1* -
gene, which encodes a major viral capsid protein that forms nanoparticles. *VP2C-Tyr1* - fusion of the
gene
encoding for *B. megaterium* tyrosinase with VP2C cargo protein that anchors tyrosinase to nanoparticles.

As a third strategy, we utilized an Aga2 yeast display system to synthesize melanin outside the yeast cell and
anchor
the melanin on the cell surface to function as a shield. We modified the yeast surface display design from (Lim
*et
al*., 2017). For this, we combined five modular protein domains to create a fusion protein with a new
function
(Fig. 5).

![](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/figure3-engenering.svg)

**Figure 5. A fusion protein of five domains to drive cell-wall-directed melanin synthesis and
accumulation.**
*GAL1* - strong galactose-inducible *S. cerevisiae* promoter. *4B4* encodes for a peptide that
selectively binds melanin. *G4S, G2S* linkers - flexible poly-Glycine-Serine linkers between different
protein
fusion domains. *SP* - synthetic α-factor prepro signal peptide. *Tyr1* - gene encoding for *B.
megaterium* tyrosinase. 3HA tag - Human influenza hemagglutinin protein tag.

These five protein modules (SP, 4B4, Aga2, Tyr1, 3HA tag) are fused together by flexible linkers. Signal peptide
from
the α-factor directs the engineered protein to the secretory pathway. Once secreted, Aga2 anchors the protein to
the
cell wall. 4B4 peptide is responsible for the binding of the synthesized melanin to accumulate it on the cell
surface
(Ballard *et al*., 2011). 3HA tag is used to test later the presence of the enzyme on the cell surface.

# Build: Assembling the expression cassettes and engineering the yeast strains

We used molecular cloning methods, including ligation, restriction, bacterial and yeast transformation, to build
our
constructs. All yeast strains generated and used are listed in Table 1.

**Table 1. Yeast strains used.** .

![](https://static.igem.wiki/teams/4440/wiki/hardware/engenering-table1.svg)

In order to build the construct for the cytoplasmic tyrosinase strategy, we used pRS306 plasmid with *GAL1*
promoter and *CYC1* terminator as a backbone. After cutting it with *BamH*I restriction enzyme, Tyr1
gene
(codon-optimized for the expression in yeast and ordered as synthetic DNA) was ligated into the pRS306-based
vector. The
pRS306 *pGAL1-TYR1-tCYC1* construct was then transformed into *E. coli* and the plasmid DNA from the
correct
clones was isolated. Finally, we inserted the construct into the genome of our background yeast strain by
homologous
recombination.

Analogous procedures were carried out to build the constructs of the second strategy using nanocompartments, and
the
third approach with cell wall localized tyrosinase. The *pGAL1-VP1*, *pGAL10-VP2C-Linker-Tyr1*, and
*pGAL10-SP-AGA2-Tyr1* constructs obtained in a ligation reaction were later isolated from bacterial cells
and
transformed into yeast.

# Test

We used several methods to experimentally test our three strains from the perspectives of cytotoxicity, melanin
production, and the identity of the synthesized products.

# Growth rate measurements reveal a slight growth defect for the Aga2-Tyr1 strain

From one side, there is always a risk that engineered proteins will have unexpected off-target effects that
cause
toxicity and need attention. Also, previous research has shown that melanin precursors can be cytotoxic due to
interfering with translation or causing the formation of free radicals (Giannopoulos *et al*., 2019);
Madhusudhan
*et al.*, 2014). To study the effect of tyrosinase expression on the yeast growth rate, we measured the
optical
density of the engineered yeast cultures while growing them in the presence of either galactose or glucose for
activation or suppression of tyrosinase expression, respectively. Most cultures reached the stationary phase
with OD600
in the range 5-9 during the course of the 60h experiment (Fig. 6). The strains expressing cytoplasmic or
particle-contained tyrosinase grew with the same rate as the background strain both in galactose- (*Tyr1*
is ON)
and glucose-containing (*Tyr1* is OFF) media (Fig. 6). However, we observed a slight growth defect in the
strain
expressing cell-wall-targeted Aga2-Tyr1 in galactose-containing media (Fig. 6).

![Fig.6. Line plot of growth rate for 4 different strains under 2 conditions, resulting in 8 measurements done at 0, 2, 4.5, 7.5, 10.5, 24, 48, 60 hours. For all strains except one growth rate are very similar to each other in both setups. Aga2-Tyr1 strain in the presence of galactose, which activates melanin production, has slightly lower OD600 than others.  Until 4.5 hours there is no significant change, but after it there is a steep slope. At 24 hours point, galactose induced Aga2-Tyr1 strain has 3.5 OD600, while others are in the 4.4-5.5 range. After that point, the slope becomes less steep, and at 48 hours galactose induced Aga2-Tyr1OD600 is 5.4 and others are in the 6-7.2 range. At 60 hours after steeper slope for galactose induced VP2C-Tyr1 and Tyr1, their OD600 reached 8.9 and 8.7 respectively; and Aga2-Tyr1 didn’t change much from the previous measurement, resulting in 5.6 OD600. ](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/lab-graph-curva.png)

**Figure 6. Growth curves of different strains in galactose-induced (*Tyr1* ON, melanin is produced)
or repressed
(*Tyr1* OFF, no melanin) conditions.** Tyr1 - strain with the tyrosinase in the cytoplasm.
*VP2C-Tyr1* -
strain with the tyrosinase in the self-assembled nanocompartments. *Aga2-Tyr1* - strain with tyrosinase
on the
surface of the cell wall.

# Tyrosinase expression drives the pigmentation of cells

We grew the yeast cultures in the presence of galactose or glucose from very low cell densities to the
stationary phase
(Fig. 6) and monitored potential melanin production by visually checking the color of the cultures. The color of
the
cells is an indication of melanin production, as melanin is a dark-colored pigment that also affects the color
of the
cells (Babaei *et al*., 2020; Gustavsson *et al*., 2016). We observed a distinct darkening of cells
expressing
Aga2-Tyr1 (cell-wall-targeted tyrosinase) 24h after the induction of tyrosinase production with galactose (Fig.
7). At
72h, the pigmentation was visible in all three strains expressing tyrosinase. The background strain (DOM90) and
cultures
grown in glucose, where tyrosinase expression is suppressed (Fig. 7), did not change their colors.
Interestingly, the
pigmentation had one tone in the culture with Aga2-Tyr1 but had a different color in cultures with cytoplasmic
tyrosinase (Tyr1) or particle-constrained tyrosinase (VP2C-Tyr1).

![Fig.7. Image comparison of different stains in flasks after 24 and 72 hours after galactose addition. For each of the 4 strains, there are samples without galactose addition, which did not show change in color. For samles with added galactose which induces tyrosinase expression, there is a change of color after 24 in Aga2-Tyr1, and after 72 hours for all 3 engineered strains.](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/engenering-colorful-bs.svg)

**Figure 7. Color change in cultures with tyrosinase expression.** The cultures grown at 30 °C
in the
presence of 2% galactose (tyrosinase expression ON) or 2% glucose (tyrosinase expression OFF) were imaged at
24h and 72h
time points.

# Fluorescence measurements confirm melanin production in the engineered yeast

![Fig.8 Bar chart of fluorescence intensity in arbitrary units for different strains using 2 time points of 0 and 60 hours after tyrosinase expression activation. In all cases except control without galactose, the peaks at 60 hours are higher than at initial time. For all engineered strains, fluorescence after 60 hours is higher in tyrosinase expression case. Aga2-Tyr1 has the highest peak around 700 AU after 60 hours with galascotse induction, with value aroun 10 AU at the beginning.](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/lab-fluor-all.png)

**Figure 8. Fluorescence quantification of extracted melanin.** Samples from 0h and 60h after activation
of
tyrosinase expression were collected and processed to extract melanin, which was then oxidized and measured
by
fluorescence. The plot shows the fluorescence measurements using 470 nm wavelength for excitation and 550 nm
for
emission.

We performed melanin extraction from our cultured yeast to identify the pigments. We collected samples from
the
stationary phase cultures at 60 hours after inducing tyrosinase expression. We implemented a protocol where
melanin is
solubilized during a 1-hour incubation at 80 °C in the presence of DMSO and alkaline conditions.
After
extraction, melanin is oxidized, and fluorescence is measured using 470 nm light for excitation and 550 nm for
emission
(Fernandes *et al*., 2016).
The fluorescence measurements showed that high levels of melanin were extracted from the galactose-induced
Aga2-Tyr1
strain at 60h (Fig. 8). The experiment is inconclusive for the cytoplasmic Tyr1 and VP2C-Tyr1 strains in terms
of
melanin production. Although we observed a slight increase in fluorescence in the samples from these strains,
it is not
clear
whether the difference is significant and whether melanin production is the cause.

# UV resistance experiments show the shielding effect of cell-wall-localized melanin

![](https://static.igem.wiki/teams/4440/wiki/proof-of-concept/frog-new.svg)

**Figure 9. Cell viability assay shows increased survival of Aga2-Tyr1 expressing cells upon UV
exposure.** Yeast
cultures grown for 24h in the presence of glucose (for a negative control) or galactose (to induce melanin
synthesis)
were exposed to varying times of UV light. Following the exposure, the cultures were serially diluted, and
the dilutions
were plated and grown for 24h to evaluate cell survival rates.

Next, we tested the photo-protective effect of pigment synthesis in our engineered yeast. We exposed cultures
that had
expressed tyrosinase for 24h to allow accumulation of melanin to different doses of UV radiation. In this
testing phase,
we used UV radiation instead of ionizing radiation due to additional safety concerns with ionizing radiation.
After
exposing the cultures to UV, we made serial dilutions of the cultures and plated these out to evaluate the
survival
rate. Importantly, we observed an increased survival of cells expressing cell-wall-localized Aga2-Tyr1 under
both tested
UV exposure conditions (3 and 10 minutes of UV) (Fig. 9). We did not observe any improved survival of cells
expressing
cytoplasmic tyrosinase or VP2C-Tyr1. Importantly, this result is supported by the melanin fluorescence
measurements. We
can speculate that the Aga2-Tyr1 strain produces eumelanin, which has brown or black color. At the same time,
red
pheomelanin is synthesized in the cytoplasm or nanocompartments of the other two strains, and its
radioprotective
properties are weaker in comparison to eumelanin (Brenner & Hearing, 2008; Pacelli *et al*., 2017).

# Learn

The first round of experiments showed that our yeast strains are capable of synthesizing melanin, as indicated
by the
culture color change (Fig. 7) and fluorescence analysis of extracted melanin (Fig. 8). These experiments also
gave us
several new directions for the investigation to improve our design.

**1.** We observed that targeting tyrosinase to the cell wall could be the most efficient approach in terms
of melanin
production. However, it causes a slight growth delay (Fig. 6). Further optimization of the strain would be
beneficial so
that the melanin synthesis would not hinder the performance of the cell factories, which are based on this
approach for
radiation protection. Possible solutions to reducing the toxicity include lowering the expression levels of the
Aga2-Tyr1 fusion protein.

**2.** These experiments revealed differences between the synthesized compounds when tyrosinase is expressed
inside the cells
or in the cell wall (Fig. 7 and 8). There are several forms of melanin with complex structures and different
absorbance
profiles (Cao *et al*., 2021). The color difference between our engineered strains indicates that different
types
of melanin are produced. Also, the observation of an earlier color change in the cells with cell-wall-anchored
tyrosinase could indicate that melanin synthesis is more efficient in an extracellular environment (Cao *et
al*.,
2021). The pathway from tyrosine to melanin includes oxidation steps, whose rate could depend on the redox
characteristics of the environment. This is supported by our finding of enhanced melanin synthesis in the
extracellular
space, which is a more oxidizing environment than the cytoplasm (López-Mirabal & Winther, 2008). This
observation led to
a new idea that intracellular melanin production could be improved by creating a more oxidizing environment
inside the
nanocompartments.

**3**
The melanin synthesis pathway is complex and can lead to the formation of diverse products. The color of the
cultures
and the results of fluorescence analysis indicate that the compound produced in Aga2-Tyr1 is, likely, eumelanin.
At the
same time, red pheomelanin can be synthesized in the cytoplasm or nanocompartments (Brenner & Hearing, 2008;
Pacelli et
al., 2017). More precise methods are necessary to determine the types of compounds produced in each strain.

**4**
UV radiation tests showed increased survival of cells expressing Aga2-Tyr1 but not of the other designs. This
observation further confirms our speculation about eumelanin synthesis in Aga2-Tyr1 since eumelanin has stronger
radioprotective properties. Further modifications might be necessary to achieve intracellular melanin
accumulation.
Also, optimization of the amount, distribution, or structure of melanin is required to accomplish greater
protection
from radiation in the Aga2-Tyr1 approach.

Babaei, M., Borja Zamfir, G. M., Chen, X., Christensen, H. B., Kristensen, M., Nielsen, J., & Borodina, I.
(2020).
Metabolic Engineering of *Saccharomyces cerevisiae* for Rosmarinic Acid Production. *ACS Synthetic
Biology,
9(8)*, 1978–1988. https://doi.org/10.1021/ACSSYNBIO.0C00048/SUPPL\_FILE/SB0C00048\_SI\_001.PDF

Ballard, B., Jiang, Z., Soll, C. E., Revskaya, E., Cutler, C. S., Dadachova, E., & Francesconi, L. C. (2011).
In vitro
and in vivo evaluation of melanin-binding decapeptide 4B4 radiolabeled with 177Lu, 166Ho, and 153Sm
radiolanthanides for
the purpose of targeted radionuclide therapy of melanoma. *Cancer Biotherapy & Radiopharmaceuticals,
26(5)*,
547–556. https://doi.org/10.1089/CBR.2011.0954

Brenner, M., & Hearing, V. J. (2008). The Protective Role of Melanin Against UV Damage in Human Skin.
Photochemistry and
Photobiology, 84(3), 539–549. https://doi.org/10.1111/J.1751-1097.2007.00226.X

Cao, W., Zhou, X., McCallum, N. C., Hu, Z., Ni, Q. Z., Kapoor, U., Heil, C. M., Cay, K. S., Zand, T.,
Mantanona, A. J.,
Jayaraman, A., Dhinojwala, A., Deheyn, D. D., Shawkey, M. D., Burkart, M. D., Rinehart, J. D., & Gianneschi,
N. C.
(2021). Unraveling the structure and function of melanin through synthesis. *Journal of the American
Chemical Society,
143(7)*, 2622–2637. https://doi.org/10.1021/JACS.0C12322/ASSET/IMAGES/MEDIUM/JA0C12322\_0012.GIF

Cheah, L. C., Stark, T., Adamson, L. S. R., Abidin, R. S., Lau, Y. H., Sainsbury, F., & Vickers, C. E. (2021).
Artificial Self-assembling Nanocompartment for Organizing Metabolic Pathways in Yeast. *ACS Synthetic
Biology,
10(12)*, 3251–3263. https://doi.org/10.1021/ACSSYNBIO.1C00045/ASSET/IMAGES/LARGE/SB1C00045\_0008.JPEG

Fernandes, B., Matamá, T., Guimarães, D., Gomes, A., & Cavaco-Paulo, A. (2016). Fluorescent quantification of
melanin.
*Pigment Cell & Melanoma Research, 29(6)*, 707–712. https://doi.org/10.1111/PCMR.12535

Giannopoulos, S., Samardzic, K., Raymond, B. B. A., Djordjevic, S. P., & Rodgers, K. J. (2019). L-DOPA causes
mitochondrial dysfunction in vitro: A novel mechanism of L-DOPA toxicity uncovered. *The International
Journal of
Biochemistry & Cell Biology, 117*, 105624. https://doi.org/10.1016/J.BIOCEL.2019.105624

Gustavsson, M., Hörnström, D., Lundh, S., Belotserkovsky, J., & Larsson, G. (2016a). Biocatalysis on the
surface of
*Escherichia coli*: melanin pigmentation of the cell exterior. *Scientific Reports 2016 6:1*, 6(1),
1–9.
https://doi.org/10.1038/srep36117

Gustavsson, M., Hörnström, D., Lundh, S., Belotserkovsky, J., & Larsson, G. (2016b). Biocatalysis on the
surface of
*Escherichia coli*: melanin pigmentation of the cell exterior. *Scientific Reports 2016 6:1*, 6(1),
1–9.
https://doi.org/10.1038/srep36117

Lim, S., Glasgow, J. E., Filsinger Interrante, M., Storm, E. M., & Cochran, J. R. (2017). Dual display of
proteins on
the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation
reactions.
*Biotechnology Journal, 12(5)*. https://doi.org/10.1002/BIOT.201600696

López-Mirabal, H. R., & Winther, J. R. (2008). Redox characteristics of the eukaryotic cytosol. *Biochimica
et
Biophysica Acta (BBA) - Molecular Cell Research*, 1783(4), 629–640.
https://doi.org/10.1016/J.BBAMCR.2007.10.013

Pacelli, C., Bryan, R. A., Onofri, S., Selbmann, L., Shuryak, I., & Dadachova, E. (2017). Melanin is effective
in
protecting fast and slow growing fungi from various types of ionizing radiation. *Environmental
Microbiology, 19(4)*, 1612–1624. https://doi.org/10.1111/1462-2920.13681

Tišma, M., Zelić, B., Vasić-Rački, D., Žnidaršič-Plazl, P., & Plazl, I. (2009). Modelling of laccase-catalyzed
l-DOPA
oxidation in a microreactor. *Chemical Engineering Journal, 149(1–3)*, 383–388.
https://doi.org/10.1016/J.CEJ.2009.01.025

References

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EXPERIMENTS

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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