{% extends "layout.html" %}
{% block title %} ENGINEERING {% endblock %}
{% block kostilj_style %}
{% endblock kostilj_style %}
{% block page_content %}
{%include 'moving-sun.html'%}
Design of melanin-shielded yeast was taken step-by-step, supported by research articles, modeling and data from
our
experiments. In this part of 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.
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 formation of free radicals that are also damaging to the cells. Another cause of L-DOPA toxicity arises from errors in translation. 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.
We took different multiple approaches to design a melanin production system in yeast cells. In the center of our 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).
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 synthesi
In the first design step we looked for the most efficient enzymes for melanin production. We found two candidate enzymes: 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 tyrosinase gene from B. megaterium for heterologous expression in yeast for melanin production (Fig. 2).
Figure 2. Melanin pathway introduced into S. cerevisiae yeast 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 cytoplasm (Fig. 3).
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. Implementation of these structures aims to increase the local concentration of enzymes, to protect the enzymes from degradation, and to 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 cargo protein) (Cheah et al., 2021). Expression of these genes is controlled by a bidirectional GAL1/GAL10 promoter (Fig. 4).
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 in order 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 together to create a fusion protein with a new function (Fig. 5).
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 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 binding of the synthesized melanin to accumulate it on the cell surface (Ballard et al., 2011). 3HA tag is used to later test the presence of the enzyme on the cell surface.
We used molecular cloning methods including ligation, restriction, bacterial transformation, and yeast transformation, in order to build our constructs. All yeast strains generated and used are listed in Table 1.
Table 1. Yeast strains used.
In order to build the construct of the cytoplasmic tyrosinase strategy, we used pRS306 plasmid with GAL1 promoter and CYC1 terminator as a backbone. After cutting it with BamHI restriction enzyme, SynDNA_Tyr1 gene 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.
We used several methods to experimentally test our three strains from the perspectives of cytotoxicity, melanin production, and the identity of the synthesized products.
From one side, there is always a risk that engineered proteins will have unexpected off-target effects that cause toxicity and need attention. Our other concern while designing these strains was that the precursors of melanin synthesis can be cytotoxic. To test this, we conducted a growth experiment and measured optical density at different time points while growing the engineered yeast cultures in the presence of either galactose or glucose for activation or suppression of melanin synthesis, respectively. During a 60-hour experiment, most of the cultures reached the stationary phase with OD600 in the range 5-9. The strains with intracellular melanin production grew very similarly to the background strain both in galactose- (Tyr1 is ON) and glucose-containing (Tyr1 is OFF media (Fig. 6). The strain expressing cell-wall-targeted Aga2-Tyr1, however, did have a slight growth defect in galactose-containing media (Fig. 6).
Figure 6. Growth curves of different strains in galactose-induced (Tyr1 ON, melanin is produced) or repressed (Tyr1 OFF, no melanin) conditions.
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. Previous studies have shown that production of melanin, which is a dark-colored pigment, also darkens the color of cells (Babaei et al., 2020; Gustavsson et al., 2016). At 24h from galactose addition we observed a significant color change in the cultures expressing Aga2-Tyr1 (cell-wall-targeted tyrosinase) but not in the other two strainsor the control DOM0090 (Fig. 7). At 72h, the color change became also apparent in the other two strainswith cytoplasmic tyrosinase (Tyr1) expression and viral-particle-directed tyrosinase (VP2C-Tyr1). At the same time, no color change occurred in the background DOM090 strain or in the cultures grown in glucose, where tyrosinase expression is suppressed (Fig. 7). Strikingly, the tone of the color was clearly different between the cultures with cytoplasmic tyrosinase (Tyr1) or particle-constrained tyrosinase (VP2C-Tyr1) and Aga2-Tyr1.
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.
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. Plot shows the fluorescence measurements using 470 nm wavelength for excitation and 550 nm for emission.
To gain further insights into the differences of the produced melanin forms between the three approaches, we
extracted
melanin from these cells for further characterization. For this, we collected cells at 60 hours after
activating
melanin
production. We used a previously developed method, where melanin is solubilized during a 1-hour incubation at
80
°C in the presence of NaOH and DMSO. In this protocol, melanin is oxidized, followed by
measurements
of
fluorescence using 470 nm light for excitation and 550 nm for emission (Fernandes et al.,
2016).
The experiment showed high levels of melanin in the galactose-induced Aga2-Tyr1 strainat 60h (Fig. 8). While
the
melanin
fluorescence levels from induced cytoplasmic Tyr1 and particle-constrained VP2C-Tyr1 were above the level of
that
measured from uninduced cultures or the background strain, the difference was quite minor (Fig. 8).
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 negative control) galactose (to induce melanin synthesis) were exposed to varying times of UV light. Following the exposure, the cultures were serially diluted, the dilutions were plated and grown for 24h to evaluate cell survival rates.
After detection of melanin in our yeast cells, we proceeded to test whether the melanin-containing cells have enhanced resistance to radiation. For the UV resistance experiments, we used cultures that were grown in media supplemented with galactose or glucose for 24 hours to accumulate sufficient amount of melanin. We irradiated the cultures with UV light for 3 and 10 minutes, made serial dilutions of the cultures and plated these out to evaluate the survival rate of different strains. Importantly, we observed a slightly increased survival of cells expressing Aga2-Tyr1 upon 3 and 10 minutes of UV exposure (Fig. 9). The experiments did not show any improved survival of cells expressing cytoplasmic tyrosinase or VP2C-Tyr1. The results of the UV resistance assay are in agreement with the melanin fluorescence measurements, which indicated that the product in Aga2-Tyr1 cells is indeed melanin, while the other two strains contain some other pigment.
The first round of experiments showed that our yeast strains are capable of synthesizing melanin, as indicated by the changes in the color of cultures (Fig. 7) and fluorescence analysis of extracted melanin (Fig. 8). These experiments also gave us several new directions for 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, but also that this process causes a small 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-tyrosinase fusion protein.
2. These experiments revealed that there are differences between the compounds that accumulate when tyrosinase is expressed in the cells or in the cell wall (Fig. 7 and 8). There are several types of melanin that have different absorbance profiles and due to the complexity of melanin structures there can also be significant diversity present in each case (Cao et al., 2021). The color difference between these two 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 Melanin synthesis pathway is complex and can lead to formation of diverse products. The results of fluorescence analysis indicate that the compound produced in Aga2-Tyr1 is melanin, while there is more uncertainty in the identity of the compounds produced intracellularly in Tyr1 and VP2C-Tyr1 strains. More precise methods are necessary to determine the types of compounds synthesized in each strain.
4 UV radiation tests showed increased survival of cells expressing Aga2-Tyr1 but not of the other designs. This indicates that further modifications are necessary to achieve intracellular melanin accumulation. Also, optimisation in terms of the amount, distribution, or structure of melanin is necessary to accomplish greater protection from radiation in the case of 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
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
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
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