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).

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).

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).

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).

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).

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. .

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 BamHI 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).

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).

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

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

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.