Melanin

Pseudomonas koreensis

Target

Isolate microorganisms capable of producing melanin from soil using different media and culture conditions, and identify them through morphological observation and molecular biology methods (such as 16S rDNA sequence analysis). Investigate the specific biochemical pathways for melanin synthesis by microorganisms, including the identification and functional study of key enzymes, as well as the regulatory mechanisms of melanin synthesis. Study the survival of melanin-producing microorganisms under UV irradiation to assess the radio protective effect of melanin.

Results

Starting with tyrosine as the substrate, melanin-producing microorganisms were screened and obtained from the environment. After separation and purification, a strain of Pseudomonas that produces melanin was isolated. PCR amplification of the 16S rRNA gene fragment of the Pseudomonas was performed using primers 1492R and 27F.

Figure 1  Electrophoresis identifying individual colonies of Pseudomonas species by colony PCR with specific primers.

The amplified 16S rRNA gene fragment was sequenced to obtain a wealth of sequence information, which was compared with the known bacterial 16S rRNA gene database, and the NJ tree is as follows, named Pseudomonas sp. QDLY500GRS.

Figure 2  The NJ tree of Pseudomonas sp. QDLY500GRS.

Figure 3  The agar plate of Pseudomonas sp. QDLY500GRS.

The UV-visible absorption of the black pigment in the liquid culture of Pseudomonas sp. QDLY500GRS was measured. True melanin exhibits absorption peaks around 220-240nm and 300-400nm, while pheomelanin has an absorption peak around 280-320nm, with a peak between 230nm and 250nm. Preliminary judgment indicates that Pseudomonas sp. QDLY500GRS may produce eumelanin.

Figure 4  The UV-visible absorption of the black pigment in Pseudomonas sp. QDLY500GRS.

Melanin was successfully crudely extracted from the liquid culture medium of Pseudomonas sp. QDLY500GRS through steps such as acid adjustment, base adjustment, and heating.

Figure 5  Crude extraction of melanin.

Summary

We have successfully isolated Pseudomonas sp. QDLY500GRS, a bacterium capable of producing melanin, from the soil and crudely extracted the melanin, laying the foundation for further study of the melanin synthesis mechanism of this bacterial strain and the application of melanin.

References

[1] Guo Jing. Cloning, expression, and molecular modification of the key enzyme tyrosinase gene for melanin synthesis in Streptomyces kathirae SC-1 [D]. Jiangnan University, 2015.

[2] Wei Hailan, Yu Libo, Yi Zhiwei, et al. Study on the melanin production conditions of the deep-sea bacterium Pseudomonas sp. blp-2 and cloning of its hppD gene [J]. Journal of Applied Oceanography, 2014, 33(04): 499-507.

Escherichia coli

Target

Escherichia coli, as a model organism, has a very cost-effective and efficient expression system. It is used as a chassis organism to clone the PkhppD and pelB genes from Pseudomonas koreensis, and the hppD gene from Pseudomonas aeruginosa, and to express the cloned hppD gene in E. coli.

Results

（1）Construct the PeLB-PkhppD-6XH plasmid

Using the designed primers, perform PCR reactions with templates containing the target genes (pelB, Pkhppd) from Pseudomonas koreensis to amplify the required gene fragments. Select appropriate restriction enzymes to double digest the pET-20b (+) plasmid, creating sticky ends that match the target gene fragment (PkhppD), PelB sequence, and 6xHis tag sequence. Connect the purified target gene fragment (PkhppD), PelB signal peptide sequence, 6xHis tag sequence, and the digested pET-20b (+) plasmid backbone to obtain the PeLB-PkhppD-6XH plasmid; transform it into the suitable host cells - Escherichia coli BL21 (DE3) / BL21 (DE3) pLys.

Figure 6  The genetic circuit diagram of PeLB-PkhppD-6XH

When attempting to induce the expression of PeLB-PkhppD-6XH for melanin production in Escherichia coli BL21 (DE3) / BL21 (DE3) pLys using L-arabinose, the expression outcome was not as effective as that of the hppD gene , with only a small amount of melanin being expressed.

Figure 7  PeLB-PkhppD-6XH is expressed with low levels of melanin in Escherichia coli BL21 (DE3) / BL21 (DE3) pLysS.

（2）Construct the PBAD-6XH-PelBPkrhdd plasmid.

Using the designed primers, perform PCR reactions with templates containing the target genes (pelB, Pkhdd) from Pseudomonas koreensis to amplify the required gene fragments. Select appropriate restriction enzymes to digest the pBAD plasmid, creating sticky ends that match the target gene fragments. Purify the target gene fragments and ligate them with the digested pBAD plasmid using DNA ligase. The target gene fragments combine with the plasmid backbone through complementary sticky ends to form the complete plasmid pBAD-pelBPkrhdd. Transform the ligation product into the suitable host cells - Escherichia coli TOP10.

Figure 8  The genetic circuit diagram of PBAD-6XH-PelBPkrhdd.

The PBAD vector contains the arabinose operon, and the absence of melanin production after induction with L-arabinose indicates that the construction of the PBAD-6XH-PelBPkrhdd plasmid has failed.

Figure 9  Transform the plasmid pBAD-pelBPkrhdd into Escherichia coli and induce with arabinose.

（3）Pahdd-6xH in pET-21b(+)

Based on Pseudomonas aeruginosa PAO1, the [gene=hppD] [protein=4-hydroxyphenylpyruvate dioxygenase] was synthesized with a 6xH tag as Pahdd-6xH in pET-21b(+), with the expected protein localization within the bacterial cells; based on Pseudomonas_koreensis_D26, the [gene=hppD][protein=4-hydroxyphenylpyruvate dioxygenase] was synthesized with the PelB signal peptide and 6His tag; the designed primer sequences were submitted to a professional primer synthesis company for chemical synthesis of the tyrosinase gene. The tyrosinase gene from Pseudomonas with a 6xHis tag was expressed in Escherichia coli in the pET-21b(+) vector, resulting in Pahdd-6xH in pET-21b(+).

Using genetic engineering techniques, the gene for 4-hydroxyphenylpyruvate dioxygenase (hppD) was heterologously expressed in Escherichia coli, achieving efficient synthesis of the target protein. The purification method for the hppD 6XHis tagged protein utilized the affinity between the histidine tag and metal ions, allowing for rapid and efficient purification of the target protein.

The genetic circuit diagram is as follows:

Figure 10  The genetic circuit diagram of Pahdd-6xH in pET-21b(+).

Express Pahdd-6xH in pET-21b(+) in Escherichia coli BL21 (DE3) / BL21 (DE3) pLys using IPTG induction; as shown in the figure, melanin production is clearly visible. This indicates that our plasmid construction was successful.

Figure 11  The liquid culture medium in which Pahdd-6xH in pET-21b(+) successfully expressed melanin.

Figure 12  We successfully expressed the Pahdd gene using IPTG induction, and the 2×BL21(DE3) strain performed better than the 2×BL21(DE3) pLyss; the expression of melanin was more effective than that of the PkhppD gene.

（4）The impact of inducers on Escherichia coli expressing melanin.

Using TPTG and copper ions to induce Pahdd@DE3, and arabinose and copper ions to induce PkhppD@DE3, it was found that the inducers (IPTG/Arabinose) and copper ions on the plate did not increase the survival rate of Pahdd@DE3, but they did promote the survival rate of PkhppD@DE3. It is possible that the original bacterial solution of Pahdd@DE3 already expresses a sufficient amount of melanin, while the bacterial solution of PkhppD@DE3 expresses relatively less melanin (see the color of the shake flask).

Figure 13  On the left is Pahdd@DE3 induced with IPTG and copper ions, and on the right is PkhppD@DE3 induced with arabinose and copper ions.

5. The survival rate of E. coli expressing melanin under UV irradiation.

After adjusting the OD value of the overnight cultured bacterial solution to a suitable level, 5 microliters were added to the plate, and then irradiated for 2 hours and 4 hours under a UV intensity of 168.5 μW/cm². After that, they were placed in a biochemical incubator for overnight cultivation at 30°C, and the survival of E. coli expressing melanin under UV irradiation was obtained through the formula: rate(%) = (CFU after irradiation / CFU before irradiation) * 100.

Figure 14  The survival rates of Pahdd@DE3 (PahppD) and PkhppD@DE3 after 2 hours and 4 hours of UV irradiation.

UV irradiation time.	Pahdd@DE3 (PahppD)	PkhppD@DE3
2h
4h

Table 1  The petri dish after the strain was cultured overnight following 2 hours and 4 hours of UV irradiation.

Summary

We have successfully utilized the Pahdd-6xH in pET-21b(+) plasmid and PeLB-PkhppD-6XH plasmidto express melanin in Escherichia coli, providing a model for subsequent research on the mechanism of melanin synthesis. Conducted UV irradiation experiments and catalyst induction experiment on Escherichia coli expressing two types of plasmids. This lays a certain foundation for the future large-scale production of melanin and the study of its mechanisms in our project.

References

[1] ChiaraScanferla EnricoCaruso Viviana TeresaOrlandi FabrizioBolognese. Bacterial melanin production by heterologous expression of 4‑hydroxyphenylpyruvate dioxygenase from Pseudomonas aeruginosa[J]. International Journal of Biological Macromolecules, 2019, 133: 1072-1080.

[2] S.eskandari Z.Etemadifar. Melanin biopolymers from newly isolated Pseudomonas koreensis strain UIS 19 with potential for cosmetics application, and optimization on molasses waste medium[J]. Journal of Applied Microbiology, 2021.

[3] DavidHörnström SusannaLundh JaroslavBelotserkovsky GenLarsson MartinGustavsson. Biocatalysis on the surface of Escherichia coli: melanin pigmentation of the cell exterior[J]. Scientific Reports, 2016, 6.

[4] Cai Kun, Chu Yindi, Huang Piying, et al. Research progress on bacterial cell surface protein display technology based on protein translocation across the outer membrane [J]. Acta Microbiologica Sinica, 2022, 62(02): 458-475.

[5] Victor EBalderas-Hernandez AídaGutiérrez-Alejandre AlfredoMartinez FranciscoBolívar GuillermoGosset María IChávez-Béjar. Metabolic engineering of Escherichia coli to optimize melanin synthesis from glucose[J]. Microbial Cell Factories, 2013, 12

Yeast

Target

Compared to Escherichia coli, yeast can perform various post-translational modifications such as glycosylation and phosphorylation, which may be necessary for the synthesis of melanin involving specific modifications of enzymes or proteins. Moreover, yeast has a strong secretion capability, allowing it to secrete synthesized melanin outside the cell, facilitating subsequent separation and purification. Although the growth rate of yeast is typically slower than that of E. coli, yeast can grow on relatively simple media and achieve high cell densities. Additionally, yeast exhibits greater tolerance to certain environmental factors. Therefore, using yeast as a chassis organism may be more advantageous.

Clone the Pkrhdd gene from Pseudomonas koreensis and express it in Pichia pastoris; clone the LAC1 gene from Cryptococcus neoformans, and using the strong inducible promoter GAL1, it can be induced in a galactose-containing medium, which can powerfully drive gene expression in Saccharomyces cerevisiae, facilitating the expression of the target gene LAC1 in brewing yeast.

Results

（1）In the Pichia pastoris yeast, the plasmid pPICZaAPkrhdd-6xH.

Choose the appropriate restriction enzymes to perform double digestion on the pPICZaA plasmid, generating sticky ends that match the target gene fragment (Pkrhdd) and the 6xHis tag sequence. Separate and purify the digested plasmid backbone. Perform a ligation reaction with the purified target gene fragment (Pkrhdd), the 6xHis tag sequence, and the digested pPICZaA plasmid backbone.

Figure 15  The genetic circuit map of pPICZaAPkrhdd-6xH

We are using chemical and electroporation methods to transform the pPICZaAPkrhdd-6xH into Pichia pastoris, and colonies have formed after cultivation, but there is no melanin expression after induction with methanol. This could be due to a failure in plasmid construction, or it could be due to other reasons.

Figure 16  Transformation of pPICZaAPkrhdd-6xH in Pichia pastoris resulted in the appearance of colonies.

（2）Brewer’s yeast GA11-gene-CYC1

The yeast galactokinase (GAL1) gene is a strongly inducible promoter and is the most commonly used promoter in the yeast recombinant protein expression system. The transcriptional activity of the GAL1 promoter is related to the carbon source in the culture medium. In the presence of glucose, the transcription of the GAL1 promoter is inhibited; galactose, on the other hand, activates the promoter. Typically, after about 4 hours of induction with galactose, the expression of recombinant proteins can be detected in cells cultured with glucose. The role of the mel gene in brewing yeast is mainly as a reporter gene. The tyrosinase enzyme it encodes can catalyze the conversion of tyrosine to dopamine, which is then further converted into melanin. This produced melanin is non-toxic and harmless, allowing for the direct observation of a black phenotype on the culture medium without the need to add color-developing compounds or use special instruments.

The melanin expressed by the mel gene in brewing yeast appears red, possibly because a larger amount of pheomelanin is produced.

Figure 17  the genetic circuit diagram of thebrewing yeast transformed with the mel gene

Figure 18  The transformation of brewing yeast with mel gene

Using Saccharomyces cerevisiae transformed with mel and non-transformed Saccharomyces cerevisiae for UV irradiation at 0h, 2h, 4h, and 8h to express radiation resistance through survival rates.

(Ultraviolet irradiation of 168.5 μW/cm²)Ultraviolet irradiation time.	Saccharomyces cerevisiae survival（transformed with the mel gene ）	Saccharomyces cerevisiae transformed with the mel gene survival(Experimental results figure)	Saccharomyces cerevisiae survival(Not transformed.)	Saccharomyces cerevisiae survival(Experimental results figure)
0h	160		88
2h	117		62
4h	115		18
8h	109		4

Table 2  The survival of yeast expressing the mel gene, which produces melanin, and normal yeast after exposure to ultraviolet light with an intensity of 168.5 μW/cm².

Survival rate
time/h	Saccharomyces cerevisiae expressing the mel gene.	Non-transformed brewer’s yeast.
0	1	1
2	0.73125	0.704545455
4	0.71875	0.204545455
8	0.68125	0.045454545

Table 3  The survival rates of Saccharomyces cerevisiae transformed with mel and non-transformed Saccharomyces cerevisiae under UV irradiation, as calculated from Table 2.

Figure 19  Survival of bacterial strains under UV irradiation

From Figure 19, it is known that the survival rate of strains expressing melanin after exposure to ultraviolet light with an intensity of 168.5 μW/cm² is higher than that of normal strains.

(3)Brewer’s yeast ADE2-gal1-egfp-CYC1+

In brewing yeast, the GAL1 promoter, tyrosine as the target gene, and the CYC1 terminator are used to express the tyrosine synthesis pathway for the production of melanin in yeast.

Figure 20  the genetic circuit diagram of the brewing yeast transformed with the tyrosinase(BM) gene

We successfully expressed melanin in Saccharomyces cerevisiae and used it for ionizing radiation experiments.

Figure 21  The culture medium for brewing yeast that has been transformed with the tyrosinase (BM) gene and expresses melanin.

Measuring the radiation resistance of brewing yeast expressing melanin using ionizing radiation.

We exposed brewing yeast expressing ADE2-gal1-egfp-CYC1+ to ionizing radiation at a photon energy of 20keV, with an irradiation intensity of 1Gy/h, for periods of 1 hour, 2 hours, 4 hours, and 8 hours; the results are as follows:

Ionizing radiation time.	Control group.（ADE2-gal1-egfp-CYC1+）	Control group: the number of petri dish colonies for (ADE2-gal1-egfp-CYC1+).	Experimental group.（ADE2-gal1-egfp-CYC1+）	Experimental group: the number of petri dish colonies for (ADE2-gal1-egfp-CYC1+).
1h		1654		810
4h		1591		454
8h		726		43
12h		1827		0

Table 4  Survival of ADE2-gal1-egfp-CYC1+ under ionizing radiation.

Figure 22  The trend chart of the survival rate of ADE2-gal1-egfp-CYC1+ under ionizing radiation.

Summary

We successfully transformed the GA11-gene-CYC1 plasmid in Saccharomyces cerevisiae.

Based on Figure 19, compared to normal brewer’s yeast, brewer’s yeast transformed with mel exhibits strong radioresistance. Based on the experimental results from Table 4, which serves as the control group in ionizing radiation, we will use the successfully engineered melanin-producing Saccharomyces cerevisiae as the experimental group for ionizing radiation exposure to further refine our project.

References

[1] Stagoj,M.N., Comino, A.,& Komel, R.(2005). Fluorescence based assay of GAL system in yeast Saccharomyces cerevisiae. FEMS Microbiology Letters, 244(1), 105-112. https://doi.org/10.1016/j.femsle.2004.12.025

[2] Guo, Z.,&Sherman, F.(1996). Signals sufficient for 3′-end formation of yeast mRNA. Molecular and Cellular Biology, 16(6), 2772-2776. https://www.semanticscholar.org/paper/Signals-sufficient-for-3’-end-formation-of-yeast-Guo-Sherman/966b6e1fc4842b4a312f466f401f6b38f0c7c217

[3] Lee, Dongpil et al.”Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans.” mBio, vol. 10, no. 5, 2019. https://doi.org/10.1128/mBio.02133-19

[4] Wang Yujie, Huang Yuping, Ruan Lifeng, Liu Nan, Shen Ping. “Cloning and Expression of the mel Gene in Saccharomyces cerevisiae.” Microbiology China 30.4 (2003). DOI: 10.3969/j.issn.0253-2654.2003.04.011

[5] https://2022.igem.wiki/sjtu-biox-shanghai/design

[6] Nan-NanLiu Guang-LeiLiu ZheChi Jian-MingWang Ly-LyZhang Zhen-MingChi HongJiang. Melanin production by a yeast strain XJ5-1 of Aureobasidium melanogenum isolated from the Taklimakan desert and its role in the yeast survival in stress environments[J]. Extremophiles, 2016, 20: 567-577.

Hydrogel

Target

Using hydrogels to encapsulate engineered bacteria (such as melanin-producing Pseudomonas, engineered strains with the hppD gene, microalgae, etc.) aims to achieve the goals of bioremediation, agriculture, and the production of engineered living materials and devices. Hydrogels provide a tunable protective environment that allows bacteria to perform their engineered functions while preventing their escape into the environment. This physical encapsulation strategy (DEPCOS) combines chemical and physical encapsulation strategies to achieve safer and more effective bioencapsulation.

Results

Algae and Pseudomonas were encapsulated in hydrogels, and observed under fluorescent conditions. After five days of growth, a distinct red fluorescence reaction was observed within the hydrogel, thereby demonstrating that algae and bacteria can grow within the hydrogel.

Figure 23  Encapsulating E. coli expressing GFP within a hydrogel.

Figure 24  The red fluorescence observed in the first well is from the encapsulated cyanobacteria-algae symbiotic culture within the hydrogel.

Summary

We have successfully cultured the target microbial strains in hydrogels. Leveraging the biocompatibility of hydrogels, we can prevent the escape of strains into the environment, which makes for a safer and more effective method of biological encapsulation, offering more possibilities for the cultivation and application of microorganisms.

References

[1] Martin, N., Bernat, T., Dinasquet, J., Stofko, A., Damon, A., Deheyn, D. D., Azam, F., Smith, J. E., Davey, M. P., Smith, A. G., Vignolini, S., & Wangpraseurt, D. (2021). Synthetic algal-bacteria consortia for space-efficient microalgal growth in a simple hydrogel system. Journal of Applied Phycology, 33(5), 2805–2815. https://doi.org/10.1007/s10811-021-02528-7

[2] Tang, T.-C., Tham, E., Liu, X., Yehl, K., Rovner, A. J., Yuk, H., de la Fuente-Nunez, C., Isaacs, F. J., Zhao, X., & Lu, T. K. (2021). Hydrogel-based biocontainment of bacteria for continuous sensing and computation. Nature Chemical Biology, 17, 724–731. https://doi.org/10.1038/s41589-021-00779-6

The construction of an algal-bacterial symbiotic system.

Target

In a greenhouse, green algae (Chlorella vulgaris/Scenedesmus obliquus) and melanin-producing Pseudomonas putida are co-cultivated under artificial bubble membranes. The green algae utilize urea, urine, and carbon dioxide, and through photosynthesis, they release oxygen and produce sugars. The melanin produced by the Pseudomonas putida helps protect against X-rays and ultraviolet rays from solar radiation. Synthetic biology is used to regulate the relationship between the two organisms to ensure their compatibility.

We co-cultivate the melanin-producing Pseudomonas (Pseudomonas sp. QDLY500GRS) with algae (Gloeocapsa), utilizing the Pseudomonas to provide CO2 and other nutrients to the Gloeocapsa, while the Gloeocapsa supplies O2 to the Pseudomonas. The melanin secreted by Pseudomonas may provide the ability to protect microalgae from radiation.

Figure 25  A simple schematic diagram of the co-cultivation system between microalgae and Pseudomonas or E. coli expressing melanin.

Results

When the algal-bacterial co-cultured liquid is placed in a hydrogel and exposed to fluorescence, the presence of red fluorescence indicates good growth of the algae; visual observation shows that the expression of melanin in the algal-bacterial co-culture is better than that in Pseudomonas alone.

Figure 26  On the left is Pseudomonas sp. QDLY500GRS.

On the right is Gloeocapsa algae with Pseudomonas sp. QDLY500GRS.

Experimental image
Experimental conditions.	Exposure to 1Gy of radiation, photographed under white light.On the left; On the right, melanin plus copper ions.	Photograph taken under white light without radiation exposure. On the right, melanin plus copper ions.
Experimental image
Experimental conditions.	Exposed to 1Gy of radiation, photographed under blue light.On the left; On the right, melanin plus copper ions.	Photograph taken under blue light without radiation exposure.On the left; On the right, melanin plus copper ions.
Experimental results.	Green algae encapsulated in hydrogel, further encapsulated in an agar environment containing melanin and metal ions, are more susceptible to death from X-ray radiation. It is possible that the surrounding melanin and metal ions absorb the radiation energy and transfer it to the hydrogel-encapsulated green algae, causing their death. In contrast, environments without melanin and metal ions are less likely to cause death from radiation.

Table 5  The interaction between microalgae and Pseudomonas putida under ionizing radiation conditions and without ionizing radiation.

From Table 5, we preliminarily speculate that melanin and metal ions may be able to absorb more radiation. If they are not separated from the protected organisms/cells/organelles, it is very likely that the energy or ions generated after being radiated will be transferred to the protected parts, which could kill the protected parts, including organisms, cells, organelles, or molecules. Therefore, the spatial distribution of melanin (metal ions) is also very important for the protected parts. In bacteria, fungal hyphae/spores, the cell walls of human skin/hair/eyes, this spatial distribution mechanism may need to be considered. In addition, melanin alone may be more about absorbing ultraviolet light, protecting the protected parts from ultraviolet damage.

Summary

We have successfully established a symbiotic system between microalgae and bacteria and cultivated it in hydrogels, which is very meaningful for studying the ways in which microalgae and bacteria promote each other in their symbiotic relationships, including nutrient exchange, signal transduction, and gene transfer.

We have modified the model in Figure 29 based on the experimental results from Table 4. The protected organisms (for example, photosynthetic algae in porous material) are separated from the protective layer (melanin + metal ions + porous material), and an insulating layer is added in between to isolate the energy/ions produced after the protective layer absorbs radiation. See figure below.

Figure 27  The revised model will be planned for the next phase.

References

[1] Nobutaka Fujieda,Kyohei Umakoshi,Yuta Ochi, et al. Copper–Oxygen Dynamics in the Tyrosinase Mechanism[J]. Angewandte Chemie, 2020, 132: 13487-13492.Martin, Noah, et al. “Synthetic algal‑bacteria consortia for space‑efficient microalgal growth in a simple hydrogel system.” Journal of Applied Phycology, vol. 33, no. 5, 2021, pp. 2805–2815. https://doi.org/10.1007/s10811-021-02528-7.

[2] Nobutaka Fujieda,Kyohei Umakoshi,Yuta Ochi, et al. Copper–Oxygen Dynamics in the Tyrosinase Mechanism[J]. Angewandte Chemie, 2020, 132: 13487-13492.