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Cloning and overexpression of a tyrosinase gene mel from Pseudomonas maltophila
1Department of Microbiology and Immunology, College of Life Sciences, Wuhan University, Wuhan, PR China. wang01@utsw.swmed.edu
Abstract:
The tyrosinase gene (mel), which is responsible for melanin formation, was isolated by shotgun cloning of SalI fragments of Pseudomonas maltophila DNA. A 0.7-kb SalI fragment in the recombinant plasmid pWSY8 imparted the ability to synthesize melanin to an Escherichia coli host HB101. The nucleotide sequence of this DNA fragment revealed an open reading frame of 504 bp, encoding a protein of 169 amino acids. The fragment containing the mel gene was then cloned into an expression plasmid pPAS1 under the control of a promoter isolated from the host, P. maltophilia AT18. This strain increased the melanin production by 70.6% compared with the strain HB101/pWSY8, in which the cloned mel gene was under the control of the lac promoter from the vector pUC18.
Insights
Researchers isolated the tyrosinase (mel) gene from Pseudomonas maltophila. This gene enables melanin synthesis in bacteria, with enhanced production achieved through optimized expression systems.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Melanin production is crucial in various biological processes.
- The tyrosinase gene (mel) from Pseudomonas maltophila is a key enzyme in melanin biosynthesis.
- Efficient genetic manipulation and expression are vital for studying microbial enzymes.
Purpose of the Study:
- To isolate and characterize the tyrosinase (mel) gene from Pseudomonas maltophila.
- To enable melanin synthesis in a heterologous host.
- To optimize melanin production through improved gene expression.
Main Methods:
- Shotgun cloning of SalI fragments from Pseudomonas maltophila DNA.
- Nucleotide sequencing of the isolated DNA fragment.
- Cloning the mel gene into expression plasmids (pWSY8 and pPAS1) with different promoters.
- Assessing melanin production in recombinant Escherichia coli strains.
Main Results:
- A 0.7-kb SalI fragment containing the mel gene was identified.
- The mel gene encodes a protein of 169 amino acids.
- Expression under a P. maltophila promoter in pPAS1 increased melanin production by 70.6% compared to lac promoter control in pWSY8.
Conclusions:
- The isolated Pseudomonas maltophila tyrosinase (mel) gene confers melanin synthesis capabilities.
- Optimized expression systems, utilizing native promoters, significantly enhance melanin yield.
- This work provides a foundation for further studies on microbial melanin production and its applications.