Heterologous production of pyomelanin biopolymer using 4-hydroxyphenylpyruvate dioxygenase isolated from Ralstonia pickettii in Escherichia coli

Abstract

Here, we report a novel melanin-type pyomelanin pigment derived from Ralstonia pickettii. Melanin-pigment producing R. pickettii was screened from soil sources; its specific growth rates were observed to be 0.143 h−1 and 0.091 h−1 in LB and NB media, respectively. The highest pigment production was 90.9 ± 5.3 mg/L (1.5 mg/L/h) in M9 minimal medium supplemented with l-tyrosine and copper ions. Purified melanin pigments exhibited a distinct spectral feature of λmax at 290 nm and a 108.9 μg/mL IC50 value for ABTS radical scavenging capacity. To identify the genes involved in pigment production, the gene sequences were annotated using sequence analysis. We identified four putative 4-hydroxyphenyl pyruvate dioxygenase (HPPD)-encoding genes responsible for pyomelanin synthesis from l-tyrosine. One hppD gene was amplified and expressed in E. coil BL21(DE3) KSYH. The HPPD protein was expressed in the soluble fraction, and the recombinant E. coli strain could produce pyomelanin at levels up to 315.5 ± 12.9 mg/L (13.1 mg/L/h) in 24 h. Pyomelanin production was highly dependent on metal ions. Additionally, the gene annotation results revealed that R. pickettii could produce eumelanin through a tyrosinase dependent pathway, and the l-DOPA (L-dihydroxyphenylalanine) intermediate was observed during LC/MS analysis, suggesting that the isolated strain produced eumelanin and pyomelanin simultaneously. These findings provide valuable information regarding melanin synthesis and applications for industrial use.