| Melanins are a class of complex, polyphenolic heteropolymers that occur as black or dark pigments in bacteria, fungi, and higher organisms. Melanins have a variety of physiological functions that include transferring electrons, resisting UV radiation, scavenging oxygen free radicals, increasing environmental stress tolerance, binding toxic heavy metal, increasing the virulence and helping pathogens to interfere with the host immune response, and melanin also have a variety of application functions in agricultural production, pharmaceutical developments, food additives, cosmetics and so on.Alteromonas stellipolaris LMG21856strain is a Gram-negative bacterium, isolated from the Antarctic sea water, and could produce red-brown pigment with high efficacy and tolerates coldness, UV radiation and oligotrophic nutrition environment. In order to determine that4-hydroxyphenylpyruvate dioxygenase (HPPD) is (not) the key enzyme for A. stellipolaris LMG21856to synthsize melanin, we cloned and expressed the HPPD gene of A. stellipolaris LMG21856in E. coli BL21(DE3); and the secondary metabolites homogentisic acid (HGA) and melanin of the recombinant E. coli BL21(DE3)/pET3Oa-AsHPPD were identified.(1) Tyrosine metabolism pathway analysis.We submitted complete genome sequences of the pre-existing Alteromonas species in GeneBank to Rapid Annotation using Subsystem Technology server (RAST) for the Kyoto Encyclopedia of Genes and Genomes (KEGG) tyrosine metabolism pathway analysis, but, tyrosinase, laccase, and Polyketide Synthase are absent in Alteromonas species genomes except HPPD.(2) Construction of expression strain E. coli BL21(DE3)/pET30a-AsHPPD.1.5kb DNA fragment product was obtained from A. stellipolaris LMG21856genome by PCR, and it could encode a polypeptide with358amino acid residues. The polypeptide has a higher identity with HPPDs of Alteromonas genus, but has a lower identity with HPPDs of animals, plants, fungi and bacteria. The complete Open Reading Frame (ORF) of AsHPPD gene were amplified by PCR, and inserted into vector pET30a to construct expression strain, named E. coli BL21(DE3)/pET30a-AsHPPD. SDS-PAGE analysis of the whole-cell protein of E. coli BL21(DE3)/pET30a-AsHPPD after4hours inducing with0.5mmol/L IPTG, and an additional protein with approximately44kDa was observed compared with the negative control E. coli BL21(DE3)/pET30a. The expressed protein with6×His·Tag was determined with Western Blotting, and purified with Ni-NTA column.(3) Observation of the red-brown pigment production of A. stellipolaris LMG21856and E. coli BL21(DE3)/pET30a-AsHPPD using eyes.The fermentation broth of E. coli BL21(DE3)/pET30a-AsHPPD after7to8hours inducing with IPTG, the red-brown water-soluble pigment production is observed in fermentation broth of E. coli BL21(DE3)/pET30a-AsHPPD, and the phenomenon is similar to A. stellipolaris LMG21856after it is cultured for72to96hours. The production of melanin is greatly enhanced by the addition of tyrosine, and the color deepens with longer incubation time, which finally turned red-brown.(4) Detection of the secondary metabolite homogentisic acid (HGA) of E. coli BL21(DE3)/pET30a-AsHPPD and A. stellipolaris LMG21856strains using UV-Vis spectrophotometryThe absorption spectrum of the fermentation broth of A. stellipolaris and E. coli transformants during various periods of incubation was measured by UV-Vis wavelength scan (190-800nm), and the result shows:1) The solution of homogentisic acid standard sample has a maximum absorption peak at290nm, and its autoxidation production has an absorption peak at350nm;2) the cell-free fermentation broth of E. coli BL21(DE3)/pET30a-AsHPPD after1to2hours inducing with0.5mmol/L IPTG, there was an additional absorption peak at290nm, and another absorption peak was observed at350nm after the fermentation broth was induced for7to8hours (begin to produce pigment), and with shaking cultured, the pigment is synthesized continuously and the absorbance was increasing.Finally, the absorption peak at290nm disappeared;3) During the late-and (or) post-stationary grow of A. stellipolaris LMG21856strain, the cell-free fermentation broth began to have an absorption value at290nm and increased with longer incubation time, but the maximum absorption peak was at300nm, and the contribution may be due to the hydroxy of lipopolysaccharide and the metabolites. With longer incubation time, there was an absorption peak at350nm when melanin appeared in fermentation broth, finaly, the red-brown color became dark, and the absorption peak at290nm disappeared. (5) Identification of the secondary metabolite HGAof A. stellipolaris LMG21856and E. coli BL21(DE3)/pET30a-AsHPPD strains by High Performance Liquid Chromatography.The cell-free fermentation broth of E. coli BL21(DE3)/pET30a-AsHPPD after4hours inducing with IPTG has a elution peak at7.066min, and the cell-free fermentation broth of A. stellipolaris LMG21856after72hours culturing has a elution peak at7.096min. Because of the presence of a variety of salt ions in fermentation broth and the diversity of pH, the elution peaks are identified with the standard sample of homogentisic acid (6.996min). Based on the above experiment results, homogentisic acid is a secondary metabolite which could be synthesized with A. stellipolaris LMG21856and E. coli BL21(DE3)/pET30a-AsHPPD; and AsHPPD is a key enzyme for A. stellipolaris LMG21856to produce melanin.(6) Separation and analysis of the secondary metabolite melanin of A. stellipolaris LMG21856and E. coli BL21(DE3)/pET30a-AsHPPD strains using Thin Layer Chromatography.Based on technology of thin layer chromatography (TLC), the melanins of A. stellipolaris LMG21856and E. coli BL21(DE3)/pET30a-AsHPPD strains were separated, respectively; but we could not obtain some obvious and insular stripes on silica gel plate, and the pigment band like a triangle. When detected in UV light, the chromatographic band of pigment all emanated white light. |
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