Tyrosinase production in recombinant E. coli containing trp promoter and ubiquitin sequence.
We have successfully expressed the active tyrosinase of Streptomyces antibioticus in Escherichia coli under the control of the trp promoter by fusing the sequence to the ORF438 gene. Because our attempt to connect the polycistronic gene of ORF438 and tyrosinase directly to the trp promoter of E. coli resulted in the expression of functionally inactive tyrosinase, we decided to fuse the COOH-terminus of ubiquitin sequence to the NH2-terminus of ORF438. Ubiquitin fusion has been shown to augment the yield of cloned gene products in E. coli by increasing the stability or translational efficiency of the fusion proteins. As a result, E. coli transformants harboring a plasmid pTRUBF that contains the ubiquitin-fused ORF438 and the tyrosinase gene produced the strong black pigment of melanin. About 300 units of tyrosinase per liter of batch culture were detected when cultivated in M9 medium containing casamino acids, L-tyrosine, and copper supplements. The black pigment, however, was not seen when grown in LB medium, suggesting that the trp promoter is well regulated. When recombinant E. coli cells grown in LB medium were transferred to a tryptophan-deficient minimal medium with phenol, we observed that phenol was removed from the solution, and the color of the medium turned black. This is due to the fact that the tyrosinase has polyphenol oxidase properties. We expect to use this recombinant E. coli for the waste treatment of phenolic compounds.
Han
K
K
Biochemical Engineering Program, School of Engineering, University of California, Irvine 92717.
Hong
J
J
Lim
H C
HC
Kim
C H
CH
Park
Y
Y
Cho
J M
JM
D001483:Base Sequence; D017931:DNA Primers / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000235:genetics*; D010636:Phenols / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics; D014364:Tryptophan / Q000235:genetics; D014452:Ubiquitins / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000378:metabolism
D017931:DNA Primers; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D010636:Phenols; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D014874:Water Pollutants, Chemical; D014364:Tryptophan; D014442:Monophenol Monooxygenase