Cloning and expression of a tyrosinase from Aspergillus oryzae in Yarrowia lipolytica: application in L-DOPA biotransformation.

Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po 1 g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po 1 g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with L-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed L-tyrosine to L-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60° C and with 3.5 mg of biomass), 0.4 mg/ml of L-DOPA was obtained.