No abstract

We have cloned and functionally expressed a tyrosinase gene from Streptomyces antibioticus in Escherichia coli under the control of an inducible bacteriophage T7 promoter. Recombinant E. coli cells containing the induced tyrosinase gene produced melanin pigments on agar plates and in liquid culture when supplemented with copper and tyrosine. The expression of an additional open reading frame from the mel gene locus of S. antibioticus was required for high-level melanin production in E. coli. Our results also show that it is possible to screen other classes of precursor compounds for incorporation into melanin pigments with unique colors and other biochemical features. In addition, it may be possible to screen for enhanced melanin production in the absence of added precursors to identify overproducing mutants in the amino acid biosynthetic pathways of E. coli. The ability to screen for a melanin phenotype in recombinant E. coli provides new opportunities for production of novel melanins and for protein engineering of tyrosinases with altered catalytic properties.

A recombinant Saccharomyces cerevisiae, expressing and secreting rice alpha-amylase, converts starch to ethanol. The rice alpha-amylase gene (OS103) was placed under the transcriptional control of the promoter from a newly described Pichia pastoris alcohol oxidase genomic clone. The nucleotide sequences of ZZA1 and other methanol-regulated promoters were analyzed. A highly conserved sequence (TTG-N3-GCTTCCAA-N5-TGGT) was found in the 5' flanking regions of alcohol oxidase, methanol oxidase, and dihydroxyacetone synthase genes in Pichia pastoris, Hansenula polymorpha, and Candida boidinii S2. The yeast strain containing the ZZA1-OS103 fusion secreted biologically active enzyme into the culture media while fermenting soluble starch.