Expression of an artificial yeast TRP-gene cluster in yeast and Escherichia coli.
All five tryptophan biosynthetic genes of Saccharomyces cerevisiae were unified on plasmid pME554, which is based on 2 micrometer DNA and pBR322 sequences allowing for autonomous replication in yeast and E. coli. Homologous and heterologous expression of this artificial yeast TRP-gene cluster was studied. Plasmid pME554 allowed for nearly normal growth of a yeast strain bearing auxotrophic mutations in all five TRP-genes. The plasmid-borne genes TRP2 to TRP5 were expressed and regulated normally in the frame of the general control. Gene TRP1, carried on an EcoRI/Bg/II fragment lacking the ARS1 function, was expressed poorly and did not respond to the general control like the chromosomally-borne TRP1 gene. Plasmid pME554 allowed for poor growth of E. coli strain W3110 tna- delta trpEA2 on minimal medium. Marked stimulation was observed, however, when anthranilic acid or indole were added. Accordingly, poor expression of the first Trp-enzyme anthranilate synthase and the last enzyme tryptophan synthase was found, whereas the other three genes were moderately well expressed in E. coli.
D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005800:Genes, Fungal; D009876:Operon; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000096:biosynthesis / Q000235:genetics
D004269:DNA, Bacterial; D004271:DNA, Fungal; D014364:Tryptophan