Cloning of the δ-aminolevulinic acid synthase structural gene in yeast

Summary

HEM1, the structural gene for δ-aminolevulinic acid synthase, has been isolated on recombinant plasmids. A yeast genomic pool constructed in the E. coli — yeast shuttle vector YEp13 was used to clone the HEM1 gene by complementation. A leu2 hem1 yeast mutants was transformed with the yeast genomic pool and hybrid YEp13 plasmids carrying the HEM1 gene were cloned by their ability to complement both the leu2 and hem1 mutations in the recipient strain. The yeast transformants, bearing the HEM1-containing plasmids pYe(HEM1), showed a 24–28 fold increase in δ-aminolevulinic acid synthase activity and in the intracellular content of δ-aminolevulinic acid (5–8 fold) as compared to wild type strains, suggesting that the p(HEM1) gene is being expressed as a catalytically active enzyme which can be transported into the mitochondria. However, the transformant strains did not present higher-than-normal content of heme or cytochromes either in glucose or in glycerol media, indicating that the production of δ-aminolevulinic acid is not the rate-limiting step in heme biosynthesis in yeast.