Two mutually exclusive biosynthetic pathways have evolved in nature for the synthesis of the essential amino acid, lysine. Biosynthesis of lysine in yeast and other higher fungi occurs via the uniquely evolved Alpha-aminoadipic acid pathway whereas in bacteria and green plants the biosynthesis of lysine occurs via the diaminopimelic acid pathway. Most genetic, biochemical, and regulatory studies of the Alpha-aminoadipate pathway including those in my laboratory have been performed in the yeast, Saccharomyces cerevisiae. There are eight enzyme catalyzed steps and more than fourteen gene loci responsible for the biosynthesis of lysine in S. cerevisiae. Lysine genes are not linked to each other and seven of these genes have been mapped on six different chromosomes of S. cerevisiae. Our studies with lysine auxotrophs have revealed that five distinct gene functions are required for the last three enzyme steps (mutants lys2 and lys5 lack Alpha-aminoadipate reductase, lys9 and lys14 lack saccharopine reductase, and lys1 lacks saccharopine dehydrogenase which catalyzes the last biosynthetic step). We have recently purified and determined the molecular properties including the subunits composition of -aminoadipate reductase and saccharopine reductase. Saccharopine dehydrogenase is a monomeric enzyme. Inspite of the availability of considerable genetic and biochemical information, little is known regarding the physical properties, expression, and regulation of cloned lysine genes except LYS2 which has been cloned in two different laboratories. Because of the simple gene enzyme relationship, we intend to clone initially LYS1 gene by functional complementation of lys1 auxotroph and develop molecular biological capabilities to investigate (a) the physical properties of cloned LYS1 gene by restriction mapping, (b) expression of cloned LYS1 gene in the transformant by determining the specific activity of saccharopine dehydrogenase, and (c) transcriptional control of LYS1 gene expression by determining the levels of LYS1 specific mRNA in appropriate transformant. Current study should pave the way for cloning other lysine genes and investigation of coding and promoter regions as well as transcriptional control of other lysine genes of S. cerevisiae.
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