Metabolic engineering of Saccharomyces cerevisiae towards increased production of terpenes and characterization of sterol biosynthetic enzymes
Hart, Elizabeth A.
Matsuda, Seiichi P. T.
Doctor of Philosophy
The yeast Saccharomyces cerevisiae possesses the endogenous precursor for diterpene production but does not biosynthesize diterpenes. Part I of this thesis describes metabolic engineering of S. cerevisiae to achieve diterpene biosynthesis at 500-fold increased production levels (5 mg/L) relative to levels observed in wild type yeast. Induced expression of S. cerevisiae genes geranylgeranyl pyrophosphate synthase (BTS1) and a truncated form of 3-hydroxy-3-methylglutaryl CoA reductase (HMG1) in yeast carrying the upc2-1 allele afforded accumulating geranylgeranyl pyrophosphate, the universal precursor for diterpene biosynthesis. The precursor demonstrated efficient cyclization to 7,13-abietadiene upon coexpression of transformed Abies grandis abietadiene synthase in a multiple copy yeast shuttle vector. Similarly, metabolic engineering of S. cerevisiae allowed investigation of achieving attenuated sesquiterpene production in vivo; those results and possible physiological implications to yeast are discussed. The recombinant strains serve as an alternative means of access to natural products via a novel in vivo production system. Part II describes the characterization of three sterol biosynthetic enzymes. Higher plants catalyze the cyclization of (S)-2,3-oxidosqualene to cycloartenol. A single point mutation demonstrated altered sterics contributing to catalytic product specificity; two point mutations are characterized and evaluated. Cycloartenol constitutes a cyclopropyl sterol structure not found in other eukaryotes. In an effort to better understand the roles served by the cyclopropyl sterols, the characterization of the gene responsible for their metabolism is described. Understanding this point of evolutionary divergence can facilitate accurate phylogenetic analysis among eukaryotes.
Molecular biology; Biochemistry