| Molecular biological techniques represent powerful tools with applications to a wide variety of biomedical problems. Described herein is the use of metabolic engineering to manipulate sterol biosynthesis for medicinal purposes. Also presented is the cloning of terpene biosynthetic genes in plants of agricultural and pharmacological importance.; Two examples of metabolic engineering of the Saccharomyces cerevisiae sterol biosynthetic pathway are illustrated in Part I. We first demonstrated the development of genetically engineered S. cerevisiae strains that efficiently produce meiosis activating sterols (MAS), which are difficult to obtain by chemical synthesis or isolation from natural sources. Homologous recombination was used to construct an erg24Δerg25ΔhemlΔ mutant, which accumulated 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol (FF-MAS), and a similar erg25ΔhemlΔ mutant produced 4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol (T-MAS). This in vivo MAS production needs no added substrate, is technically simpler than chemical synthesis, and provides an inexhaustible source of MAS in relatively high purity and yield. In a different approach to metabolic engineering, we generated recombinant S. cerevisiae strains to screen drugs against the pathogenic parasite Trypanosoma cruzi. The native sterol Δ8-Δ7 isomerase in S. cerevisiae was replaced by the heterologous T. cruzi sterol isomerase and the corresponding human enzyme respectively. The relative effectiveness of the tested compounds on these two strains was compared using simple plate assay and validated by chromatographic methods. This differential screening method identifies inhibitors that specifically target the T. cruzi sterol isomerase with minimal side-effect on the parallel human enzyme and avoids the direct handling of deadly T. cruzi cells.; Part II of this thesis describes the characterizations of two plant terpene biosynthetic genes. The saponins in model legume Medicago truncatula may negatively affect the forage digestibility of ruminants, and the aglycones of these saponins are most likely derived from triterpene β-amyrin. A putative β-amyrin synthase clone uncovered from M. truncatula Expressed Sequence Tag libraries was expressed in S. cerevisiae , and the recombinant enzyme cyclized 2,3-oxidosqualene cleanly into β-amyrin. Also, a monoterpene synthase gene from Artemisia annua L., a medicinal plant with anti-malaria activity, was expressed in Escherichia coli, and the in vitro enzymatic reaction with geranyl pyrophosphate afforded (−)-β-pinene and (−)-α-pinene (94:6) as the cyclization products. |
