| Abstract |
Plants produce an enormous variety of low molecular weight compounds called secondary metabolites, through various biosynthetic pathways. Terpenoids and isoprenoids contribute more than 50,000 compounds to this chemical diversity which include numerous commercial flavors, fragrances and medicines. Artemisinin and taxol are such terpene-based drug compounds. In general, most useful terpenoids are produced in small quantities in plants, which has slowed considerably their commercial utilization. Research has recently targeted the development of microbial fermentative processes as an alternative approach. Saccharomyces cerevisiae is an amenable organism for metabolic engineering and the diversion of the metabolic machinery towards the overproduction of terpenoids compounds. Terpene synthases, the enzymes synthesizing terpenes utilize geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranyl-geranyl pyrophosphate (GGPP) as substrates. In yeast they are synthesized by the sterol biosynthetic pathway. Although, the whole genetic pathway is present in yeast, it is tightly regulated and the precursors are present in limited quantities. To overcome this problem, a yeast strain producing high sterol levels was identified and tested for its capacity to produce a monoterpene cineole. This was achieved by stable transformation of a plasmid carrying the cineole synthase gene, a monoterpene synthase from Salvia fruticosa, under the control of an inducible promoter, and facile detection of the terpene products as volatiles by Head Space - Solid Phase Microextraction (HS-SPME) coupled with Gas Chromatography/ Mass Spectrometry (GC/MS) analysis. The selected strain was targeted for further modifications. A mutant stabilized version (K6R) of HMG2 under the control of the inducible Galactose promoter was stably integrated into the HO locus to generate strain AM63. The modified strain produces on average 1.5 fold more cineole than the parental strain and exhibited reduced background volatile metabolites when transformed with Sf-CinSyn (RC).
Further molecular engineering of AM63 yeast strain aimed to maximize terpene productivity without sacrificing cell viability which could hamper the biofermentation process. Two different modifications targeted the upregulation of FPP synthase encoded by ERG20 gene by GAL promoter integration into the yeast chromosome of AM68 strain, and deletion of one allele of erg9 gene encoding for a squalene synthase in the diploid AM70 strain resulting in further enhancement in terpene production. AM68 strains produces 3 fold more cineole, while AM70 cells produced 3.5 fold more sesquiterpenes than the parental strain. Additional modifications targeted the HMG1 gene by truncation of the N-terminus and expression of the one allele under a stable constitutive promoter. The increased yield of terpenes in yeast enabled the identification of several novel terpene synthases isolated from Salvia fruticosa (Greek sage) and Salvia pomifera. Four of them failed to yield any products when tested as bacterially expressed proteins. One clone was a monoterpene synthase producing mostly cineole, distinct from the previously identified canonical Salvia cineole synthase, while another one was a naturally truncated form which failed to yield any products. The two other genes encoded for sesquiterpene synthases, the first producing beta-farnesene and nerolidol and the second was a multiproduct enzyme synthesizing alpha-cubebene, alpha-copaene, trans-caryophyllene and delta-cadinene. Focusing on the terpene synthase molecule we tested whether additional modifications in the N-terminus of CS in the chloroplastic targeting sequence could further enhance product yield. The truncated SfCinS1(RR) and SfCinS1(RC) catalyzed the formation of multiple monoterpenes using the endogenous GPP pool as substrate with a significant peak of 1,8-cineole. However, SfCinS1(RC) was found to be more stable, efficient, with high activity during long incubation times. Additionally, using the two-hybrid system we screened a Salvia fruticosa glandular trichome library to identify interacting proteins to the Cineole monoterpene synthase. One of the interactors an, HSP90 when co-expressed with cineole synthase (CS) reproducibly increased product yield by 20%. Parallel work in the context of the PENED funded project requirements focused on the in vivo characterization of two recently isolated enzymes involved in secondary metabolism and plant defense by expressing them in Arabidopsis transgenic plants. When exposed to acrolein-induced oxidative stress Arabidopsis plants overexpressing a thioredoxin-peroxidase transgene, LeTpx1, exhibited less sensitivity than wild type plants. Correspondingly, the BI-GST, a plant GST-like protein inhibiting Bax lethality in yeast cells, and the LeTpx1 transgenes significantly increased plant resistance to the microbial pathogen, Pseudomonas syringe pv.tomato DC3000.
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Engineering yeast for the production of plant terpenoids and characterization of their biosynthetic enzymes from Salvia sp. : arabidopsis plants overexpressing select antioxidant genes exhibit resistance to oxidative stress and bacterial infection
