| Isoprenoids with more than 40,000 described compounds are the largest and most structurally diverse group of plant metabolites. Isoprenoids play various biological roles in plants. Depending on the number of isoprene units, isoprenoids can be classified into several groups, such as monoterpenes, sesquiterpenes and diterpenes (respectively 2, 3 and 4 C5 units).Isoprenoids are functionally important in many different parts of cell metabolism such as photosynthesis (carotenoids, chlorophylls, plastoquinone), respiration (ubiquinone), hormonal regulation of metabolism (sterols), regulation of growth and development (gibberellic acid, abscisic acid, brassinosteroids, cytokinins, prenylated proteins), defense against pathogen attack, intracellular signal transduction (Ras proteins), vesicular transport within the cell (Rab proteins) as well as defining membrane structures(sterols, dolichols, carotenoids). Many isoprenoids also have considerable medical and commercial interest as flavors, fragrances (such as limonene, menthol, camphor), food colorants (carotenoids) or pharmaceuticals (such as bisabolol, artemisinin, lycopene, taxol).Isoprenoids are widely present in plant tissues, and extraction from plants has been the traditional option for the large-scale production of these compounds. However, in many cases this method is neither feasible nor economical. Among the drawbacks in using plants as a source for isoprenoid production are influence of geographical location and weather on the composition and concentration of isoprenoids in the plant tissues, low concentration and poor yields for the recovery of isoprenoids from plants, and the high costs associated with extraction and purification. Chemical synthesis of isoprenoids has also been reported, and currently most of the industrially interesting carotenoids are produced via chemical synthesis. However, because of the complex structures of isoprenoids, chemical synthesis, involving many steps, is difficult. Side reactions, unwanted side products, and low yield are other disadvantages. In vitro enzymatic production of isoprenoids through the action of plant isoprenoid synthases is also impractical due to the dependency on the expensive precursors, as well as poor in vitro conversion. There is therefore much interest in using microorganisms as cell factories for the production of isoprenoids. The intracellular pools of isoprenoid precursors in microorganisms appear, to be however, not enough to provide high level production. It may therefore be necessary to deregulate the pathways involved in the biosynthesis of isoprenoid precursors in order to improve production.Yeast therefore has a high inherent capacity for the biosynthesis of isoprenoid precursors that may be directed to the production of heterologous compounds. Besides, tools from other related areas are being incorporated into the metabolic engineer's repertoire. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites, quantification of these metabolites using modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (GC-MS, LC-MS), as well as the mathematical analyses.In this study, we chose S. cerevisiae as a host cell for the accumulation of isoprenoid precursors to extend an understanding of the mechanisms by which the flux through the pathway is controlled. The results are as follows:(1) In order to enhance the flux to isoprenoid precursors, both the erg9 gene which is responsible for conversion of FPP to squalene and the coq1 gene which lies in downstream pathway of GPP, FPP, GGPP were knockout by two disruption cassettes for gene replacement.(2) Upon genetic modification, the change in the concentration of GGPP was measured via LC-MS at batch cultivations time. LC separations were performed on a 4.6 mm×25 cm SB-Aq C18 5-μm column (Agilent Tech., USA) with a mobile phase consisted of 75% eluent A (10mM tributylamine aqueous solution adjusted pH to 4.95 with 15mM acetic acid) and 25% eluent B (methanol), and mass spectra were operated in negative ion mode over a range of m/z 50–600, and selective ion monitors (SIM) were at m/z 449.1-449.2 for GGPP ([M–H]?). The calibration curves were linear over the range of 0.1–50 ng/ml. In this range, relative standard deviations (R.S.D.) were <10% for intra-day precision and <14% for interday precision. The accuracy was within the range of 96.5-105.4%. The disruption of both erg9 and coq1 at 72 h of the fermentation period improved the accumulation of GGPP at most(3) A precise and sensitive nonradioactive method was developed for the simultaneous quantification of the isoprenoid precursors, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), squalene, ergosterol and lanosterol in recombinant and wild-type S. cerevisiae. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–selective ion-monitoring mass spectrometry (GC-SIM-MS). The analysis of GOH, FOH, GGOH, squalene, ergosterol and lanosterol illustrates robustness and reliability of this method outlined. Quantification of the analytes was performed by external calibration with reference substances and internal standardization. The recovery of the procedure has been evaluated.(4) The application of fluorescence and chemiluminescence analysis to the measurement of metabolites and the changes in metabolite concentrations under the molecular manipulation characterized cellular physiology and gene function, which would help us illuminate the effects of perturbation in pathways of interest, as well as unbiased characterizations of microbial stress responses as a whole. Functional analysis revealed greater energy-dependent efflux activity of membrane transporters, lower intracellular ATP level and mitochondrial membrane potential, more endogenous reactive oxygen species generation in response to gene disruption.The current work is based on the integration of genetic engineering, chemical analysis and data processing, which may provide a convenient format for considerable knowledge of metabolic pathway, as well as a platform for the production of a broad range of high value isoprenoids in yeast. This combination of rigorous analysis and quantitative molecular biology methods has endowed metabolic engineering with an effective synergism that crosses traditional disciplinary bounds. Meantime, the application of metabolic flux analysis contributes to reflect the activities of organisms, understand the underlying regulation mechanisms.
|
PDF Full Text Request