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Novel Synthetic Biology Tools for Metabolic Engineering of Saccharomyces cerevisiae

Siavash Partow (Institutionen för kemi- och bioteknik, Systembiologi)
Göteborg : Chalmers University of Technology, 2012. ISBN: 978-91-7385-727-7.- 199 s.
[Doktorsavhandling]

The most well characterized eukaryote model organism Saccharomyces cerevisiae is not only preferred as a microbial cell factory for synthesis of industrial products, e.g. bioethanol, but this eukaryote host system is also defined as a robust scaffold for commercial production of diverse chemicals e.g. isoprenoids. Therefore, a number of tools in different emerging fields e.g. systems biology, evolutionary engineering and synthetic biology have been developed. Synthetic biology offers an alternative approach that is becoming more accessible as a tool for better performing metabolic engineering of yeast. Due to the fact that the regulations of gene dosage and gene transcription are the first two key steps allowing control of metabolic pathways, improve of both gene expression and gene dosage through modulating promoter choice and plasmid copy number were pursued. The strength of seven different constitutive or glucose based promoters, TEF1, PGK1 TPI1, HXT7, PYK1, ADH1 and TDH3, was compared at different stages of a batch cultivation using LacZ as reporter. A new divergent promoter was developed, containing two strong and constitutive promoters, TEF1 and PGK1, to support high level gene expression. Furthermore, this bidirectional promoter was used to construct new episomal plasmids, the pSP series, to optimize the endogenous mevalonate (MVA) pathway through gene overexpression and also to construct integration cassettes containing the synthetic methylerithritol phosphate (MEP) pathway genes. The last two studies showed the successful implementation of synthetic biology tools in metabolic engineering in terms of pathway optimization and pathway reconstruction in order to improve sesquiterpene production in S. cerevisiae. Optimization of the MVA pathway was performed in two steps, modulating the FPP branch point and modulating the possible nodes which are directly involved or related to the MVA pathway including overexpression of tHMG1, ERG20, GDH2 and upc2-1 and deletion of GDH1, DPP1 and LPP1. Combination of all these modifications led to a 4-fold improvement of α-santalene yield over the reference strain. In the second study, the bacterial MEP pathway, containing 8 genes, was reconstructed through stable integration into the yeast genome in two steps. However, a functional MEP pathway was not obtained even after reconstruction of the possible bacterial Fe/S trafficking routes and the bacterial electron transfer system in order to circumvent lack of the enzyme activity. In another approach, improvement of gene dosage via modulating plasmid copy number was investigated. Here, two strategies, individually and in combination,were applied in order to reduce the maker gene at both protein and RNA levels, and their impact on plasmid copy number of pSP-GM1was investigated. Both methods, destabilization of the marker protein using a ubiquitin/N-degron tag and down-regulation of the marker gene employing weak promoters, elevated the plasmid copy number. Combination of the weak promoter and ubiquitin tag showed a synergistic effect and increased the plasmid copy number by 3 fold. A proof-ofconcept study was performed to determine if the enhancement in plasmid copy number could affect patchoulol production when patchoulol synthase was expressed from the modified plasmid. The result showed that while the final biomass concentration was unchanged, patchoulol production reached about 30 mg/L when employing modified plasmid, which was more than 3 times higher compared to when the synthase gene was expressed from the original plasmid.

Nyckelord: S. cerevisiae, Synthetic biology, Metabolic engineering, Yeast promoter, MVA pathway, MEP pathway, Fe – S clusters, Multi-copy plasmid.



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Denna post skapades 2012-09-03. Senast ändrad 2013-09-25.
CPL Pubid: 162789