Metabolic Engineering of Saccharomyces cerevisiae for Improved Biosynthesis of S-Adenosylmethionine
Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Publicerad
2009
Författare
Thörn, Christian
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
S-Adenosylmethionine (SAM) is an important molecule for normal cell functions, it is involved in a number of biological processes and it mainly acts as methyl donor. SAM biosynthesis, in Saccharomyces cerevisiae is regulated by the gene product of MET13 through feedback inhibition by SAM itself. The expression of the chimeric gene C1-MET13, in which the sequence coding the C-terminal domain of MTHFR has been substituted by the sequence of the homologous gene from Arabidopsis thaliana, results in a yeast strain able to synthesize high levels of SAM. In this work, a strain expressing C1-MET13 was constructed. The overproduction of SAM in this strain was demonstrated to be independent of the supply of glycine and formate that were previously reported to be required for a robust overproduction of SAM in a C-MET13 strain. In yeast expressing C1-MET13, SAM levels showed a 10-fold increase during exponential growth phase on glucose in batch cultivations. Interestingly, SAM biosynthesis varied depending on the yeast growth conditions, increasing significantly when yeast was growing on ethanol or exposed to high concentrations of selenate. Although the mechanisms behind this metabolic regulation need to be further studied the results obtained here gave important information to define more precisely the most favorable conditions for SAM biosynthesis. The present work was included in a broader on-going project for the production of selenobioactive compounds in S. cerevisiae through metabolic engineering. Among all selenometabolites that have been shown to have beneficial effects in preventing and treating cancer, Seleno-methylselenocysteine (SeMCys) has been proven to be most effective for humans. The increased methylating capacity that results from higher concentration of SAM intended to improve the biosynthesis of SeMCys. To this aim, a strain co-expressing C1-MET13 with two plant genes coding Selenocysteine methyltransferase (SMT) and Methioninemethyltransferase (MMT) was constructed. SAM concentration was still relatively high, though lower compared to SAM levels found in the yeast strain expressing only C1-MET13 as heterologous gene, indicating that SAM was consumed. In order to improve the uptake of selenate for the biosynthesis of seleno compounds, the strain expressing the three heterologous genes was cultivated in a fed-batch system under sulfur-limited condition and in the presence of selenate. Under these conditions, SAM concentration was higher compared to the concentration determined in batch cultivations, although the cells did not grow as expected due to toxicity from high selenate level in the medium. Analysis of selenometabolites showed SeMCys being synthesized during these inhibitory conditions. Although the conditions and the setting for this kind of cultivation require a better refinement, this represented an exploratory study for the design of optimal bioprocess towards the biotechnological production of SAM and SeMCys.
Beskrivning
Ämne/nyckelord
Livsvetenskaper , Industriell bioteknik , Life Science , Industrial Biotechnology