School of Biological Sciences, Univeristy of Bristol
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Samenvatting eindwerk 2011-2012: Reconstruction of the tropolone biosynthetic pathway in Aspergillus oryzae
This thesis describes the reconstruction of the tropolone biosynthetic pathway in the new host Aspergillus oryzae. The genes used to do this were obtained from Talaromyces stipitatus and Acremonium strictum.
The tropolone biosynthetic pathway was recently researched and published by the Schools of Chemical and Biological Sciences at the University of Bristol (Davidson et al. 2012). Some tropolones have interesting properties and are researched as antimalarial or antibacterial agents.
Now the biological pathway for tropolone synthesis is unraveled researchers want to construct a strain which produces it in high quantities so that it would be possible to obtain more tropolone product at lower costs. The first steps and methods for construction of this strain are described in this thesis.
The first step is the construction of a multigene expression system which contains the first four genes (AsA, Trop B, C, D) of tropolone synthesis. Herefore the following methods are used: PCR, homologous recombination in yeast, Escherichia coli transformation, Gateway® site-specific recombination and ligation.
Once the multigene expression system is assembled, it is used to transform A. oryzae for expression. In order to investigate the production of the new metabolites, the mould is grown in liquid medium, chemical extraction is performed and the extract is analyzed by LC-MS.
The LC-MS traces show the transformants have production of novel metabolites in comparison with the untransformed A. oryzae. The mass and UV spectra of the novel metabolites are consistent with the compounds produced when the first two genes are active in the new host. Two unidentified compounds are also produced. The first step in the biosynthetic pathway is the production of 3-metylorcinaldehyde, the next step is done by TropB.
The third gene, TropC, is needed to make a ring expansion and so produce the specific seven carbon ring structure which defines a tropolone. This gene appears not to be working properly.
Further work in this research is to find out why the third gene is not working and solve the problem. Characterization of the metabolites is also ongoing.