Manipulation of the Shikimate Pathway in Streptomyces coelicolor

McKernan, Philip Andrew (1995) Manipulation of the Shikimate Pathway in Streptomyces coelicolor. PhD thesis, University of Glasgow.

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The work in this thesis has been performed as part of an investigation into the upregulation of flux through the shikimate pathway of Streptomyces coelicolor, in order to produce higher levels of substrates for the synthesis of secondary metabolites. Disruption of the aroQ gene, which encodes the type II dehydroquinase (the third enzyme in this pathway) was attempted to block the pathway, and allow analysis of flux through the first two steps of the pathway. However, these experiments were not successful. Overexpression of the aroQ gene in S.coelicolor may cause an upregulation of this step, and possibly the whole pathway. To achieve such overexpression, a 3 kb fragment containing the aroQ gene was subcloned into a high copy-number streptomycete vector, and transformed into S.lividans prior to transformation into S.coelicolor. The plasmid produced causes a 7-fold increase in the activity of dehydroquinase. Since only a single protein appears to be overexpressed from this plasmid, it appears that the aroQ gene is not transcriptionally coupled to any other genes. However, this plasmid could not be isolated from S.lividans cells for transformation into S.coelicolor. The aroA gene encodes EPSP synthase, the enzyme which catalyses the fifth step of the pathway. Cloning of this gene was attempted by designing oligonucleotide primers against regions of similarity from the amino acid sequences of EPSP synthases from other species, with the oligonucleotides being used to clone the gene via PCR and probing of genomic blots and a lambda-library. All efforts at cloning aroA were unsuccessful. The aroD gene from E.coli, which encodes the type I dehydroquinase, was subcloned onto a bifunctional vector under the control of the hybrid tac promoter. The plasmid produced was transformed into S.coelicolor. It caused an approximately 40-fold increase in the activity of dehydroquinase. The overexpressed protein (the type I dehydroquinase) was thermolabile as expected, but showed much higher resistance to inhibition by Cl- anions than anticipated. It appears to be overexpressed only around the point of transition from exponential to stationary phase, presumably due to the protein containing the rare TTA codon (encoding a leucine). The incorporation of leucine at this point in the polypeptide is controlled at the translational level by the presence of the tRNAUUA, which is encoded by the bldA gene, and is apparently only expressed at significant levels at the end of the growth phase.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Iain Hunter
Keywords: Genetics
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-75501
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:37
Last Modified: 19 Nov 2019 19:37

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