title: The gene encoding the glyphosate-tolerant EPSP synthase from Anabaena variabilis
Creators: name: Muir, Gillian Morag
Publisher: name: University of Glasgow
Year: 1996
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Glyphosate is a broad-spectrum, non-selective, post-emergence herbicide, active against a variety of weed and crop species. The primary site for herbicidal action is 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, the penultimate enzyme of the shikimate pathway. In higher plants and microorganisms, this pathway provides a metabolic route to the synthesis of the aromatic amino acids - phenylalanine, tyrosine and tryptophan - plus other aromatic compounds. EPSP synthase has been purified from various sources to investigate its kinetic characteristics and the inhibition properties of glyphosate. The cyanobacteria are the largest, most diverse and widely distributed group of photosynthetic prokaryotes. The physiological and biochemical effects of glyphosate on certain cyanobacterial strains have been examined. The filamentous, nitrogen fixing strain Anabaena variabilis ATCC 29413, like other cyanobacteria, is tolerant to glyphosate. Tolerance is due to an EPSP synthase that is uninhibited by the herbicide. A detailed kinetic and molecular study of this enzyme has been hindered by the consistently low yield of EPSP synthase protein purified from A. variabilis cells. As such, the purpose of the work described in this thesis was to isolate the A. variabilis EPSP synthase gene (termed aroA) and overexpress the encoded enzyme. A number of cloning methods were undertaken in an attempt to reach this goal. The polymerase chain reaction was employed to amplify a defined segment of the aroA gene from A. variabilis genomic DNA using different sets of degenerate oligonucleotide primers. These primers were designed from conserved regions of EPSP synthase sequence from various plants and microorganisms. A truncated fragment of an aroA gene was synthesised, however, this piece of DNA did not originate from A. variabilis DNA. The source of this contaminating PCR product has yet to be identified. The design of the PCR primers may have affected the specificity of the amplification reaction and could explain why other primer sets failed to amplify the sequence of interest. Subsequently, a library of A. variabilis genomic DNA was constructed in the phagemid vector, pBluescript SK-. After evaluating its size and quality, the library was screened with an aroA probe from the unicellular, non-nitrogen fixing cyanobacterium Synechocystis sp. PCC 6803 to isolate the clone of interest. Prior to screening. Southern blot experiments had demonstrated that the Synechocystis probe hybridised to specific fragments of restriction digested A. variabilis DNA. This heterologous probe was, therefore, considered suitable for screening purposes. Persistent problems with non-specific hybridisation between the probe and the genetic material of the host cell harbouring the library frustrated the attempts made to locate an aroA clone. Reducing the level of background hybridisation required a slightly different approach. It was established that plaque hybridisation was more sensitive than colony hybridisation. Consequently, another A. variabilis library was made, but on this occasion the phage, lambda FIX II, was used as the vector system. Heterologous aroA probes from Synechocystis, E. coli and pea were employed to maximise the possibility of finding the A. variabilis EPSP synthase clone. Despite control experiments signifying the good quality of the newly constructed library, not one of the heterologous probes pulled out the clone of interest. The final strategy involved isolating the A. variabilis EPSP synthase gene by phenotypic complementation of an aroA- auxotrophic mutation of the E. coli strain AB2829. A control experiment showed that expression of the E. coli EPSP synthase from cloned DNA complemented the deficiency of the host and enabled the mutant to grow on selective medium. The E. coli mutant was then transformed with a library of A. variabilis genomic DNA made from EcoRI restriction fragments thought to contain the entire aroA coding sequence. Succeeding experiments showed that such a library could not complement the aroA mutation of E. coli AB2829. The advantages and disadvantages of each of the above techniques are discussed in detail with specific regard to cloning the A. variabilis EPSP synthase gene. Other gene cloning strategies not used in this work are described and the practical reasons for not employing these techniques are debated. The possible exploitation of the EPSP synthase gene from A. variabilis by genetic engineers for the construction of a glyphosate-tolerant crop plant is discussed, as is the contribution the gene sequence could have made to the debate regarding the cyanobacterial origin of higher plant and algal plastids.