Pyrimidine metabolism in Brevibacterium helvolum and genetic manupulation of this organism to overproduce pyrimidine nucleosides

Kalirai, S.K., 1993. Pyrimidine metabolism in Brevibacterium helvolum and genetic manupulation of this organism to overproduce pyrimidine nucleosides. MPhil, Nottingham Trent University.

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Studies were carried out on Brevibacterium helvolum in order to isolate a thymidine overproducing strain. This required a knowledge of the pyrimidine metabolic pathway in this bacterial species.

A mutant strain of B. helvolum from IGI was employed that had already undergone some genetic manipulation and was found to be capable of producing and excreting more thymidine than the wild type.

Studies on the wild type strain show that B. helvolum can utilise both purine and pyrimidine (deoxy)nucleosides as alternative sources of carbon. These studies suggest that the organism possesses the enzymes necessary to degrade thymidine, deoxyuridine, uridine, cytidine, guanosine, adenosine and deoxyadenosine, but not the sugar deoxyribose. It was also shown to be sensitive to the pyrimidine analogues 5-fluorouracil, 5-fluoro-deoxyuridine and, to a limited extent, 2-thiouracil.

The ICI mutant strain is thought to be a dUMP overproducer. It was found to be resistant to high levels of fluorouracil and fluorodeoxyuridine. It could not utilise uridine as a sole source of carbon implicating the absence of a functional uridine phosphorylase. Resistance to fluorouracil was proposed to be a combination of three factors; a defective UMP pyrophosphorylase and uridine phosphorylase and, substrate competition between uracil (produced as an end product of dUMP degradation) and fluorouracil for thymidine phosphorylase.

In the course of the studies undertaken a thymidine overproducing strain was isolated in which the breakdown of thymidine was found to be reduced. HPLC studies showed this strain could produce and excrete 100% more thymidine and deoxyuridine than the original mutant strain. The thymidine auxotroph (probable genotype thyA deoC deoA) from which the thymidine overproducing strain was derived was found to produce and excrete almost seven times the level of deoxyuridine produced by the original mutant.

Studies also suggest the existence of an activity viz deoxyuridine phosphorylase in B. helvolum which is not known to be present in E. coli.

In addition, a super high thymine requirer of the probable genotype thyA deoD deoR (analogous to the super high thymine requirer of E. coli) was isolated during the course of the investigations undertaken.

Item Type: Thesis
Creators: Kalirai, S.K.
Date: 1993
ISBN: 9781369323269
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 01 Oct 2020 15:47
Last Modified: 27 Sep 2023 09:36

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