Biological production of thymidine
Novel organisms, including DNA construct host cell combinations, are disclosed. The organisms comprise a transcription unit (e.g. operon) comprising DNA sequences encoding for enzymes which promote the supply of single carbon units for the conversion of dUMP to dTMP. Examples include: dihydrofolate reductase genes e.g. T4 frd; Serine Hydroxymethyltransferase genes e.g. glyA; 3-phosphoglycerate dehydrogenase genes e.g. serA; and THF synthase genes e.g. ADE3. The organisms are used in a biological method of producing thymidine with significantly reduced levels of uridine.
1. A bacterium capable of producing thymidine when grown in a culture medium, the bacterium comprising one or more genetic modifications to its DNA, or some extra-chromosomal DNA necessary for its growth, characterized in that the bacterium comprises one or more genetic modifications which result in the thymidine biosynthetic pathway preferentially producing TdR at the expense of UdR as a consequence of one or more genetic modifications which increases the availability of a single carbon unit to dUMP for conversion to dTMP, wherein at least one genetic modification comprises introducing a transcriptional unit capable of expressing a dihydrofolate reductase (EC 1.5.1.3) and
wherein the bacterium comprises a host strain and at least one construct comprising extra-chromosomal DNA and wherein a host strain gene encoding a nucleoside diphosphate kinase (EC 2.7.4.6) of the bacterium has been knocked out or otherwise inactivated or excised.
2. The bacterium as claimed in claim 1 wherein the host strain is stabilized such that if the one or more constructs are lost, the host bacterium is unable to make DNA and dies.
3. The bacterium as claimed in claim 1 wherein the dihydrofolate reductase is a T even phage dihydrofolate reductase.
4. The bacterium as claimed in claim 3 wherein the T even phage is T4.
5. The bacterium as claimed in claim 1 wherein the transcriptional unit is a frd gene.