Vectors, cells and processes for pyrimidine deoxyribonucleosides production
Novel DNA constructs and host cells comprising the same are disclosed. DNA constructs comprise a transcription unit (e.g. operon) comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin or a uridine kinase gene and/or a dCTP deaminase gene. In preferred embodiments the constructs comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin further comprise DNA sequences encoding for thymidylate synthase and/or transcription units comprising sequences encoding for uridine kinase preferably together with dCTP deaminase. In particularly preferred embodiments, the host cells comprise constructs having all of the above characteristics wherein the host cell displays repressed or no uracil DNA glycosylase activity. This may be achieved by removal of the host cell ung gene. Use of host cells in the manufacture of pyrimidine deoxyribonucleotides e.g. thymidine is also disclosed.
1. A DNA construct comprising a transcriptional unit which comprises a wild type T4 ribonucleotide reductase gene, an E. coil uridine kinase gene and a dCTP deaminase gene.
2. A modified host cell comprising a DNA construct, which construct comprises a transcription DNA unit, which unit comprises a modified T4 nrdA gene comprising SEQ ID NO: 9 and a thioredoxin gene, and wherein said host cell further comprises one or more of the following features:
(a) a transcription unit located on said DNA construct, comprising a thymidylate synthase gene heterologous to the thymidylate synthase gene of the host cell;
(b) a transcription unit located on said DNA construct, comprising a uridine kinase gene; and
(c) a transcription unit located on said DNA construct, comprising a dCTP deaminase gene.
3. The modified host cell of claim 2 wherein the host cell is an E. coli cell modified such that the host cell's endogenous ung gene is inactivated by an insertion in the ung gene.
4. A DNA construct comprising a transcriptional unit which comprises a modified T4 nrdA gene, an E. coli uridine kinase gene, and a dGTP deaminase gene, wherein the modified T4 nrdA gene comprises SEQ ID NO: 9.