In vivo fluorination

The cell according to any one of the preceding claims, wherein the second nucleic acid further comprises a second promoter and a gene encoding an activator of transcription, said gene being under the control of the second promoter, wherein transcription of the activator of transcription can be induced by the inducer, wherein the activator of transcription upon expression activates transcription from the first promoter.

The cell according to any one of the preceding claims, wherein the second nucleic acid further comprises a second promoter and a gene encoding an activator of transcription, said gene being under the control of the second promoter, wherein transcription of the activator of transcription can be induced by the inducer, wherein the activator of transcription upon expression activates transcription from the first promoter.

The cell according to any one of the preceding claims, wherein the cell can tolerate toxic compounds, such as fluorinated compounds.

The cell according to any one of the preceding claims, wherein the cell is a non-pathogenic organism.

The cell according to any one of the preceding claims, wherein the cell is a mammalian cell, a plant cell, an insect cell, a yeast cell or a bacterial cell.

The cell according to any one of the preceding claims, wherein the cell is a bacterial cell of the Pseudomonas genus, the Bacillus genus, the Vibrio genus or the Escherichia genus or a yeast cell of the genus Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon or Lipomyces.

The cell according to any one of the preceding claims, wherein the cell is a bacterial cell selected from Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas taiwanesis Pseudomonas syringae, Pseudomonas stutzeri, Pseudomonas oleovorans, Pseudomonas mendocina, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Vibrio natriegens and Escherichia coli, or a yeast cell selected from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofer, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica, preferably the cell is Pseudomonas putida KT2440.

The cell according to any one of the preceding claims, wherein the cell is Pseudomonas putida KT2440.

The cell according to any one of the preceding claims, wherein the fluorinase gene encodes a fluorinase derived from a Streptomyces such as Streptomyces sp. MA37, Streptomyces cattleya, Streptomyces xinghaiensis, Nocardia brasiliensis or Actinoplanes sp. N902-109.

The cell according to any one of the preceding claims, wherein the fluorinase is capable of catalysing fluorination of a substrate such as adenosine to obtain a fluorinated compound, such as 5′-deoxy-5′-fluoroadenosine.

The cell according to any one of the preceding claims, wherein the substrate is selected from the group consisting of S-adenosyl-L-methionine (SAM) or a derivative thereof, such as a methylaza derivative, 5′-chloro-5′-deoxyadenosine, a 2-deoxyadenosine analogue, an L-methionine analogue, a di-cyclic peptide conjugate of 5′-chlorodeoxy-2-ethynyladenosine, a tri-cyclic peptide conjugate of 5′-chlorodeoxy-2-ethynyladenosine, fluoride, 18F, and chloride.

The cell according to any one of the preceding claims, wherein the fluorinase is the fluorinase as set forth in SEQ ID NO:

• 1, or a functional variant thereof having at least 80% homology thereto, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence homology to SEQ ID NO:
• 1.

The cell according to any one of the preceding claims, wherein the fluorinase gene is codon-optimised.

The cell according to any one of the preceding claims, wherein the first nucleic acid further comprises a phosphorylase gene such as flB1 or deoD, said gene encoding a phosphorylase (EC 2.4.2.28) capable of catalysing the phosphorylation of a fluorinated substrate such as a fluorinated adenosine, said gene being under control of the first promoter, whereby the cell is capable of producing a phosphorylated and fluorinated compound such as 5′-deoxy-5′-fluoro-D-ribose 1-phosphate

The cell according to any one of the preceding claims, wherein the phosphorylase gene is codon optimised.

The cell according to any one of the preceding claims, wherein the first nucleic acid further comprises a gene encoding a nucleosidase (EC 3.2.2.9) such as Pfs, said nucleosidase being capable of catalysing the conversion of 5′-fluoro-5′-deoxyadenosine to 5′-fluorodeoxyribose, said nucleosidase gene being under control of the first promoter, optionally wherein the gene encoding the nucleosidase is codon-optimised.

The cell according to any one of the preceding claims, wherein the activator of transcription is a polymerase such as a T7 RNA polymerase and the first promoter is the native promoter of said polymerase such as a T7 promoter.

The cell according to any one of the preceding claims, wherein the polymerase is a T7 RNA polymerase and the first promoter is a T7 promoter.

The cell according to any one of the preceding claims, wherein the riboswitch is the fluoride-responsive riboswitch (FRS) from Pseudomonas syringae as set forth in SEQ ID NO:

• 4, or a functional variant thereof having at least 90% homology to SEQ ID NO:
• 4, such at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence homology to SEQ ID NO:
• 4.

The cell according to any one of the preceding claims, wherein the inducer is a fluoride salt such as NaF or KF.

The cell according to any one of the preceding claims, further comprising a mutation in at least one fluoride transporter gene encoding a fluoride transporter, said mutation resulting in a partial or total loss of function of said fluoride transporter.

An expression system for expression in a cell, comprising:

• a) a first nucleic acid comprising a first promoter and a fluorinase gene encoding a fluorinase (EC 2.5.1.63) capable of catalysing the formation of a C—F bond, said fluorinase gene being under the control of the first promoter;
• b) a second nucleic acid comprising a riboswitch,
• wherein transcription of the fluorinase gene from the first promoter is induced in the presence of an inducer, wherein said riboswitch is responsive to said inducer, whereby the cell is capable of producing a fluorinated product such as 5′-fluoro-5′-deoxyadenosine or 5′-deoxy-5′-fluoro-D-ribose 1-phosphate.