Patent Application
20240263207
The present invention relates to a cell capable of producing a fluorinated, a chlorinated or a brominated compound, methods for producing fluorinated, chlorinated or brominated compounds in a cell and expression systems therefor.
The present application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 17, 2023, is named “16DMTU-HO78402PA_SEQ.txt” and is 24,576 bytes in size.
Halogenated molecules represent a third of the drugs currently on clinical trials. Specifically, almost 25% of pharma molecules contain fluorine (F) and latest figures indicate that the market of fluorinated compounds for the pharma industry will continue expanding in the upcoming years (Zhou 2016). F-containing organic compounds are extremely relevant from a pharmaceutical and industrial point of view, since F enhances lipophilicity, helping cell membrane penetration and increasing drug bioavailability (Zhang 2012). Although most of the pharmaceutically-relevant drugs that we rely on are provided, directly or via a precursor molecule, by nature, a missing key structure in this picture is F-containing organic molecules. Only 12 naturally-occurring organofluorines have been identified (e.g., fluoroacetate, found in tropical plants). This is due to the huge thermodynamic constraints that biochemical reactions involving F need to overcome, and the toxicity of low-molecular-weight fluorinated compounds (O'Hagan 2008).
There is just one microbial enzymatic reaction that occurs in the high-G+C content bacterium Streptomyces (Deng 2004). The fluorination pathway of Streptomyces cattleya uses S-adenosyl-L-methionine (SAM) and inorganic fluoride (F−) as substrates, generating 5′-fluoro-5′-deoxyadenosine (5′-FDA) and L-methionine in the first step catalysed by the fluorinase, which is encoded by flA. Although the fluorinase of S. cattleya was the first enzyme to form a C—F bond to be identified and isolated (O'Hagan 2002), other fluorinases have been identified and described in other actinomycetes. For example, fluorinases from Streptomyces sp. MA37, Nocardia brasilensis, and Actinoplanes sp. were identified by genome mining by comparison to the fluorinase from S. cattleya (Deng 2014). A fluorinase was also identified in Streptomyces xinghaiensis (Ma et al., 2016). Whereas this enzyme has been thoroughly characterized in vitro (O'Hagan 2002, Zhu 2007), its wide biotechnological exploitation in vivo has been limited mostly because of the high toxicity of the resulting fluorinated molecules and the complexity of the C—F bond chemistry. The only successful in vivo fluorometabolite production was performed in Salinospora tropica, where the chlorinase from this organism was replaced with FlA to produce fluorosalinosporamide (Eustaquio 2010). After that, further engineering of the fluorinases to improve their efficiency, activity and increase of their substrate range has been done (Sun 2016, Yeo 2017).
Moreover, successful protein expression for production of chemicals requires significant efforts to optimize the chassis and tools to achieve sufficiently high titers and yields. One of the challenges as well is the reduction of costs derived, for example, of the additives that need to be used in the process, such as inducers or antibiotics. To avoid the use of inducers, some strategies have been proposed, such as the use of thermosensitive or quorum sensing based expression systems. Another alternative is the use of riboswitches that recognize a specific molecule that is involved in the production process and triggers the expression of the pathway when it is present. Riboswitches are regulatory elements that are found in the 5′-UTR of bacterial mRNA and that post-transcriptionally control the gene expression upon binding to a specific metabolite. They have been used widely as biosensors, which respond with a detectable output to the presence of a certain molecule, providing easy screening methods of high-producing strains (Liu 2015). However, the use of riboswitches has been limited by their contextual effect on the expression gene (Kent 2018), although they have been used successfully in inducible expression systems (Morra 2015). A fluoride responsive riboswitch (FRS) have been described in numerous bacterial and archaeal organisms, controlling the expression of genes involved in the detoxification of the ion, typically transporters (Stockbridge 2012, Clayton Speed 2018). The sequence, structure, and mechanism of action have been studied for the riboswitch from Pseudomonas syringae and Bacillus subtilis, which have been used as well as biosensors to detect the intracellular concentration of NaF in Escherichia coli (Baker 2012).
Finally, the election of a suitable host for production presents a challenge that has considerable impact in the field of metabolic engineering. Environmental bacteria, that naturally thrive under rough physicochemical conditions, are evolutionarily pre-endowed with many physiological traits (e.g., stress endurance and metabolic plasticity) desirable in industrial settings (Danchin 2012, Calero and Nikel 2018).
In a first aspect, is provided a cell capable of producing a fluorinated, a chlorinated or a brominated compound from a substrate in the presence of fluoride, chloride or bromide, respectively, said cell comprising:
Also provided is a cell capable of producing a fluorinated compound from a substrate in the presence of fluoride, said cell comprising:
Also provided is an expression system for expression in a cell, comprising:
Also provided is an expression system for expression in a cell, comprising:
Also provided is a method for in vivo fluorination, chlorination or bromination of a substrate, comprising the steps of:
Also provided is a method for in vivo fluorination of a substrate, comprising the steps of:
Also provided is a fluorinated, a chlorinated or a brominated product obtainable by the methods described herein.
Also provided is a fluorinated compound obtainable by the methods described herein.
Also provided is a composition comprising a fluorinated, a chlorinated or a brominated product obtainable by the methods described herein.
Also provided is a composition comprising a fluorinated product obtainable by the methods described herein.
Also provided is a method for manufacturing a fluorinated, a chlorinated or a brominated compound of interest, said method comprising the steps of:
Also provided is a method for manufacturing a fluorinated compound of interest, said method comprising the steps of:
Also provided herein is the fluorinated, chlorinated or brominated product or the composition described herein for use as a medicament.
Also provided herein is the fluorinated product or the composition described herein for use as a medicament.
The present disclosure relates to cells capable of producing a fluorinated, a chlorinated or a brominated compound, methods for producing fluorinated, chlorinated or brominated compounds in a cell and expression systems therefor.
The fluorinase enzyme (EC 2.5.1.63, also known as adenosyl-fluoride synthase) catalyses the reaction between fluoride ion and the co-factor S-adenosyl-L-methionine (SAM) to generate L-methionine and 5′-fluoro-5′-deoxyadenosine (5′-FDA), the first committed product of the fluorometabolite biosynthesis pathway. Fluorinase was originally isolated from the soil bacterium Streptomyces cattleya, and is the only known enzyme capable of catalysing the formation of a carbon-fluorine bond, the strongest single bond in organic chemistry.
Fluorinase catalyses the reaction:
S-adenosyl-L-methionine+fluoride<=>5′-fluoro-5′-deoxyadenosine+L-methionine.
It can however also act on other substrates besides SAM, for example methylaza-SAM derivatives. Fluorinase can also catalyse the following reactions:
Fluorinase can thus catalyse fluorination reactions (addition of an F atom to a compound using fluoride as co-substrate), chlorination reactions (addition of a CI atom on a compound using chloride as co-substrate) and bromination reactions (addition of a Br atom on a compound using bromide as co-substrate).
The term refers herein to S-methyl-5′-thioadenosine phosphorylase (EC 2.4.2.28), which is capable of catalysing the following reaction:
The enzyme is also capable of catalysing phosphorylation of fluorinated adenosine.
In a first aspect, is provided a cell capable of producing a fluorinated, a chlorinated or a brominated compound from a substrate in the presence of fluoride, chloride or bromide, respectively, said cell comprising:
Also provided is a cell capable of producing a fluorinated, a chlorinated or a brominated compound from a substrate in the presence of fluoride, chloride or bromide, respectively, said cell comprising:
Throughout the disclosure, a cell capable of producing a fluorinated, a chlorinated or a brominated compound from a substrate in the presence of fluoride, chloride or bromide, respectively, will refer to a cell capable of converting a substrate into a fluorinated, a chlorinated or a brominated product in the presence of fluoride, chloride or bromide, respectively.
Preferably, the cell can tolerate toxic compounds, such as fluorinated compounds. In some embodiments, the cell is a cell of a non-pathogenic organism. For example, the cell is a cell of a GRAS (Generally Regarded As Safe) organism.
Cells useful for performing the methods disclosed herein may be mammalian cells, plant cells, insect cells, yeast cells or bacterial cells.
In embodiments where the cell is a yeast cell, preferably the yeast is of the genus Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon or Lipomyces. For example, the yeast is selected from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofer, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica.
In other embodiments, the cell is a bacterial cell of the Pseudomonas genus, the Bacillus genus, the Vibrio genus or the Escherichia genus. For example, the bacterial cell is 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. In preferred embodiments, the bacterial cell is a Pseudomonas putida cell, for example Pseudomonas putida KT2440.
The cell is used as production organism for fluorinated, chlorinated or brominated products as described herein.
The cell comprises a first nucleic acid comprising a first promoter and a fluorinase gene encoding a fluorinase, and a second nucleic acid comprising a riboswitch. In some embodiments the first nucleic acid and the second nucleic acid are comprised within the same nucleic acid molecule. An example of such an embodiment is shown on
In other embodiments, the first nucleic acid and the second nucleic acid are different, i.e. they are not comprised within the same nucleic acid molecule. In such embodiments, the cell may comprise a first nucleic acid comprising a first promoter and a fluorinase gene encoding a fluorinase, and a second nucleic acid comprising a riboswitch, a second promoter and a gene encoding an activator of transcription. An example of such an embodiment is shown on
The fluorinase gene encodes a fluorinase (EC 2.5.1.63). Fluorinases are enzymes which are capable of catalysing the formation of a C—F bond in the presence of fluoride. Fluorinases can also under some circumstances catalyse the formation of a C—Cl bond or of a C—Br bond. In the absence of fluoride, but in the presence of chloride, fluorinases can catalyse the formation of a C—Cl bond. In the absence of fluoride (and chloride) but in the presence of bromide, fluorinases can catalyse the formation of a C—Br bond.
Expression of the fluorinase gene is under the control of the first promoter comprised in the first nucleic acid. The fluorinase gene encodes a fluorinase or a functional variant thereof, such as a mutant, which retains the activity of the fluorinase but may have modified properties, such as modified substrate preferences, modified efficiency, and others. For example, fluorinases have been modified by directed evolution and rational design approaches to improve catalytic efficiency on non-native substrates (Sun et al., 2016; Thomsen et al., 2013). Such mutant fluorinases may also be used in the context of the present disclosure.
Fluorinases expressed in the cell of the present disclosure are capable of catalysing fluorination, chlorination or bromination of a substrate to obtain a fluorinated, a chlorinated or a brominated compound, respectively, as will be detailed further below. The fluorinase thus is capable of catalysing fluorination of a substrate to obtain a fluorinated compound.
In some embodiments, the fluorinase gene encodes the fluorinase of Streptomyces sp. MA37 as set forth in SEQ ID NO: 1 (FlA1), 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.
In some embodiments, the fluorinase gene encoding the fluorinase is as set forth in SEQ ID NO: 2, or a homologue 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: 2.
As the fluorinase gene may be a heterologous gene, it may be codon-optimised to improve transcription in the cell in which it is to be expressed, as is known in the art.
Accordingly, in some embodiments, the fluorinase gene encoding the fluorinase is as set forth in SEQ ID NO: 3, or a homologue 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: 3. SEQ ID NO: 3 encodes FlA1 of Streptomyces sp. MA37 (SEQ ID NO: 1) and has been codon optimised for expression in P. putida.
Fluorinase can convert S-adenosyl-L-methionine (SAM) to 5′-fluoro-5′-deoxyadenosine (5′-FDA) in the presence of inorganic fluoride (F−). The reaction is reversible. The enzyme can also convert other substrates to yield other products. For example, it can act on derivatives of SAM, such as a methylaza derivative, 5′-chloro-5′-deoxyadenosine, a 2-deoxyadenosine analogue, or L-methionine analogues, di-cyclic peptide conjugates of 5′-chlorodeoxy-2-ethynyladenosine, tri-cyclic peptide conjugates of 5′-chlorodeoxy-2-ethynyladenosine, fluoride, 18F, and chloride.
In some embodiments, the substrate is a SAM derivative of formula (I):
The SAM derivative may thus be of formula (II):
wherein X is selected from the group consisting of: S, Se and NMe; R is selected from the group consisting of: Me and propargyl; and A is a heterocycle.
In some embodiments, A is selected from the group consisting of
In some embodiments, the SAM derivative of formula (I) is of formula (III):
wherein R is selected from the group consisting of: Me and propargyl.
In preferred embodiments, the substrate is SAM.
In the presence of fluoride (F−) the fluorinase catalyses fluorination of the substrate. This is the preferred reaction catalysed by the enzyme. However, in the absence of fluoride, the enzyme is capable to catalyse other reactions. For example, in the absence of fluoride and in the presence of chloride, the enzyme is capable of chlorinating the substrates. In the absence of fluoride and chloride and in the presence of bromide, the enzyme is capable of brominating the substrates.
Hence, the cell disclosed herein which expresses a fluorinase can be used to obtain fluorinated compounds, chlorinated compounds or brominated compounds. The type of reaction can be directed by providing the appropriate co-substrates, fluoride, chloride or bromide.
The co-substrates may be provided in the form of salts. Preferred salts are soluble salts. In some embodiments, the reaction is a fluorination reaction and the co-substrate is a fluoride salt. Preferred fluoride salts are NaF or KF; less soluble salts such as CaF2 may also be used. In other embodiments, the reaction is a chlorination reaction and the co-substrate is a chloride salt. Preferred chloride salts are NaCl or KCl; less soluble salts such as CaCl2) may also be used. In other embodiments, the reaction is a bromination reaction and the co-substrate is a bromide salt. Preferred bromide salts are NaBr or KBr; less soluble salts such as CaBr2 may also be used.
The cell may in some embodiments further comprise an activator of transcription, which can bind the first promoter. The first promoter controls expression of the fluorinase gene. In some embodiments, the activator of transcription is the T7 RNA polymerase and the first promoter is a T7 promoter, which is recognised by the T7 RNA polymerase. Accordingly, the fluorinase gene is expressed in the cell provided that the activator of transcription is expressed and active.
The present inventors have found that in order to obtain tight and precise regulation of the expression of the fluorinase gene, it is advantageous to tightly regulate expression of the activator of transcription. This surprisingly results in an efficient system which can be tightly regulated to precisely control the fluorination, chlorination or bromination reaction. As shown in the examples, the use of a riboswitch together with a second promoter to control expression of the activator of transcription is particularly advantageous. However, the riboswitch may control expression of the fluorinase gene from the first promoter by activating transcription from the first promoter directly.
The riboswitch is activated by an inducer, as described herein below. In some embodiments, the inducer is a co-substrate of the reaction. For example, for fluorination of a substrate, the inducer is the co-substrate, i.e. fluoride. For chlorination of a substrate, the inducer may be the co-substrate chloride. For bromination of a substrate, the inducer may be the co-substrate bromide.
In other embodiments, the inducer is not a co-substrate of the reaction. For example, for chlorination of a substrate, the inducer is fluoride or bromide, preferably fluoride. For bromination of a substrate, the inducer is fluoride or chloride.
Preferably, a fluoride-responsive riboswitch (FRS) is used. For example, the FRS from Pseudomonas syringae is used. In its natural context this riboswitch drives the expression of the gene coding for the F− transporter EriCF in the presence of F−.
In embodiments where it is desirable to obtain fluorinated products, the riboswitch is fluoride-responsive, i.e. it is activated by the presence of fluoride. In embodiments where it is desirable to obtain chlorinated products, the riboswitch may be fluoride-responsive, i.e. activated by the presence of fluoride, and chloride is provided as co-substrate; or the riboswitch may be chloride-responsive, i.e. activated by the presence of chloride, and chloride is provided as inducer. In embodiments where it is desirable to obtain brominated products, the riboswitch may be fluoride-responsive, i.e. activated by the presence of fluoride, or it may be chloride-responsive, i.e. activated by the presence of chloride, and bromide is provided as co-substrate; or the riboswitch may be bromide-responsive, i.e. activated by the presence of bromide, and bromide is provided as inducer.
In some embodiments, the riboswitch is a fluoride-responsive riboswitch, such as the FRS riboswitch from P. syringae (SEQ ID NO: 4). In some embodiments, the cell is a Pseudomonas putida cell, in particular a P. putida KT2440 cell. In some embodiments, the activator of transcription is the T7 RNA polymerase and the first promoter is a T7 promoter.
In some embodiments, the second nucleic acid thus comprises a riboswitch having the sequence as set forth in SEQ ID NO: 4, or a functional variant thereof having at least 80% homology, such as at least 81% homology, such as at least 82% homology, such as at least 83% homology, such as at least 84% homology, such as at least 85% homology, such as at least 86% homology, such as at least 87% homology, such as at least 88% homology, such as at least 89% homology, such as at least 90% homology, 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% homology, such as 100% homology thereto. The term “functional variant” of an FRS refers to a sequence which retains the ability to drive expression of a gene downstream of it in response to fluoride.
The second nucleic acid in some embodiments may also comprise a second promoter. The second promoter may be the native promoter normally found with the riboswitch used. For example, the riboswitch is the FRS riboswitch from P. syringae as described above, and the second promoter is the native promoter of the eriCF gene of P. syringae. In other embodiments, the second promoter is a constitutive promoter, such as a synthetic promoter, for example a PEM7 promoter, a PBG42 promoter, a PtetA promoter, or any constitutive promoter as known in the art.
Without being bound by theory, the use of a two-step regulation system, i.e. a second nucleic acid comprising a riboswitch and a promoter controlling expression of a first nucleic acid comprising a first promoter and a fluorinase gene, is expected to amplify the transcriptional signal and result in increased expression of the fluorinase gene in an inducible manner. However, as can be seen from the examples below, a one-step regulation system, i.e. wherein the riboswitch directly controls expression of the fluorinase from the first promoter, also leads to expression of fluorinase in an inducible manner.
In some embodiments, the second nucleic acid comprises or consists of the FRS sequence as set forth in SEQ ID NO: 4 or a functional variant thereof and of the native promoter of the eriCF gene of P. syringae. The sequence of the corresponding second nucleic acid is set forth in SEQ ID NO: 6. In some embodiments, the second nucleic acid comprises or consists of SEQ ID NO: 6 or a functional variant thereof having at least 80% homology, such as at least 81% homology, such as at least 82% homology, such as at least 83% homology, such as at least 84% homology, such as at least 85% homology, such as at least 86% homology, such as at least 87% homology, such as at least 88% homology, such as at least 89% homology, such as at least 90% homology, 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 thereto.
In other embodiments, the second nucleic acid comprises or consists of the FRS sequence as set forth in SEQ ID NO: 4 or a functional variant thereof and of the native promoter of the eriCF gene of P. syringae, and further comprises one or more of the 5′-terminal codons of the eriCF gene, such as one, two, three, four, five, six, seven, eight, nine or ten 5′-terminal codons of the eriCF gene. In a particular embodiment, the second nucleic acid comprises or consists of the FRS sequence as set forth in SEQ ID NO: 4 or a functional variant thereof and of the native promoter of the eriCF gene of P. syringae, and further comprises the eight 5′-terminal codons of the eriCF gene. The corresponding sequence is as set forth in SEQ ID NO: 5. The second nucleic acid may comprise a sequence having at least 80% homology, such as at least 81% homology, such as at least 82% homology, such as at least 83% homology, such as at least 84% homology, such as at least 85% homology, such as at least 86% homology, such as at least 87% homology, such as at least 88% homology, such as at least 89% homology, such as at least 90% homology, 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: 5.
In some embodiments, the second nucleic acid comprises or consists of the FRS sequence as set forth in SEQ ID NO: 4 or a functional variant thereof and of the PEM7 promoter. The sequence of the corresponding second nucleic acid is set forth in SEQ ID NO: 8. In some embodiments, the second nucleic acid comprises or consists of SEQ ID NO: 8 or a homologue thereof having at least 80% homology, such as at least 81% homology, such as at least 82% homology, such as at least 83% homology, such as at least 84% homology, such as at least 85% homology, such as at least 86% homology, such as at least 87% homology, such as at least 88% homology, such as at least 89% homology, such as at least 90% homology, 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: 8.
In other embodiments, the second nucleic acid comprises or consists of the FRS sequence as set forth in SEQ ID NO: 4 or a functional variant thereof and of the PEM7 promoter, and further comprises one or more of the 5′-terminal codons of the eriCF gene, such as one, two, three, four, five, six, seven, eight, nine or ten 5′-terminal codons of the eriCF gene. In a particular embodiment, the second nucleic acid comprises the FRS sequence as set forth in SEQ ID NO: 4 or a functional variant thereof and of the PEM7 promoter, and further comprises the eight 5′-terminal codons of the eriCF gene. The corresponding sequence is set forth in SEQ ID NO: 7. The second nucleic acid may thus have at least 80% homology to SEQ ID NO: 7, such as at least 81% homology, such as at least 82% homology, such as at least 83% homology, such as at least 84% homology, such as at least 85% homology, such as at least 86% homology, such as at least 87% homology, such as at least 88% homology, such as at least 89% homology, such as at least 90% homology, 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: 7.
Accordingly, in some embodiments the second nucleic acid is as set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, preferably as set forth in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably as set forth in SEQ ID NO: 5 or SEQ ID NO: 6; or the nucleic acid comprises or consists of a sequence having at least 80% homology to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, such as at least 81% homology, such as at least 82% homology, such as at least 83% homology, such as at least 84% homology, such as at least 85% homology, such as at least 86% homology, such as at least 87% homology, such as at least 88% homology, such as at least 89% homology, such as at least 90% homology, 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: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, preferably to SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably to SEQ ID NO: 5 or SEQ ID NO: 6.
In some embodiments, the second nucleic acid comprises or consists of SEQ ID NO: 5.
The intracellular availability of the co-substrate (fluoride, chloride or bromide) or inducer may have an influence on the efficiency of reaction. In some embodiments, it may thus be advantageous to use a cell which has reduced ability to transport the co-substrate out of the cell, in order to increase intracellular availability of the co-substrate.
Accordingly, in some embodiments, the cell further comprises a mutation in at least one fluoride, chloride or bromide transporter gene encoding a fluoride, chloride or bromide transporter, respectively, said mutation resulting in a partial or total loss of function of said fluoride, chloride or bromide transporter, respectively.
In embodiments where the cell is used to perform in vivo fluorination, the cell thus may comprise 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.
In embodiments where the cell is used to perform in vivo chlorination, the cell thus may comprise a mutation in at least one chloride transporter gene encoding a chloride transporter, said mutation resulting in a partial or total loss of function of said chloride transporter.
In embodiments where the cell is used to perform in vivo bromination, the cell thus may comprise a mutation in at least one bromide transporter gene encoding a bromide transporter, said mutation resulting in a partial or total loss of function of said bromide transporter.
The mutation may be a partial or total deletion, as is known in the art.
For example, in a specific embodiment, the cell is a Pseudomonas putida cell used for in vivo fluorination of a substrate as described herein, in particular a P. putida KT2440 cell, and the cell has a mutation in the crcB gene as set forth in SEQ ID NO: 9. In one embodiment, the crcB gene has been deleted.
In specific embodiments, the cell is capable of producing a fluorinated compound from a substrate in the presence of fluoride, said cell comprising:
In another embodiment, the cell is capable of producing a fluorinated compound from a substrate in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of producing a chlorinated compound from a substrate in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing a brominated compound from a substrate in the presence of bromide, said cell comprising:
In specific embodiments, the cell is capable of producing a fluorinated compound from a substrate in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of producing a chlorinated compound from a substrate in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing a brominated compound from a substrate in the presence of bromide, said cell comprising:
In some embodiments, the cell may further express a phosphorylase (EC 2.4.2.28), and is thus capable of producing a phosphorylated compound from a fluorinated, chlorinated or brominated compound. In some embodiments, the cell is capable of producing a phosphorylated fluorinated, chlorinated or brominated compound from a substrate in the presence of fluoride.
In specific embodiments, the cell is capable of phosphorylating a fluorinated compound in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of phosphorylating a chlorinated compound in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of phosphorylating a brominated compound in the presence of bromide, said cell comprising:
In some embodiments, the cell is capable of producing a fluorinated, a chlorinated or a brominated compound and is further capable of phosphorylating said compound. In some embodiments, the phosphorylase gene and the fluorinase gene are both controlled by the riboswitch. In other words, the phosphorylase and the fluorinase gene are present within a same nucleic acid molecule (here the first nucleic acid), for example a vector.
In some embodiments, the cell is capable of producing a fluorinated and phosphorylated compound from a substrate in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of producing a chlorinated and phosphorylated compound from a substrate in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing a brominated and phosphorylated compound from a substrate in the presence of bromide, said cell comprising:
In specific embodiments, the cell is capable of producing a fluorinated and phosphorylated compound from a substrate in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of producing a chlorinated and phosphorylated compound from a substrate in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing a brominated and phosphorylated compound from a substrate in the presence of bromide, said cell comprising:
In some embodiments, the phosphorylase gene is the flB1 gene from Streptomyces sp. MA37 as set forth in SEQ ID NO: 12 or SEQ ID NO: 13, encoding the FlB1 phosphorylase as set forth in SEQ ID NO: 11. In some embodiments, the phosphorylase gene is a homologue of flB1 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: 12 or SEQ ID NO: 13.
In some embodiments, the phosphorylase is a functional variant of FlB1 as set forth in SEQ ID NO: 11, such as a functional variant having at least 80% homology, 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: 11.
In some embodiments, the phosphorylase gene is the deoD gene from Escherichia coli as set forth in SEQ ID NO: 14, encoding the DeoD phosphorylase as set forth in SEQ ID NO: 15. In some embodiments, the phosphorylase gene is a homologue of deoD 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: 14.
In some embodiments, the phosphorylase is a functional variant of DeoD as set forth in SEQ ID NO: 15, such as a functional variant having at least 80% homology, 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: 15.
The phosphorylase gene, such as the flB1 gene or the deoD gene, may be codon-optimised as is known in the art.
Thus in embodiments where the cell is capable of expressing a phosphorylase as described above, such as an FlB1 phosphorylase or a DeoD phosphorylase, the cell is capable of converting 5′-fluoro-5′-deoxyadenosine to 5′-deoxy-5′-fluoro-D-ribose-1-phosphate. The cell is thus capable of producing 5′-deoxy-5′-fluoro-D-ribose-1-phosphate.
In some embodiments, the cell may further express a nucleosidase (EC 3.2.2.9), and is thus capable of producing a deoxyribose compound from a fluorinated, chlorinated or brominated compound. The nucleosidase catalyses the reaction:
In some embodiments, the cell is capable of producing a 5′-fluorodeoxyribose, a 5′-chlorodeoxyribose or a 5′-bromodeoxyribose from a substrate in the presence of fluoride.
In specific embodiments, the cell is capable of producing 5′-fluorodeoxyribose in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of producing 5′-chlorodeoxyribose in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing 5′-bromodeoxyribose in the presence of bromide, said cell comprising:
In some embodiments, the cell is capable of producing a fluorinated, a chlorinated or a brominated compound and is further capable of converting said compound into a 5′-fluorodeoxyribose, a 5′-chlorodeoxyribose or a 5′-bromodeoxyribose. In some embodiments, the nucleosidase gene and the fluorinase gene are both controlled by the riboswitch. In other words, the nucleosidase and the fluorinase gene are present within a same nucleic acid molecule (here the first nucleic acid), for example a vector.
In some embodiments, the cell is capable of producing 5′-fluorodeoxyribose in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of producing 5′-chlorodeoxyribose in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing 5′-bromo-5′-deoxyadenosine to 5′-bromodeoxyribose in the presence of bromide, said cell comprising:
In specific embodiments, the cell is capable of producing 5′-fluorodeoxyribose in the presence of fluoride, said cell comprising:
In other embodiments, the cell is capable of 5′-chloro-5′-deoxyadenosine to 5′-chlorodeoxyribose in the presence of chloride, said cell comprising:
In other embodiments, the cell is capable of producing 5′-bromodeoxyribose in the presence of bromide, said cell comprising:
In some embodiments, the nucleosidase gene is the pfs gene from Escherichia coli as set forth in SEQ ID NO: 16, encoding the Pfs nucleosidase as set forth in SEQ ID NO: 17. In some embodiments, the nucleosidase gene is a homologue of pfs 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: 16.
In some embodiments, the nucleosidase is a functional variant of Pfs as set forth in SEQ ID NO: 17, such as a functional variant having at least 80% homology, 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: 17.
The nucleosidase gene, such as the pfs gene, may be codon-optimised as is known in the art.
Thus in embodiments where the cell is capable of expressing a nucleosidase as described above, such as Pfs nucleosidase, the cell is capable of converting 5′-fluoro-5′-deoxyadenosine to 5′-fluorodeoxyribose. The cell is thus capable of producing 5′-fluorodeoxyribose.
The present disclosure further relates to an expression system for expression in a cell, said system comprising:
In some embodiments, the disclosure further relates to an expression system for expression in a cell, said system comprising:
The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, the second nucleic acid, the riboswitch, the second promoter and the gene encoding an activator of transcription, may be as described herein.
The expression system is suitable for expression of the first nucleic acid and of the second nucleic acid in a cell, where the cell may be as described herein. In particular embodiments, the cell is a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell.
The first and the second nucleic acids may be independently comprised in a vector or integrated in the genome of the cell. In some embodiments, the first and the second nucleic acids are provided in a vector, such as in one vector. In some embodiments, the first nucleic acid is provided in a vector, and the second nucleic acid is provided in another vector. In some embodiments, the first nucleic acid is provided in a vector and the second nucleic acid is integrated in the genome of the cell. In some embodiments, the second nucleic acid is provided in a vector and the first nucleic acid is integrated in the genome of the cell. In some embodiments, the first and the second nucleic acids are integrated in the genome of the cell.
In some embodiments, the second nucleic acid comprises a riboswitch, and the second nucleic acid and the first nucleic acid are the same. Thus the present disclosure further relates to an expression system for expression in a cell, said system comprising:
The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, the second nucleic acid and the riboswitch may be as described herein.
The expression system is suitable for expression in a cell where the cell may be as described herein. In particular embodiments, the cell is a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell.
The first nucleic acid may be comprised in a vector or integrated in the genome of the cell. In some embodiments, the first nucleic acid is provided in a vector. In some embodiments, the first nucleic acid is integrated in the genome of the cell.
The present disclosure further relates to an expression system for expression in a cell, said system comprising:
The above expression system is thus suitable for phosphorylating a fluorinated, a chlorinated or a brominated compound as described herein. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the gene encoding a phosphorylase, the second nucleic acid, the riboswitch, the second promoter, and the gene encoding an activator of transcription, may be as described herein.
The present disclosure further relates to an expression system for expression in a cell, said system comprising:
The above expression system is thus suitable for producing a 5′-fluorodeoxyribose, a 5′-chlorodeoxyribose or a 5′-bromodeoxyribose as described herein. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the gene encoding a nucleosidase, the second nucleic acid, the riboswitch, the second promoter, and the gene encoding an activator of transcription, may be as described herein.
The expression system is suitable for expression of the first nucleic acid and of the second nucleic acid in a cell, where the cell may be as described herein. In particular embodiments, the cell is a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell.
The first and the second nucleic acids may be independently comprised in a vector or integrated in the genome of the cell. In some embodiments, the first and the second nucleic acids are provided in a vector, such as in one vector. In some embodiments, the first nucleic acid is provided in a vector, and the second nucleic acid is provided in another vector. In some embodiments, the first nucleic acid is provided in a vector and the second nucleic acid is integrated in the genome of the cell. In some embodiments, the second nucleic acid is provided in a vector and the first nucleic acid is integrated in the genome of the cell. In some embodiments, the first and the second nucleic acids are integrated in the genome of the cell.
The present disclosure further relates to an expression system for expression in a cell, said system comprising:
In other embodiments, the expression system comprises:
Such expression systems comprising both a fluorinase gene and a gene encoding a phosphorylase when introduced in the cell allow fluorination and phosphorylation of compounds in the presence of inducer. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, and the phosphorylase gene encoding a phosphorylase, the second nucleic acid, the riboswitch, the second promoter and the gene encoding an activator of transcription, may be as described herein.
The present disclosure further relates to an expression system for expression in a cell, said system comprising:
In other embodiments, the expression system comprises:
Such expression systems comprising both a fluorinase gene and a gene encoding a nucleosidase when introduced in the cell allow production of a 5′-fluorodeoxyribose, a 5′-chlorodeoxyribose or a 5′-bromodeoxyribose in the presence of inducer. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, and the nucleosidase gene encoding a nucleosidase, the second nucleic acid, the riboswitch, the second promoter and the gene encoding an activator of transcription, may be as described herein.
The expression system is suitable for expression of the first nucleic acid and of the second nucleic acid in a cell, where the cell may be as described herein. In particular embodiments, the cell is a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell.
The first and the second nucleic acids may be independently comprised in a vector or integrated in the genome of the cell. In some embodiments, the first and the second nucleic acids are provided in a vector, such as in one vector. In some embodiments, the first nucleic acid is provided in a vector, and the second nucleic acid is provided in another vector. In some embodiments, the first nucleic acid is provided in a vector and the second nucleic acid is integrated in the genome of the cell. In some embodiments, the second nucleic acid is provided in a vector and the first nucleic acid is integrated in the genome of the cell. In some embodiments, the first and the second nucleic acids are integrated in the genome of the cell.
Methods for introducing vectors in the cell and methods for integrating nucleic acids in the genome of a cell are known in the art.
Also provided herein is a method for in vivo fluorination, chlorination or bromination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo fluorination, chlorination or bromination of a substrate, comprising the steps of:
In this embodiment, the first nucleic acid and the second nucleic acid may be the same molecule, i.e. the riboswitch controls transcription from the first promoter directly.
In other embodiments, the method is a method for producing a phosphorylated and fluorinated, chlorinated or brominated compound, and the expression system comprises a gene encoding a phosphorylase (EC 2.4.2.28).
In one such embodiment, the method is a method for in vivo phosphorylation of a fluorinated, chlorinated, or brominated substrate, comprising the steps of:
In another such embodiment, the method is a method for in vivo phosphorylation of a fluorinated, chlorinated, or brominated substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo fluorination and phosphorylation, chlorination and phosphorylation or bromination and phosphorylation of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo fluorination and phosphorylation, chlorination and phosphorylation or bromination and phosphorylation of a substrate, comprising the steps of:
In some embodiments, the inducer and the co-substrate are identical.
In some embodiments, the fluorinated, the chlorinated or the brominated substrate such as a fluorinated adenosine, a chlorinated adenosine or a brominated adenosine, are provided in the medium. In other embodiments, the cell is capable of synthesising the fluorinated, the chlorinated or the brominated substrate which is to be phosphorylated.
In other embodiments, the method is a method for producing a 5′-fluorodeoxyribose, a 5′-chlorodeoxyribose or a 5′-bromodeoxyribose, and the expression system comprises a gene encoding a nucleosidase (EC 3.2.2.9).
In one such embodiment, the method is a method for in vivo production of 5′-fluorodeoxyribose, 5′-chlorodeoxyribose and/or 5′-bromodeoxyribose, comprising the steps of:
In another such embodiment, the method is a method for the production of 5′-fluorodeoxyribose, 5′-chlorodeoxyribose and/or 5′-bromodeoxyribose, comprising the steps of:
In one embodiment, the method is a method for in vivo production of 5′-fluorodeoxyribose, 5′-chlorodeoxyribose and/or 5′-bromodeoxyribose, comprising the steps of:
In one embodiment, the method is a method for in vivo production of 5′-fluorodeoxyribose, 5′-chlorodeoxyribose and/or 5′-bromodeoxyribose, comprising the steps of:
In preferred embodiments, the method is for producing 5′-fluoro-5′-deoxyadenosine and converting it to 5′-fluorodeoxyribose.
In some embodiments, the inducer and the co-substrate are identical.
In one embodiment, the method is a method for in vivo fluorination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo fluorination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo phosphorylation of a fluorinated substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo fluorination and phosphorylation of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo fluorination and phosphorylation of a substrate, comprising the steps of:
In some embodiments, the inducer is a fluoride salt.
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In one embodiment, the cell comprises:
In another embodiment, the method is a method for in vivo chlorination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo chlorination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo phosphorylation of a chlorinated substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo chlorination and phosphorylation of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo chlorination and phosphorylation of a substrate, comprising the steps of:
In some embodiments, the inducer is a chloride salt. In other embodiments, the inducer is a fluoride salt.
In another embodiment, the method is a method for in vivo bromination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo bromination of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo phosphorylation of a brominated substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo bromination and phosphorylation of a substrate, comprising the steps of:
In one embodiment, the method is a method for in vivo bromination and phosphorylation of a substrate, comprising the steps of:
In some embodiments, the inducer is a bromide salt. In other embodiments, the inducer is a fluoride salt.
The cell is preferably as described herein; in particular, the cell may be a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, the second nucleic acid, the riboswitch, the second promoter and the gene encoding an activator of transcription, may be as described herein.
The cell is preferably as described herein; in particular, the cell may be a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, and the riboswitch, may be as described herein.
The cell is preferably as described herein; in particular, the cell may be a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the gene encoding a phosphorylase, the second nucleic acid, the riboswitch, the second promoter and the gene encoding an activator of transcription, may be as described herein.
The cell is preferably as described herein; in particular, the cell may be a Pseudomonas cell, such as a Pseudomonas putida cell, such as a Pseudomonas putida KT2440 cell. The elements of the expression system, i.e. the first nucleic acid, the first promoter, the fluorinase gene encoding a fluorinase, the gene encoding a phosphorylase, the second nucleic acid, the riboswitch, the second promoter and the gene encoding an activator of transcription, may be as described herein.
The substrate and co-substrate may be as described herein.
The method may be performed by using an expression system suitable for expression of the first nucleic acid and of the second nucleic acid in a cell, as described herein. The method may be performed by using an expression system suitable for expression of the first nucleic acid in a cell, as described herein.
The method may be performed by using an expression system suitable for expression of the first nucleic acid and of the second nucleic acid in a cell, as described herein.
The method may be performed by using an expression system suitable for expression of the first nucleic acid and of the second nucleic acid in a cell, as described herein.
The type of medium used for propagating the cell will depend on the type of cell used for the method. The medium used is as known in the art.
The in vivo fluorination, chlorination or bromination of a substrate as described herein may be achieved by adding an inducer, which activates transcription of the gene encoding the activator of transcription. This in turn induces transcription of the fluorinase gene, as explained in detail herein above. As a result, the fluorinase is expressed, and can fluorinate, chlorinate or brominate the substrate in the presence of the appropriate co-substrate. In some embodiments, the co-substrate and the inducer are identical.
The inducer may be provided in the form of salts. Preferred salts are soluble salts. In some embodiments, the reaction is a fluorination reaction and the inducer is a fluoride salt. Preferred fluoride salts are NaF or KF; less soluble salts such as CaF2 may also be used. In other embodiments, the reaction is a chlorination reaction and the inducer is a chloride salt. Preferred chloride salts are NaCl or KCl; less soluble salts such as CaCl2) may also be used. In other embodiments, the reaction is a bromination reaction and the inducer is a bromide salt. Preferred bromide salts are NaBr or KBr; less soluble salts such as CaBr2 may also be used.
The inducer may be added at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM, such as at least 0.6 mM, such as at least 0.7 mM, such as at least 0.75 mM, such as at least 0.8 mM, such as at least 0.9 mM, such as at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM, such as at least 11 mM, such as at least 12 mM, such as at least 13 mM, such as at least 14 mM, such as at least 15 mM, such as at least 20 mM, such as 25 mM or more.
The time of adding the inducer may be optimised as is known in the art, and may depend on the choice of cell and/or on the choice of the fluorinase gene. In some embodiments, the inducer is added at culture onset or in mid-exponential phase. It may occur that the presence of inducer will inhibit or slow cell growth, why in some embodiments it may be advantageous to propagate the cells until mid-exponential phase prior to adding the inducer.
The choice of co-substrate will typically be dictated by the type of in vivo reaction which is to be performed. In embodiments where in vivo fluorination of a substrate is desired, the co-substrate is fluoride. In embodiments where in vivo chlorination of a substrate is desired, the co-substrate is chloride. In embodiments where in vivo bromination of a substrate is desired, the co-substrate is bromide.
The co-substrates may be provided in the form of salts. Preferred salts are soluble salts. In some embodiments, the reaction is a fluorination reaction and the co-substrate is a fluoride salt. Preferred fluoride salts are NaF or KF; less soluble salts such as CaF2 may also be used. In other embodiments, the reaction is a chlorination reaction and the co-substrate is a chloride salt. Preferred chloride salts are NaCl or KCl; less soluble salts such as CaCl2) may also be used. In other embodiments, the reaction is a bromination reaction and the co-substrate is a bromide salt. Preferred bromide salts are NaBr or KBr; less soluble salts such as CaBr2 may also be used.
The co-substrate may be added at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM, such as at least 0.6 mM, such as at least 0.7 mM, such as at least 0.75 mM, such as at least 0.8 mM, such as at least 0.9 mM, such as at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM, such as at least 11 mM, such as at least 12 mM, such as at least 13 mM, such as at least 14 mM, such as at least 15 mM, such as at least 20 mM, such as 25 mM or more.
The time of adding the co-substrate may be optimised as is known in the art, and may depend on the choice of cell and/or on the choice of the fluorinase gene. In some embodiments, the co-substrate is added at culture onset or in mid-exponential phase. It may occur that the presence of co-substrate will inhibit or slow cell growth, why in some embodiments it may be advantageous to propagate the cells until mid-exponential phase prior to adding the co-substrate. In other embodiments, the co-substrate is present in the medium used for propagation from the beginning of the culture.
In some embodiments, the inducer is the co-substrate.
Once the inducer and/or co-substrate has been added to the medium, the cell is incubated for a duration such that fluorination, chlorination or bromination of the substrate is achieved to obtain the desired fluorinated, chlorinated or brominated product, respectively.
The duration of the incubation may depend on the choice of cell and of the type of reaction. In some embodiments, the cell is incubated in the presence of inducer for at least 4 hours, such as at least 8 hours, such as at least 12 hours, such as at least 16 hours, such as at least 20 hours, such as at least 24 hours, such as at least 36 hours, such as at least 48 hours, such as at least 72 hours, or more. Such incubation times, which are relatively short, are particularly relevant for embodiments where the cell is a bacterial cell, such as a P. putida cell, or a eukaryotic cell such as a yeast cell. In embodiments where the cell is a eukaryotic cell such as an insect cell, a mammalian cell or a plant cell, longer incubation times may be required. The skilled person knows how to determine which incubation times are suitable.
In some embodiments, the method further comprises a step of recovering the product, i.e. the fluorinated, chlorinated or brominated product.
The product will typically be present intracellularly and can be recovered as is known in the art, for example by solvent extraction from the cells (e.g. with an ethanol solution or a methanol solution). Preparative liquid chromatography can also be used to recover the product.
Also provided herein is a fluorinated, a chlorinated or a brominated product obtainable by the methods described herein, and compositions comprising a fluorinated, a chlorinated or a brominated product obtainable by the methods described herein.
Also provided herein is a method for manufacturing a fluorinated, a chlorinated or a brominated compound of interest, said method comprising the steps of:
In some embodiments, the fluorinated, chlorinated or brominated product obtained by the methods described herein can be used directly, i.e. without further modification.
In other embodiments, however, the fluorinated, chlorinated or brominated compound is further converted into a fluorinated, chlorinated or brominated compound of interest. Indeed, the fluorinated, chlorinated or brominated product obtained by the present method may be used as a drug precursor. The methods of manufacturing a fluorinated, a chlorinated or a brominated compound of interest may thus comprise a step of converting the product obtained by the present methods into a compound of interest, such as a drug or a therapeutic compound. The compound of interest may be a fluorinated nucleoside, a deoxy-fluoronucleoside, a chlorinated nucleoside, a deoxy-chloronucleoside, a brominated nucleoside, a fluoro-ribose phosphate or a deoxy-bromonucleoside.
Also provided herein is the use of a fluorinated, a chlorinated or a brominated product obtainable by the present methods or of a composition comprising such product as a medicament.
P. putida KT2440 was grown at 30° C. and routinely cultured in LB medium or on LB agar plates according to standard protocols unless otherwise stated. Modified M9 minimal medium containing 5 g L−1 of glucose as carbon source was used for all the expression and production assays. E. coli DH5a was used for cloning and plasmid maintenance and was grown in LB at 37° C.
Antibiotics and other supplements were used at the following concentrations: chloramphenicol (Cm) 30 μg·mL−1, kanamycin (Km) 50 μg·mL−1, gentamicin (Gm) 10 μg·mL−1, and streptomycin (St) 100 μg·mL−1. 300 mM sucrose was used for preparing electrocompetent P. putida cells. Overnight pre-cultures were grown in modified M9 supplemented with 0.02% yeast extract. 3-methylbenzoate (3-MB) was used for induction of XylS/Pm promoter. Sodium fluoride (NaF) was purchased from Sigma-Aldrich (201154) and used as inducer, co-substrate and stressor.
Plasmids and strains construction pFB-PmflA1 was constructed by cloning the synthetic codon optimized gene flA1 from Streptomyces sp. MA37 into pPS23 vector, containing the expression system XylS/Pm, using the restriction sites AvrII and EcoRI. The expression vector pFB-PT7flA1 was constructed by cloning the gene flA1 into vector pSEVA231-T7pr, containing the expression system PT7, using the same restriction sites. For the construction of plasmid pFB-PT7flB1(NHis) the synthetic codon optimized gene flB1 from Streptomyces sp. MA37 (SEQ ID NO: 13) was amplified using a forward primer containing a 6× histidine sequence. The amplified gene was cloned into the plasmid pSEVA231-T7pr using AvrII and EcoRI restriction sites, as well. The construction of plasmid pFB-PT7flA1B1 was performed by USER cloning. For this purpose, plasmid pFB-PT7flA1 was amplified using the USER primers flA1_RSv1_U_rv and pSEVA_USER_flB, and gene flB1 was amplified. The amplified fragments were digested with DpnI and 100 ng of the corresponding DNA fragments were mixed with the USER enzyme in a final volume of 12 μl, and incubated at 37° C. for 25 minutes and 25° C. for 25 minutes. 5 μl of this mix was subsequently transformed by heat-shock into DH5α chemically competent cells and plated into LB agar plates with the corresponding antibiotics and incubated at 37° C. overnight. The correct constructions were checked by colony PCR and sequencing.
The four versions of the P. syringae riboswitch, FRSv1-4 (SEQ ID NO: 5, 6, 7 and 8, respectively), were synthesized by IDTDNA and cloned into plasmid pSEVA441, together with the msfGFP. The amplified fragments were digested with DpnI and 100 ng of the corresponding DNA fragments were mixed with the USER enzyme in a final volume of 12 μl, and incubated at 37° C. for 25 minutes and 25° C. for 25 minutes. 5 μl of this mix was subsequently transformed by heat-shock into DH5α chemically competent cells and plated into LB agar plates with the corresponding antibiotics and incubated at 37° C. overnight. The correct constructions were checked by colony PCR and sequencing. These clonings yielded plasmids pSEVA441-FRSv1, pSEVA441-FRSv2, pSEVA441-FRSv3, and pSEVA441-FRSv4.
Plasmid pFB-FRSflA1 was constructed by cloning the gene flA1, amplified with USER primers compatible with the riboswitch USER fragments, into plasmid pSEVA231, amplified with compatible USER primers as well. The fragments were treated with DpnI and 100 ng of the flA1, riboswitch FRSv1, and pSEVA231 fragments were mixed with the USER enzyme in a final volume of 12 ml, and incubated at 37° C. for 25 minutes and 25° C. for 25 minutes. 5 μl of this mix was subsequently transformed by heat-shock into DH5α chemically competent cells and plated into LB agar plates with the corresponding antibiotics and incubated at 37° C. overnight. The correct constructions were checked by colony PCR and sequencing.
The E. coli purine nucleotide phosphorylase, coded by the gene deoD (SEQ ID NO: 14), as well as the nucleosidase pfs (SEQ ID NO: 16), were amplified by PCR from the genome of the strain MG1655 using reverse primers containing the sequence to introduce a 6×His tag, in order to allow purification if needed. These fragments were respectively mixed with a PCR fragment amplified with USER primers of the pPS23 vector, containing the XylS/Pm expression system. The cloning was performed by USER cloning as described above.
The plasmid for integration of the T7 RNA polymerase under the regulation of the fluoride responsive riboswitch, pTn7::FRS-T7RNAP, was constructed by cloning the synthetic fragment FRSv1, containing the riboswitch and the original promoter from P. syringae, as well as the first amino acid of the gene eriCF, to yield a translational fusion to the T7 RNA polymerase. The synthetic gene was amplified and cloned by USER cloning into the backbone pTn7::11_T7RNAP. Both DNA fragments were cloned by USER cloning as explained above.
For the deletion of crcB gene using CRISPR/Cas9, plasmid pSEVA231-CRISPRcrcB was constructed. Primers were phosphorylated by incubating 1 μl both primers at 100 μM with the enzyme T4 PNK and annealed simultaneously by incubating 30 minutes at 37° C., 4 minutes at 95° C., and decreasing the temperature 5° C. per minute until 25° C. were reached. The annealed primers were then ligated with plasmid pSEVA231-CRISPR, previously digested with the enzyme BsaI, using a T4 ligase enzyme. The ligation was then transformed by heat-shock into DH5α chemically competent cells and plated into LB agar plates with the corresponding antibiotics and incubated at 37° C. overnight. The correct constructions were checked by colony PCR.
To delete the crcB gene, an overnight culture of P. putida KT2440 containing the plasmids pSEVA658::SSR and pSEVA421::Cas9-tracrRNA was diluted to a A600 of 0.1, and grown until the A600 reached 0.4-0.5. Cells were subsequently induced to express the SSR recombinase with 1 mM of 3-MB for 3 hours at 30° C. After the induction, electrocompetent cells of the P. putida KT2440 induced strains were propagated using 300 mM sucrose, and were electroporated using 100 ng of pSEVA231-CRISPRcrcB plasmid and 1 μl of 100 M of crcBdeI-Rec, a recombineering oligo containing 45 bp of the upstream and downstream region including the start and stop codons of the gene crcB for its deletion. Cells were recovered in LB at 30° C. for 2 hours, plated in LB with the antibiotics needed, and incubated overnight at 30° C. The successful recombineering was checked by colony PCR and sequencing.
The integration of the T7 RNA polymerase regulated by the fluoride responsive riboswitch was achieved by electroporating the plasmid pTn7::FRS-T7RNAP together with the helper plasmid pTNS2, into freshly prepared P. putida KT2440 and P. putida KT2440ΔcrcB electrocompetent cells. The integration of the mini Tn7 module with the polymerase into the genome was checked by colony PCR.
Overnight cultures of P. putida KT2440 with plasmids pSEVA441-FRSv1, pSEVA441-FRSv2, pSEVA441-FRSv3, pSEVA441-FRSv4, or pSEVA441 as autofluorescence control, were diluted 40 times in 10 ml of minimal medium. Cells were grown in 96-well microtiter plates (flat bottom, Greiner Bio-one) for five hours at 30° C. and 300 rpm, after which the cultures were induced using the following concentrations: 0, 1, 5 and 15 mM of NaF. Subsequently, kinetics of the growth and fluorescence, using wavelengths of excitation and emission of 485 nm and 528 nm, respectively, were performed using a SynergyMX (BioTek) microtiter plate reader for 20 hours. Measurements for kinetics were carried out every ten minutes and three independent biological replicates were carried out. The same procedure was carried out with P. putida KT2440::FRS-T7RNAP and P. putida KT2440ΔcrcB::FRS-T7RNAP strains containing the plasmid pSEVA231-T7pr::msfGFP as well as with the plasmid pSEVA231-T7pr, used as an autofluorescence control. In this case 0, 1, and 15 mM of NaF was used for induction.
In vivo biofluorination in P. putida KT2440 harboring the plasmids pFB-PmflA1 was performed by diluting overnight cultures to an A600 of 0.1 in fresh minimal medium, containing NaF 10 mM, in 50 ml in 250-ml shake flasks. Cells were grown for five to six hours at 30° C. with shaking of 200 rpm, when they reached an A600 between 0.4-0.6, after which they were induced with 3-MB 1 mM. After 24 hours of incubation, samples were retrieved for supernatant analysis. The same strain with empty vector pPS23 was used as negative control. Two independent biological replicates were carried out for each condition.
In vivo biofluorination in P. putida KT2440ΔcrcB harboring the plasmids pFB-PmflA1 was performed by diluting overnight cultures to an A600 of 0.1 in fresh minimal medium in 50 ml in 250-ml shake flasks. Cells were grown for one two hours at 30° C. with shaking of 200 rpm, when they reached an A600 0.2, after which they were induced with 3-MB 1 mM. After 6 hours of incubation, when cultures reached an A600 0.6, 0.5 nM of NaF was added. After 24 hours and 48 hours of incubation, samples were retrieved for supernatant analysis. The same strain with empty vector pPS23 was used as negative control. Two independent biological replicates were carried out for each condition.
In vivo biofluorination in P. putida KT2440 harboring the plasmids pFB-PT7flA1, pFB-FRSflA1, or pFB-FRSflA1B1 was performed by diluting overnight cultures to an A600 of 0.1 in fresh minimal medium in 50 ml in 250-ml shake flasks. Cells were grown for four to five hours at 30° C. with shaking of 200 rpm, when they reached an A600 between 0.4-0.6 or adding NaF 15 mM. After 24 hours of incubation, samples were retrieved for supernatant analysis and metabolite extractions. The same strain with empty vector pSEVA231-T7Pr was used as negative control. Two independent biological replicates were carried out for each condition.
For sample processing, cells were centrifuged at 5000 rpm for 10 minutes, and the supernatants and pellets were separated. For supernatant analysis, a second centrifugation was performed, and 1 ml of supernatant was used for the analysis in a LC-MS system. For the metabolite extraction from the pellets, cells were washed with a phosphate buffer, and incubated with 0.5 ml of extraction solution (ethanol 60% (v/v), ammonium acetate 10 mM, pH 7.2) at 78° C. for 1 minute. Cells were subsequently centrifuged at maximum speed for 1 minutes and the supernatant was placed in a new Eppendorf tube. This extraction was repeated three times for each sample and then evaporated in a Speed-vac for at least 6 hours. Pellets were eluted in 100 μl of water and analysed in a LC-MS system.
In vitro biofluorination was performed with the strain P. putida KT2440 harboring the plasmids pFB-PmflA1, pFB-PT7flB1(NHis), and pFB-PT7flA1, and pPS23 or pSEVA231-PT7pr as negative controls, respectively. Overnight cultures of the strains were diluted to an OD600 of 0.1 in fresh minimal medium, in 250-ml shake flasks. Cells were grown for six hours at 30° C. with shaking of 200 rpm, when they reached an OD600 between 0.4-0.6, after which they were induced with 3-MB 1 mM for the XylS/Pm system or 15 mM of NaF for the FRS-PT7. After 20 hours of incubation, all the volume of culture was centrifuged at 5000 rpm for 10 minutes at 4° C., and the supernatant was discarded. Cellular pellets were washed with Tris-HCl 50 mM pH7.8 and resuspended in a final volume of 3 ml. Cells were disrupted using glass beads in a bead beater, using a program of 6000 rpm for 20 seconds. The mix was centrifuged at 17000 g for 2 minutes at 4° C., and the supernatant was transferred to a new tube. Protein concentrations were determined using a Bradford assay, yielding a concentration of at least 1 mg/ml.
For the fluorinase activity assays, 1 ml of the protein fraction was then mixed with 0.2 mM of SAM and 5 mM of NaF, in Tris-HCl 50 mM pH7.8. This reaction was incubated for 20 hours at 30° C., and then inactivated by boiling the samples at 95° C. for 5 minutes and centrifuging at 17000 g for 10 minutes. The supernatant was taken and analysed by LC-MS.
Similarly, for phosphorylase activity assays, 1 ml of the protein fraction from cultures expressing the phosphorylase was then mixed with 0.2 mM of 5′-FDA, in Tris-HCl 50 mM pH7.8. 0.5 ml of the initial volume was inactivated by boiling the samples at 95° C. for 5 minutes and centrifuging at 17000 g for 10 minutes, yielding the initial concentration of 5′-FDA in the reaction (0 hours). After 20 hours of incubation at 30° C., the rest of the reaction was inactivated as described before and the supernatant was taken and analyzed by HPLC.
Overnight cultures of P. putida KT2440 and P. putida KT2440ΔcrcB were diluted 20 times in 10 mL of modified M9 minimal medium containing different concentrations of NaF. Cells were grown in 96-well microtiter plates (flat bottom, Greiner Bio-one) at 30° C. and 200 rpm in an ELx808 microtiter plate reader (BioTek). Cells exposed to NaF at various concentrations were incubated in a microtiter plate reader for 24 hours, and growth was followed by measuring OD630 every 30 minutes. The concentrations used for each strain were 0, 25, 50, 75, 125, 200 and 250 mM for P. putida KT2440, and 0, 0.1, 0.5, 1 and 5 mM for P. putida KT2440ΔcrcB.
Growth arrest by NaF in P. putida KT2440ΔcrcB was performed by diluting 20 times the overnight cultures in 10 mL of modified M9 minimal medium. Cells were grown in 96-well microtiter plates (flat bottom, Greiner Bio-one) at 30° C. and 200 rpm in an ELx808 microtiter plate reader (BioTek). After six hours of incubation, the cultures had reached an OD630 of 0.5, and the NaF was added at different concentrations. Cells exposed to the stressor were incubated in a microtiter plate reader for 24 hours, and growth was followed by measuring OD630 every 30 minutes. The concentrations of NaF used were 0, 0.05, 0.1, 0.5, 0.75, 1, 2 and 5 mM.
Detection of 5′-FDA were carried out by LC-MS measurements on a Dionex UltiMate 3000 UHPLC (Fisher Scientific, San Jose, CA) connected to an Orbitrap Fusion Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA). The system used a Cortecs T3, 2.1×150 mm, 1.6 μm column kept at 30° C. The flow rate was 0.3 mL/min with a mobile phase of 100% formic acid (0.1%) for 4.2 min followed by a linear gradient of to 25% formic acid (0.1%)/75% acetonitrile (0.1% formic acid) over 1.3 min. This gradient was held for 1 min after which it was changed immediately to 100% formic acid (0.1%) and 0% acetonitrile (0.1% formic acid) and held for 6.5 min. The sample was passed on to the MS equipped with a heated electrospray ionization source (HESI) in positive-ion mode with nitrogen as nebulizer gas (40 a.u.). The cone and probe temperature were 317° C. and 335° C., respectively. Probe gas flow was 12 a.u. and spray voltage was 3500 V. Scan range was 50 to 400 Da and time between scans was 100 ms. All reagents used was of analytical grade. The detection of 5′-FDA was tested against an in-house synthesized standard.
Example 2—Fluorinase selection for its expression in P. putida KT2440 The canonical fluorination pathway, described for the first time in S. cattleya, utilizes SAM and NaF as substrates, which are converted by the fluorinase FlA into 5′-FDA. 5′-FDA is afterwards phosphorylated by a purine nucleotide phosphorylase (flB), and finally converted to fluoroacetaldehyde by an isomerase and an aldolase.
Fluoroacetaldehyde is subsequently transformed into fluoacetate or 4-fluorothreonine as the final products (Murphy 2003). The key step of the pathway, the formation of a C—F bond, is performed by the fluorinase in a very inefficient process due to the F chemistry. A comparison of several fluorinases has shown the inefficiency of such enzymes, which present turnovers lower than 0.3 min−1. The fluorinase encoded by gene flA1 from Streptomyces sp. MA37 was selected for expression in P. putida for biofluorination. Due to the high G+C content of Streptomyces genes, a codon optimization was synthesized and, as a first approach to check the expression of the gene, was cloned under the regulation of the XylS/Pm system, a well-known high expression system in P. putida KT2440 (Calero 2016), yielding the plasmid pFB-PmflA1.
The performance of the plasmid pFB-PmflA1 in P. putida KT2440 was tested in minimal medium supplemented with glucose and inducing the cells in exponential phase. After 24 hours of induction, we prepared a protein extract from the cultures and performed in vitro assays in which the extracts of P. putida KT2440 containing the fluorinase FlA1 were incubated with high concentrations of SAM and NaF. After 20 hours of incubation, a peak corresponding with the mass of 5′-FDA was found by LC-MS. The peak detected in these samples was then compared to a chemically synthesized 5′-FDA standard (data not shown). Protein extracts of the empty plasmid pPS23, containing only the XylS/Pm system, were incubated with SAM and NaF as well, but no 5′-FDA peak was found in those conditions. These results show that the fluorinase is being produced in P. putida KT2440 and that it is active enough for detection of the product of the enzyme.
To test the activity of the fluorinase in vivo, P. putida KT2440 cells harboring plasmid pFB-PmflA1 were grown in the presence of NaF 10 mM as substrate of the reaction. In this case, the same peak of the product of the fluorination reaction, 5′-FDA, that was found in the in vitro assays, was found in in the supernatants of P. putida KT2440 cultures. This peak presented the same mass as the custom chemically synthesized 5′-FDA standard peak as well. No trace of 5′-FDA was found in the supernatant of the cultures of P. putida KT2440 with plasmid pFB-PmflA1 that were not induced or when the empty plasmid pPS23 was used instead (data not shown). These results showed that the fluorinase FlA1 was being expressed and is was functional in vivo as well, although at much lower levels (data not shown). No difference in the growth was found between the strain harboring the pFB-PmflA1 when they were induced or not, although they were slower that the strains with the empty plasmid pPS23, showing a burden of the fluorinase plasmid even when this is not being expressed (data not shown).
We explored the option of having an expression system that could respond to the addition of substrate, removing as many components of the system as possible. For this, we used the F− responsive riboswitch (FRS) from P. syringae, which in its natural context drives the expression of the gene coding for the F− transporter EriCF in the presence of F−. To check the performance of the riboswitch in P. putida KT2440 we cloned four different versions of the riboswitch before the fluorescent reporter protein msfGFP. In the constructions FRSv1 and FRSv2 we kept the original promoter of gene eriCF, whereas in FRSv3 and FRSv4 we changed the promoter to a constitutive heterologous promoter, PEM7. In the constructions FRSv1 and FRSv3 we conserved the first 8 amino acids of the original eriCF gene as well, whereas FRSv2 and FRSv4 started with the coding sequence of the msfGFP (
It has been shown previously that the genetic context of riboswitches affects critically the performance of such RNA regulatory devices (Kent 2018). Therefore, in order to create a system based on the FRS that is independent of the gene of interest, we cloned the promoter region of eriCF gene that showed the best performance, FRSv1, controlling the expression of the T7 RNA polymerase. This construction allows the expression of the polymerase while keeping This FRS-PT7 system was therefore integrated it in single copy into the genome of P. putida KT2440, in the neutral region downstream glmS gene, using a mini-Tn7 delivery vector for this purpose. The resulting strain, P. putida KT2440::FRS-T7RNAP, was used as hosts for introduction of plasmids containing the PT7 promoter controlling any gene for their expression. To test its performance in P. putida, we cloned the gene coding for the fluorescent reporter protein msfGFP under the regulation of the PT7 promoter (
The intracellular availability of NaF in P. putida KT2440 cells was hypothesised to play an important role on the biofluorination. A gene annotated as a F− transporter was found in the genome of P. putida KT2440. This gene, crcB, has also been shown to detoxify F− in E. coli by extruding the ion out of the cells, keeping its intracellular concentration low (Baker 2012). In order to demonstrate the extruding action of CrcB in P. putida KT2440, an in phase scarless deletion of the gene crcB was constructed. The effect of the deletion of crcB on the growth in the presence of high concentrations of NaF in P. putida KT2440 was assessed by calculating the minimal inhibitory concentrations (MICs) of NaF needed to suppress the growth in both a wild-type and a ΔcrcB mutant. It was found that, whereas P. putida KT2440 was able to thrive in minimal medium supplemented with 75 mM of NaF (
The transporter CrcB in P. putida KT2440 seems to keep low levels of intracellular concentrations of F−, as shown above. This could have an effect on the high concentrations of NaF needed to activate the FRS-T7. To test this hypothesis, we used the FRS-PT7 system as a biosensor for the intracellular concentration of NaF. Therefore, we constructed the strain P. putida KT2440ΔcrcB::FRS-T7RNAP, and introduced the PT7-msfgfp vector, to test the induction levels and concentrations of NaF needed. The concentrations of inducer used for this purpose were lower than in the P. putida KT2440 background, due to the toxic effect that is produced by the internal accumulation of F− in the cells when the transporter is not present. However, even though the concentrations were considerably lower, the expression levels achieved in this background were almost two times higher than in the wild-type background (
To explore the possibility of having a growth-decoupled platform for biofluorination, we tested the effect of NaF in the growth when added in mid-exponential phase. In this context, cells were expected to tolerate higher concentrations than when the stressor was added from the beginning during lag phase. Effectively, in this case when 0.5 mM of NaF was added in mid-exponential phase, a decrease in the growth phase was found, although a not so low final growth was seen. That was not the case when 1 mM of NaF was added, a condition that stopped cell growth almost completely (data not shown). In addition to the harsh effect on growth that F− has in cell growth when the homeostasis is disrupted, these results also show that by changing the addition of NaF, the growth arrest can be modulated during the production process.
We therefore tested the production of 5′-FDA in a P. putida KT2440ΔcrcB background using the pFB-Pmfla1 plasmid. In contrast with the biofluorination assays using the wild-type P. putida KT2440 when the NaF was present in the medium from the beginning, in the biofluorination assays using P. putida KT2440ΔcrcB the NaF was added when a A600 of approximately 0.5 was reached. We also observed that the simultaneous addition of the inducer 3 MB and the substrate NaF affected negatively the growth as well as the production, and no biofluorination was detected (data not shown). However, when the inducer 3 MB was added early (A600 of about 0.2) and then the NaF was added in mid exponential phase, an effect on the growth was seen but production of 5′-FDA was achieved after 24 hours (data not shown). After 48 hours of production, cells are able to grow up to A600 of 2.5 (data not shown).
The FRS-T7 system was shown to work in P. putida KT2440 in presence of the inducer NaF. In order to give a further application to the system than a biosensor of the substrate NaF, we used it as an expression system using NaF, an inorganic salt, as the inducer. For this purpose, we cloned the fluorinase gene flA1 under the regulation of promoter PT7 and transformed it into the strains harbouring the FRS-T7 RNA polymerase system (
We tested the possibility of using the F− responsive riboswitch as an expression system for the in vivo fluorination, which would additionally use the NaF as inducer. For this purpose, we constructed plasmid pFB-FRSflA1 (
The organofluorine 5′-FDA can be further converted into the fluorinated sugar 5′-fluorodeoxyribose phosphate (5′-FDRP) by the action of a purine nucleotide phosphorylase (PNP) from S. cattleya, coded by gene flB. In order to assess the ability of P. putida KT2440 to express this gene, the codon optimized gene flB1 from Streptomyces sp. MA37 (SEQ ID NO: 13) was cloned under the regulation of the T7 promoter, to be used in the F− inducible expression system based on the T7 RNA polymerase that we described before for the fluorinase expression (
The expression of flB1 (SEQ ID NO: 11) in P. putida KT2440::FRS-T7RNAP was tested in minimal medium supplemented with glucose and inducing the cells in exponential phase. After 24 hours of induction, we prepared a protein extract from the cultures and performed in vitro assays in which the extracts of P. putida KT2440::FRS-T7RNAP containing the PNP FlB1 were incubated with high concentrations of 5′-FDA. After 20 hours of incubation, a decrease in the concentration of 5′-FDA compared to the initial concentration added to the reactions could be seen in the cell extracts from cultures with plasmid pFB-T7FlB1(NHis). No decrease in the 5′-FDA concentration could be detected when cell extracts of the empty plasmid pSEVA231 were used (
We furthermore explored the possibility that 5′-FDA could be utilized for other enzymes different to the endogenous of the fluorination pathway from Streptomyces. For this purpose, we used another PNP and a nucleosidase of E. coli. The E. coli PNP, coded by the gene deoD (SEQ ID NO: 14), is able to perform the same reaction as FlB1, and produce 5′-FDRP, whereas the nucleosidase, coded by the gene pfs (SEQ ID NO: 16), removes the adenosine of 5′-FDA, yielding the sugar 5′-fluorodeoxyribose. These three enzymes share a similarity of 26% in their amino acidic sequence, and contain a common domain, the PNP_UDP_1 domain (PF0148), belonging to the Phosphorylase superfamily. To test the activity of these two enzymes, they were overexpressed in P. putida and the cell extracts were incubated with the substrate 5′-FDA. After 20 hours, the substrate of the reaction was reduced or depleted completely, and the products could be detected by LC-MS (
We finally tested the ability of the cells to synthesize in vivo 5′-FDRP using F− as the inducer and substrate of the reaction. For this purpose, we constructed the plasmid pFB-PT7flA1B1, which contains genes flA1 and flB1, both under the regulation of the promoter PT7. This plasmid was transformed into the strain that contains the T7 RNA polymerase under the regulation of the riboswitch FRS, so the whole expression system responds to F− (
Streptomyces
Streptomyces
Streptomyces
Pseudomonas
syringae
Pseudomonas
syringae FRS
Pseudomonas
syringae FRS
Pseudomonas
syringae FRS
Pseudomonas
syringae FRS
Pseudomonas
putida
Pseudomonas
putida
Streptomyces
Streptomyces
E. coli
E. coli
E. coli
E. coli
1. A cell capable of producing a fluorinated, a chlorinated or a brominated compound from a substrate in the presence of fluoride, chloride or bromide, respectively, said cell comprising:
2. The cell according to item 1, 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.
3. The cell according to any one of the preceding items, wherein the first nucleic acid and the second nucleic acid are the same nucleic acid.
4. The cell according to any one of the preceding items, 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 or a chlorinated substrate such as a fluorinated or chlorinated adenosine, said phosphorylase gene being under control of the first promoter.
5. The cell according to any one of the preceding items, 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.
6. The cell according to any one of the preceding items, 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.
7. A cell capable of producing a fluorinated and phosphorylated compound, a chlorinated and phosphorylated compound, or a brominated and phosphorylated compound from a substrate, said cell comprising:
8. A cell capable of converting 5′-fluoro-5′-deoxyadenosine to 5′-fluorodeoxyribose in the presence of fluoride, said cell comprising:
9. The cell according to any one of the preceding items, wherein the cell can tolerate toxic compounds, such as fluorinated compounds.
10. The cell according to any one of the preceding items, wherein the cell is a non-pathogenic organism.
11. The cell according to any one of the preceding items, wherein the cell is a mammalian cell, a plant cell, an insect cell, a yeast cell or a bacterial cell.
12. The cell according to any one of the preceding items, 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 and Lipomyces.
13. The cell according to any one of the preceding items, 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.
14. The cell according to any one of the preceding items, wherein the cell is Pseudomonas putida KT2440.
15. The cell according to any one of items 1 to 14, 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.
16. The cell according to any one of the preceding items, wherein the fluorinase is capable of catalysing fluorination, chlorination or bromination of a substrate to obtain a fluorinated, a chlorinated or a brominated compound, such as 5′-deoxy-5′-fluoroadenosine, 5′-chloro-5′-deoxyadenosine, 5′-bromo-5′-deoxyadenosine, 5′-deoxy-5′-fluoro-D-ribose 1-phosphate, 5′-chloro-5′-deoxy-D-ribose 1-phosphate, or 5′-bromo-5′-deoxy-D-ribose 1-phosphate.
17. The cell according to any one of the preceding items, 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, chloride, 5′-deoxy-5′-fluoroadenosine, 5′-chloro-5′-deoxyadenosine, and 5′-bromo-5′-deoxyadenosine.
18. The cell according to any one of the preceding items, wherein the substrate is a SAM derivative of formula (I):
19. The cell according to item 18, wherein the SAM derivative of formula (I) is of formula (II):
20. The cell according to any of the preceding items wherein the substrate is selected from the group consisting of
21. The cell according to any one of items 18 to 20, wherein A is selected from the group consisting of
22. The cell according to any one of items 18 to 21, wherein the SAM derivative of formula (I) is of formula (III):
23. The cell according to any one of items 18 to 22, wherein the substrate is selected from the group consisting of
24. The cell according to any one of the preceding items, 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.
25. The cell according to any one of the preceding items, wherein the fluorinase gene is codon-optimised.
26. The cell according to any one of the preceding items, wherein the fluorinase gene is as set forth in SEQ ID NO: 2 or SEQ ID NO: 3, or a homologue 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: 2 or SEQ ID NO: 3.
27. The cell according to any one of the preceding items, wherein the phosphorylase is the phosphorylase as set forth in SEQ ID NO: 11 or SEQ ID NO: 15, 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: 11 or SEQ ID NO: 15.
28. The cell according to any one of the preceding items, wherein the nucleosidase is the nucleosidase as set forth in SEQ ID NO: 17, 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: 17.
29. The cell according to any one of the preceding items, wherein the phosphorylase gene is as set forth in SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14, or a homologue 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: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
30. The cell according to any one of the preceding items, wherein the nucleosidase gene is as set forth in SEQ ID NO: 16, or a homologue 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: 16.
31. The cell according to any one of the preceding items, wherein the phosphorylase gene, the nucleosidase gene and/or the fluorinase gene is codon-optimised.
32. The cell according to any one of the preceding items, 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.
33. The cell according to any one of the preceding items, wherein the polymerase is a T7 RNA polymerase and the first promoter is a T7 promoter.
34. The cell according to any one of the preceding items, 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.
35. The cell according to any one of the preceding items, wherein the second nucleic acid is as set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, preferably as set forth in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably as set forth in SEQ ID NO: 5 or SEQ ID NO: 6; or wherein the nucleic acid comprises or consists of a sequence having at least 90% homology to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, 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: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8, preferably to SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8, most preferably to SEQ ID NO: 5 or SEQ ID NO: 6.
36. The cell according to any one of the preceding items, wherein the inducer is a fluoride salt such as NaF or KF, or a chloride salt such as NaCl or KCl.
37. The cell according to any one of the preceding items, further comprising a mutation in at least one fluoride, chloride or bromide transporter gene encoding a fluoride, chloride or bromide transporter, respectively, said mutation resulting in a partial or total loss of function of said fluoride, chloride or bromide transporter, respectively.
38. The cell according to item 37, wherein the mutation is a deletion.
39. The cell according to any one of items 37 to 38, wherein the cell is a Pseudomonas putida cell and the fluoride transporter gene is the crcB gene as set forth in SEQ ID NO: 9.
40. An expression system for expression in a cell, comprising:
41. The expression system in a cell according to item 40, 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.
42. The expression system in a cell according to any one of items 40 to 41, wherein the first nucleic acid and the second nucleic acid are the same nucleic acid.
43. The expression system in a cell according to any one of items 40 to 42, 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 or a chlorinated adenosine, said phosphorylase gene being under control of the first promoter.
44. The expression system in a cell according to any one of items 40 to 43, 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.
45. The expression system according to any one of items 40 to 44, 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.
46. An expression system for expression in a cell, comprising:
47. An expression system for expression in a cell, comprising:
48. The expression system according to any one of items 46 to 47, 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 an inducer, wherein the activator of transcription upon expression activates transcription from the first promoter.
49. The expression system of items 46 to 48, wherein the cell is as defined in any one of items 1 to 39.
50. The expression system of any one of items 46 to 49, wherein the first nucleic acid and/or the second nucleic acid are independently comprised in a vector or are integrated in the genome of the cell.
51. A method for in vivo fluorination, chlorination or bromination of a substrate, comprising the steps of:
52. A method for in vivo production of 5′-fluorodeoxyribose, 5′-chlorodeoxyribose and/or 5′-bromodeoxyribose, comprising the steps of:
53. The method according to any one of items 51 to 52, 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, thereby inducing transcription of the fluorinase gene.
54. The method according to any one of items 51 to 53, wherein the first nucleic acid and the second nucleic acid are the same nucleic acid.
55. The method according to any one of items 51 to 54, 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 or a chlorinated substrate such as a fluorinated or chlorinated adenosine, said gene being under control of the first promoter.
56. The method according to any one of items 51 to 55, 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, thereby inducing transcription of the fluorinase gene, the nucleosidase gene and/or the phosphorylase gene.
57. A method for in vivo phosphorylation of a fluorinated, a chlorinated, or brominated substrate, comprising the steps of:
58. The method according to item 57, 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 an inducer, wherein the activator of transcription upon expression activates transcription from the first promoter.
59. The method according to items 51 to 58, wherein the cell is as defined in any one of items 1 to 39, or wherein the cell comprises an expression system as defined in any one of items 40 to 50.
60. The method according to any one of items 51 to 59, wherein the inducer is the co-substrate.
61. The method according to any one of items 51 to 60, wherein the method is for in vivo fluorination of the substrate, and the co-substrate or inducer is a fluoride salt such as NaF or KF.
62. The method according to any one of items 51 to 61, wherein NaF or KF is added at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM, such as at least 0.6 mM, such as at least 0.7 mM, such as at least 0.75 mM, such as at least 0.8 mM, such as at least 0.9 mM, such as at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM, such as at least 11 mM, such as at least 12 mM, such as at least 13 mM, such as at least 14 mM, such as at least 15 mM, such as at least 20 mM, such as 25 mM or more.
63. The method according to any one of items 51 to 62, wherein the method is for in vivo chlorination of the substrate, and the co-substrate or inducer is a chloride salt such as NaCl or KCl.
64. The method according to any one of items 51 to 63, wherein NaCl or KCl is added at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM, such as at least 0.6 mM, such as at least 0.7 mM, such as at least 0.75 mM, such as at least 0.8 mM, such as at least 0.9 mM, such as at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM, such as at least 11 mM, such as at least 12 mM, such as at least 13 mM, such as at least 14 mM, such as at least 15 mM, such as at least 20 mM, such as 25 mM or more.
65. The method according to any one of items 51 to 64, wherein the method is for in vivo bromination of the substrate, and the co-substrate or inducer is a bromide salt such as NaBr or KBr.
66. The method according to any one of items 51 to 65, wherein NaBr or KBr is added at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM, such as at least 0.4 mM, such as at least 0.5 mM, such as at least 0.6 mM, such as at least 0.7 mM, such as at least 0.75 mM, such as at least 0.8 mM, such as at least 0.9 mM, such as at least 1 mM, such as at least 2 mM, such as at least 3 mM, such as at least 4 mM, such as at least 5 mM, such as at least 6 mM, such as at least 7 mM, such as at least 8 mM, such as at least 9 mM, such as at least 10 mM, such as at least 11 mM, such as at least 12 mM, such as at least 13 mM, such as at least 14 mM, such as at least 15 mM, such as at least 20 mM, such as 25 mM or more.
67. The method according to any one of items 51 to 66, wherein the inducer and/or co-substrate is added at the culture onset or in mid-exponential phase.
68. The method according to any one of items 51 to 67, wherein the cell is incubated in the presence of inducer and/or co-substrate for at least 4 hours, such as at least 8 hours, such as at least 12 hours, such as at least 16 hours, such as at least 20 hours, such as at least 24 hours, such as at least 36 hours, such as at least 48 hours, or more.
69. The method according to any one of items 51 to 68, further comprising a step of recovering the fluorinated, chlorinated or brominated product.
70. A fluorinated, a chlorinated or a brominated product, or 5′-fluorodeoxyribose, 5′-chlorodeoxyribose or 5′-bromodeoxyribose obtainable by the method according to any one of items 51 to 69.
71. A composition comprising a fluorinated, a chlorinated or a brominated product according to item 70.
72. A method for manufacturing a fluorinated, a chlorinated or a brominated compound of interest, said method comprising the steps of:
73. The fluorinated, chlorinated or brominated product or the 5′-fluorodeoxyribose, 5′-chlorodeoxyribose or 5′-bromodeoxyribose according to item 70 or the composition according to item 71 for use as a medicament.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18201983.6 | Oct 2018 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/078790 | 10/23/2019 | WO |
