WO2015165840A1

WO2015165840A1 - Antibiotic-free method for selection of transformed bacteria

Info

Publication number: WO2015165840A1
Application number: PCT/EP2015/059031
Authority: WO
Inventors: Domenico Maione; Maria GIULIANI
Original assignee: Novartis Ag
Priority date: 2014-04-28
Filing date: 2015-04-27
Publication date: 2015-11-05

Abstract

An antibiotic-free selection method based on the use of a FabV gene as a selection marker. The FabV gene encodes a triclosan-resistant ACP-enoyl reductase. The FabV gene functionally replaces Fabl in vivo and renders triclosan-sensitive bacteria resistant to triclosan. It can therefore be used as selection marker for plasmid maintenance when triclosan is added as selective agent into the culture medium. This selection method does not require the use of mutant strains or particular media. Moreover triclosan is stable, easy to handle, inexpensive, and approved as safe for humans.

Description

ANTIBIOTIC-FREE METHOD FOR SELECTION OF TRANSFORMED BACTERIA

FIELD OF THE INVENTION

The present invention relates to antibiotic-free selection of transformed bacteria for recombinant protein production or the propagation of a vector. BACKGROUND ART

An essential requirement for genetic engineering of bacteria is the ability to select the cells which have been modified by the uptake of a vector containing a particular gene of interest and maintain the growth of the transformed bacteria so that the gene of interest can be expressed. Most commonly a selection marker is included in the vector. The selection marker can be gene or DNA sequence that allows separation of the bacteria containing the marker and those not containing it. The combination of the selection marker and the selection medium can allow for the growth of bacteria that have been transformed with the vector, while prohibiting the growth of bacteria that have not been transformed.

An antibiotic resistance gene as a selection marker, in combination with a medium containing the antibiotic can be used in order to achieve selection. However, the use of antibiotic resistance genes contributes to the problem of widespread antibiotic resistance (Levy S.B., 2002). In addition the increasing regulatory requirements to which biological agents are subjected have a great impact in the field of industrial protein expression and production.

Previously disclosed antibiotic-free selection systems include the use of auxotrophic strains, for instance, amino acid auxotrophic Escherichia coli strains, that can be used in combination with genes that overcome the specific auxotrophy, as selection markers. These selection systems are based on the use of mutant, auxotrophic bacteria strains, and chemically defined selection media.

Goh and Good (2008) disclose use of a growth essential target gene, FabI, as a plasmid-borne marker in combination with triclosan as a selective agent. However, when FabI is overexpressed in the absence of triclosan, it is toxic for the cell such that the level of triclosan during growth needs to be carefully optimised and balanced with expression of FabI.

Thus there is a need for improved methods of bacterial selection that are antibiotic-free, wherein the growth of transformed bacteria can occur in complex media, and wild-type bacteria can be used for selection. Ideally the selection methods will provide a high plasmid copy number and a comparable amount of recombinant protein production to antibiotic-based selection methods.

SUMMARY OF THE INVENTION

The present invention relates to an improved method for selection of transformed bacteria utilising FabV enoyl-acyl carrier protein reductase. In a first aspect there is provided a method for antibiotic-free selection of transformed bacteria comprising the steps of (i) transforming a triclosan-sensitive bacterium with a vector carrying a functional FabV gene as a selection marker to give a transformed bacterium and (ii) growing the transformed bacteria in a growth medium which contains triclosan in a concentration sufficient to suppress the growth of triclosan-sensitive bacteria.

In a related aspect there is provided a method for selection of transformed bacteria comprising a nucleic vector which comprises a nucleic acid sequence of interest, which method comprises the steps of;

i) transforming triclosan-sensitive bacteria with a vector comprising (a) as a selection marker a nucleic acid sequence encoding a functional FabV protein; and (b) the nucleic acid sequence of interest, which does not encode a functional FabV protein, to give transformed bacteria, and; ii) growing the transformed bacteria in a growth medium containing triclosan at a concentration sufficient to suppress the growth of triclosan-sensitive bacteria; and

iii) selecting one or more bacteria that are able to grow in the growth medium.ated aspect there is provided

As used herein, the term "antibiotic-free selection" refers to the use of conditions that enable the discrimination of cells displaying a required phenotype without the use of antibiotics, e.g., the growth of bacteria in medium wherein antibiotic(s) are not used to select for cells containing a nucleic acid of interest. The term "antibiotic" as used herein takes the usual meaning in the art and refers to an organic substance produced by a microorganism, and/or a semisynthetic equivalent organic substance, that either inhibits or kills other microorganisms. As used herein the term "triclosan-sensitive bacterium" is used to mean a bacterium that cannot survive and/or actively grow and divide in the presence of triclosan (at a particular inhibitory concentration defined below), for example, the bacteria does not demonstrate significant growth (p < 0.05) of cells in the presence of triclosan within an 8 hour time period as determined by optical density measurement at 600 nm (OD600).

In one embodiment the growth medium is a complex medium, for example, LB broth, comprising triclosan at a concentration sufficient to suppress the growth of triclosan-sensitive bacteria. The minimum inhibitory concentration (MIC) is the lowest concentration of Triclosan that inhibits visible growth of a particular microorganism after overnight incubation. MICs may be determined following DIN 58940-81 using a micro-dilution assay (Beuth 1998). The skilled person will understand that different strains and types of bacteria may have different MICs. Particularly the concentration of triclosan in the medium is greater than 0Λμg/mL, about 0.2μg/mL, particularly at least O^g/mL or more particularly greater than 0.2 μg/mL, for example, about or greater than 0.3μg/mL, about or greater than 0Aμg/mL, about or greater than 0.5μg/mL, about or greater than O^g/mL about or greater than ^g/mL, about or greater than l ^g/mL, about 2μ§/ηιΙ, or about 2^g/mL. Particularly between O^g/mL and 2^g/mL, between O^g/mL and 2μg/mL, between O^g/mL and 2μ§/ητΙ,, between O^g/mL and 2μ§/ηιΙ,, between O^g/mL and 2μg/mL or between ^g/mL and 2μg/mL.

Accordingly the triclosan-sensitive bacteria will have a MIC of less than ^g/mL, less than O^g/mL, less than 0.8 μg/mL, less than 0.7 μg/mL, less than 0.6 μg/mL, less than 0.5 μg/mL, less than 0.4 μg/mL, less than 0.3 μg/mL, less than or about 0.2 μg/mL or less than or about 0.1 μg/mL. In certain embodiments the triclosan-sensitive bacteria strain is a strain of Escherichia coli, for example BL21(DE3). The skilled person will be able to select other suitable strains for use in the present invention, for example, using the micro-dilution assay referred to above.

In a second aspect of the invention, there is provided an expression vector for complementing triclosan-sensitive bacteria, particularly E. coli bacteria, comprising an isolated functional FabV gene as a selection marker.

In a related aspect the present invention provides an expression vector for complementing triclosan-sensitive bacteria, comprising (a) as a selectable marker a nucleic acid sequence encoding a functional FabV protein; and (b) a nucleic acid sequence of interest which does not encode a functional FabV protein.

The term "FabV protein" refers to a FabV enoyl-acyl carrier protein reductase. Thus a nucleic acid sequence encoding a FabV protein (a "FabV gene") is one that, when expressed, is capable of providing resistance or increased resistance to Triclosan, particularly, as demonstrated by an increased MIC when compared to a non-complemented or non-transformed cell. Typical expression vectors contain, at the minimum, a promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator. Expression of the FabV gene will generally be under control of a promoter, for example, the P3 promoter from the ampC gene although the skilled person will be aware of other suitable promoters.

Particularly the expression vector will comprise an origin of replication, for example the Fl, ColEl or Ml 3 origins of replication. The expression vector may also comprise a multiple cloning site (MCS). In certain embodiments the expression vector comprises a nucleic acid of interest. Yet more particularly, the nucleic acid of interested is cloned into the multiple cloning site. Particularly the vector is a non-integrative vector. The term "non-integrative" as used herein in reference to a vector indicates that the vector does not integrate into the host DNA, i.e. it does not integrate into the bacterial chromosome, and replicates extra-chromosomally.

In a third aspect, the invention provides a bacterium containing the expression vector of the second aspect, wherein expression of the nucleic acid sequence encoding a FabV enoyl-acyl carrier protein reductase renders the bacterium (which would otherwise be sensitive to triclosan) resistant, or more resistant, to triclosan when compared to a non-complemented or non- transformed bacterium of the same type. In a fourth aspect of the invention there is provided a method of producing a protein of interest comprising a step of growing a transformed bacterium of the third aspect under conditions such that (a) the growth of triclosan-sensitive bacteria is suppressed and (b) the protein of interest is produced. Particularly the protein of interest is encoded by a nucleic acid sequence of interest on the expression vector.

In a fifth aspect, the invention provides a method of producing a nucleic acid of interest comprising a step of growing a transformed bacterium of the invention under conditions such that (a) the growth of triclosan-sensitive bacteria is suppressed and (b) the nucleic acid of interest is propagated.

In a sixth aspect there is provided a method for preparing a pharmaceutical composition, comprising purifying a protein or nucleic acid of interest produced in said methods of the fourth and fifth aspects, and preparing a pharmaceutical composition comprising the purified protein or nucleic acid of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure la shows a map of the pFabVk plasmid.

Figure lb shows a map of the pFabVk-GFP plasmid (6908bp).

Figure lc shows a map of the pFabV plasmid (5450bp) which lacks the kanamycin resistance gene.

Figure 2a shows the growth of the E.coli transformed with the pFabVk-GFP vector (left) compared to the growth of E.coli transformed with the pET24-GFP vector (right), in LB+Triclosan (O^g/mL) media.

Figure 2b shows the positive control experiment where the transformed E.coli of figure 2a (pFabVk-GFP (left) and pET24-GFP (right) are grown on LB+ kanamycin (50μg/mL) plates.

Figure 3 shows the growth (OD₆₀o_nm) against time (h) for E.coli transformed with the pFabVk- GFP vector (left), and E.coli transformed with the pET24-GFP vector (right) in different concentrations of triclosan. The data reported in figures 3 and 4 represent averages of 3 replicas.

Figure 4 shows the specific growth rate (h ¹) of E.coli transformed with either pET24-GFP (diamonds) or pFabVk-GFP (squares) at various triclosan concentrations ^g/mL). The minimum inhibitory concentration (MIC) is shown as 0.2 μg/mL. The data reported in figures 3 and 4 represent averages of 3 replicas.

Figure 5 shows the specific production fluorescence/OD (a.u.) against time after induction (h) for; E.coli transformed with pET24-GFP in LB broth (squares), E.coli transformed with pET24- GFP grown in LB broth supplemented with kanamycin (diamonds), and E.coli transformed with pFabVk-GFP grown in LB broth supplemented with triclosan (triangles). The data reported are average of triplicates.

Figure 6 shows the specific production fluorescence/OD (a.u.) against time after induction (h) for; E.coli transformed with pET24-GFP (circles), pET24-GFP with kanamycin (diamonds) or pFabVk-GFP with triclosan (triangles) grown under high cell density conditions. The data reported are average of triplicates.

Figure 7 shows GFP production (specific fluorescence/OD (a.u.) for E.coli transformed with pFabVk-GFP (left-hand bar) compared to E.coli transformed with pET24-GFP (right-hand bar) after up to 15 cultivation cycles. Data reported are average of duplicates. DETAILED DESCRIPTION

The inventors have discovered that the use of the FabV gene as a selection marker, combined with the addition of triclosan to the media provides a robust and efficient selection system for antibiotic-free selection of transformed bacteria.

Triclosan-sensitive bacteria can be complemented by the FabV gene to restore growth in the presence of triclosan. For example wild-type E.coli contain the ACP-enoyl reductase isozyme Fabl making them highly sensitive to triclosan (minimum inhibitory concentration (MIC) < 0.2 μg/mL) (Zhu et al, 2010). Triclosan specifically inhibits fatty acid synthesis in E.coli by binding to the Fabl substrate site, forming a stable FabI/NAD+/triclosan ternary complex (Heath et al., 1999). In contrast P. aeruginosa is resistant to triclosan even at very high concentration (>2 g/L) due to the presence of the ACP-enoyl reductase isozyme FabV, that is not inhibited by the molecule. The P. aeruginosa FabV gene dominates E. coli Fabl and renders E. coli resistant to triclosan. It can therefore be used as selection marker for plasmid maintenance when triclosan is added into the culture medium.

In contrast to amino acid based auxotrophy systems or systems which rely on the deletion or "knock-out" of endogenous genes, the selection method of the invention does not require the skilled person to generate or use mutant bacterial strains.

In amino acid auxotrophy based selection methods, the untransformed mutant amino acid auxotrophic strains are able to grow in complex media, as complex media provide sufficient concentrations of the amino acids that the strains are auxotrophic for. Selection and growth in amino acid auxotrophy based systems are therefore limited to chemically defined media. The selection method of the invention allows selection to occur in both chemically defined and complex growth media to which triclosan has been added to a sufficient concentration to suppress the growth of triclosan-sensitive bacteria. The selection method of the invention is therefore not restricted to chemically defined growth media, although they are also suitable for use with the invention. Chemically defined growth media generally provide lower yields and are more expensive than complex media, especially if the list of media components includes growth factors and vitamins. Therefore the development of an expression system that allows reproducible results in both complex media and chemically defined growth media is highly desirable.

The method of the present invention is capable of providing a high plasmid copy number, which can be maintained over 20 cycles of cultivation, and which is comparable to that of conventional antibiotic based systems, as demonstrated in Example 6.

Example 3 demonstrates that triclosan-sensitive bacteria, when complemented with the FabV expressing plasmid and cultivated in LB broth with triclosan at a concentration of 0.2mg/L (equivalent to 0.2μg/mL), produce significantly more protein than triclosan-sensitive bacteria transformed with a control plasmid, grown in LB broth and not subject to any selection method.

This increase in recombinant protein production exhibited by the FabV selection method has also been shown (Example 4) to be comparable to the protein production in an antibiotic-based selection method, when cultivation occurred at high cell density.

FabV

Transformation and complementation of any triclosan-sensitive bacteria with an expression vector comprising a functional FabV gene allows transformed bacteria to grow in the presence of triclosan. Selection of transformed bacteria occurs when the triclosan-sensitive bacteria are grown in a media which contains triclosan at a concentration sufficient to suppress the growth of untransformed triclosan-sensitive bacteria.

P. aeruginosa is resistant to triclosan even at very high concentration (>2 g/1) due to the presence of the ACP-enoyl reductase isozyme, FabV, that is not inhibited by triclosan. The P. aeruginosa FabV gene dominates E. coli Fabl and renders E. coli resistant to triclosan. It is therefore ideal for use as selection marker for plasmid maintenance when triclosan is added into the culture medium.

FabV, was discovered in Vibrio cholerae. FabV is unlike other ACP-enoyl reductases in that it is completely refractory to triclosan inhibition, is 60% longer than the typical short-chain dehydrogenase/reductase (SDR) family member (which are generally about 250 residues long), and has an eight-residue space between the active-site tyrosine and lysine residues (Tyr-X⁸-Lys). This spacing has two more residues than those in the active site of Fabl and one more than the maximum reported for other SDR proteins (Zhu et al, 2010), (Massengo-Tiasse et al, 2008). Docking studies discussed in Hirsbeck et al, 2012, have shown that triclosan is unlikely to adopt the binding mode in FabV that is observed in other ACP-enoyl reductases. The functional FabV gene comprises a polynucleotide sequence that is capable of expressing an active ACP-enoyl reductase that is resistant to inhibition by triclosan and is able to confer resistance to triclosan-sensitive bacteria by complementation.

Particularly the functional FabV gene comprises the sequence identified in SEQ ID NO: 1 (NCBI Reference Sequence: NC 002516.2; PA2950 FabV trans-2-enoyl-CoA reductase), and encodes a triclosan-resistant ACP-enoyl reductase as identified in the following SEQ ID NO:2:

MI IKPRVRGFICVTTHPAGCEANVKQQIDYVEAKGPWNGPKKVLVIGSSTGYGLAARITAAFGSGADT LGVFFERPGSESKPGTAGWYNSAAFEKFAHEKGLYARSINGDAFSDEVKRLTIETIKRDLGKVDLWYS LAAPRRTHPKSGEVFSSTLKPIGKSVSFRGLDTDKEVIKDWLEAASDQEVADTVAVMGGEDWQMWIDA LLEADVLADGAKTTAFTYLGEKITHDIYWNGSIGAAKKDLDQKVLGIRDKLAPLGGDARVSVLKAWTQ ASSAIPMMPLYLSLLFKVMKEQGTHEGCIEQVDGLYRESLYGAEPRLDEEGRLRADYKELQPEVQSRVE ELWDKVTNENLYELTDFAGYKSEFLNLFGFEVAGVDYEQDVNPDVQIANLIQA

The FabV gene preferably encodes a protein with a sequence identity that is greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more) with respect to SEQ ID NO: 2. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=l . The gene can be modified according to codon preference of the host cell without modifying the encoded protein. Active ACP-enoyl reductases encoded by the FabV gene can take various forms, including fusion proteins. The functional FabV gene can also comprise a fragment of a FabV gene, which encodes an active ACP-enoyl reductase.

Triclosan-sensitive bacteria

Any bacterium which is sensitive to triclosan inhibition is suitable for use in the invention as the subject for transformation with the expression vector. For example E.coli are highly sensitive to inhibition by triclosan (MIC < 0.2 mg/1) (Zhu et al 2010). E.coli are therefore suitable for use in the invention as the subject for transformation with the expression vector, for example, the E.coli BL21 strains such as E.coli BL21(DE3).

Expression vector

The selection method of the invention comprises transforming triclosan-sensitive bacteria with an expression vector comprising a functional FabV gene as a selection marker. Any expression vector commonly used in the production of therapeutic products can be used, where the functional FabV gene is inserted into the vector using methods generally known in the art. Suitable vectors include episomal vectors, such as plasmids, for example, expression vectors derived from the pET24 expression vector. The pET24 derived vector of figure la is 6908bp in length, but the expression vector can be of any size. The pFabV expression vector of figure lc is also suitable for use in the selection method of the invention. The vector may be a circular or linear nucleic acid molecule capable of replication in a cell, and may be episomal or integrating. Other suitable vectors include, but are not limited to, phagemids, cosmids, and bacterial artificial chromosomes (BACs).

The expression vector preferably comprises an origin of replication that is functional in the host bacterium. Any origin of replication commonly known to those skilled in the art can be used, for example Fl, ColEl or Ml 3 origins of replication, as in figure la, b and c.

The expression vector will also comprise a promoter or regulatory region for the expression of the functional FabV gene. The promoter or regulatory region can include a constitutive promoter or an inducible promoter. Many such bacterial promoters are commonly known to those skilled in the art, for example LacO. In one embodiment of the present invention, the FabV gene is under control of the weak constitutive P3 promoter from the ampC gene. A weak promoter is preferable in order to provide the appropriate quantity of the FabV protein for its metabolic role, without producing excess FabV.

The expression vector can also comprise a multiple cloning site (MCS) for insertion of the nucleic acid of interest. An MCS is a sequence having several restriction enzyme sites to facilitate insertion of a sequence of interest at this position in the vector. The multiple cloning site according to the present invention may for example be derived from the multiple cloning sites of any commercially available plasmid.

The expression vector can also comprise a gene for antibiotic resistance, such as the Kanamycin resistance gene KanR in figure la, although this is not intended for use as a selection marker in methods of the invention.

The expression vector will usually include a nucleic acid of interest. It is preferable that the nucleic acid of interest is under control of a different promoter to FabV, such that high levels of expression of the nucleic acid of interest can be achieved without impacting FabV levels.

Nucleic acids and proteins of interest

The nucleic acid of interest can be a gene of interest which encodes a protein that can be produced and isolated. As used herein, a "nucleic acid of interest" refers to a nucleic acid sequence, such as a DNA sequence, that encodes and is used to produce a product, such as a protein, of interest typically for purification from the cell in which the protein is expressed. In certain embodiments, the nucleic acid of interest encodes a polypeptide particularly a heterologous polypeptide, i.e., a protein that is not naturally expressed by the host bacterium by way of non-limiting example, bacterial proteins from other bacterial species, viral genes, eukaryotic genes, artificial polypeptide sequences, non-wild-type proteins, mutant proteins, truncated proteins and the like. In a preferred embodiment, the expression of the nucleic acid of interest is not required for the growth of the cell in which it is expressed. In certain embodiments, the nucleic acid of interest is expressed under the control of a strong promoter (e.g., a T7 promoter). In certain embodiments, the nucleic acid of interest is expressed under the control of an inducible promoter. In certain embodiments, the nucleic acid of interest is expressed under the control of a strong, inducible promoter. In certain embodiments, the nucleic acid of interest is expressed under the control of a weak, inducible promoter. The nucleic acid of interest can be for use in any bioprocess, for example, biochemical characterisation, biopharmaceutical research or protein crystallisation. The skilled person will be aware of other suitable promoters.

In a preferred embodiment, the nucleic acid of interest does not encode an active FabV enoyl- acyl carrier protein reductase, i.e. it is different to or separate from the FabV gene of the vector. In certain embodiments, the nucleic acid of interest is a nucleic acid sequence from a prokaryotic organism. In certain embodiments, the nucleic acid of interest is a nucleic acid sequence from a prokaryotic organism other than E. coli. In certain embodiments, the nucleic acid of interest is a nucleic acid sequence from a eukaryotic organism. In certain embodiments, the nucleic acid of interest is an artificial sequence. In certain embodiments, the expression vector comprises a multiple cloning site. In certain embodiments, the nucleic acid of interest is inserted into the multiple cloning site in the vector. As discussed above, in certain embodiments the vector includes a promoter sequence to control the expression of the nucleic acid of interest. In a preferred embodiment, expression of the FabV gene and the nucleic acid of interest are under the control of separate promoters. In certain embodiments, at least one of the promoter sequences controlling the expression of the FabV gene and the promoter sequence controlling the expression of the nucleic acid of interest are from different organisms.

In certain embodiments, the vector comprises a coding sequence for expression of a fusion construct with the product of expression of the nucleic acid of interest. In certain embodiments, the coding sequence for expression as a fusion construct with the nucleic acid of interest is a reporter construct. In certain embodiments, the coding sequence for expression as a fusion construct with the nucleic acid of interest is a tag to facilitate purification of the expression product of the nucleic acid of interest, for example a 'his' tag.

In certain embodiments, the nucleic acid of interest can be for use in the preparation of a pharmaceutical composition, such as a vaccine. For example, the nucleic acid of interest can be a gene which expresses an immunogen or antigen using the selection method of the invention. Thus, particularly the nucleic acid of interest is a gene which codes for a protein of interest such as an immunogen or antigen. An antigen refers to a molecule containing one or more epitopes (e.g., linear, conformational or both) capable of eliciting an immunological response, more particularly a protective immune response, when administered to a subject such as an animal. By "elicit" is meant to induce, promote, enhance or modulate an immune response or immune reaction. A particular protein of interest is CRM 197 (NCBI protein Accession number 1007216A), for example, expressed from a nucleic acid sequence of interest such as nucleic acid sequence NCBI Accession number HW071379 and described in EP2445930.

Alternatively, the nucleic acid of interest can express a reporter protein such as blue fluorescent protein (BFP) or green fluorescent protein (GFP), as described in the examples below. The nucleic acid of interest can be inserted into the expression vector at an MCS. For example, a reporter gene such as BFP or GFP can be inserted at the MCS of pFabV to provide pFabV-BFP or pFabV-GFP respectively. In certain embodiments, the reporter protein can be an enzymatic reporter construct including, but not limited to, horse radish peroxidase (HRP), luciferase, or alkaline phosphatase.

In certain embodiments, the expression vector can include a coding sequence for an affinity tag or an epitope tag adjacent to the MCS to allow for generation of a fusion protein with the expression product of the nucleic acid of interest to facilitate detection or purification of the polypeptide expressed from the nucleic acid of interest. Such tags include, but are not limited to a 6 X His tag, a GST-tag, an HA1 tag, and a myc tag.

In particular embodiments, the nucleic acid of interest can be isolated and purified, for example for use in the preparation of a pharmaceutical composition, such as a vaccine. The nucleic acid of interest can take the form of DNA or RNA, but usually it will be DNA. For instance, in one embodiment the nucleic acid of interest is an expression vector of the invention and the invention therefore provides a method of producing and purifying the expression vector which is propagated by the selection method of the invention.

Triclosan and the media

Triclosan, [5-chloro-2-(2,4,-dichlorophenoxy) phenol] also known as Irgasan DP-300 (CAS 3380-34-5) is a chemical compound that specifically inhibits fatty acid synthesis in many triclosan-sensitive bacteria including E.coli by forming a stable FabI/NAD+/triclosan ternary complex (Heath et al 1999). Triclosan is a biocide that fulfils the criteria of a non-antibiotic (Russel et al, 2003). Triclosan was approved in 1986 by the European Community Cosmetic Directive for preservative in cosmetics products at concentrations up to 0.3%. Triclosan was evaluated also by SCF and EFSA for use in food contact materials and classified with a restriction of 5 mg/kg of food (reference 10).

Example 2 shows that at 37°C in LB medium, E.coli BL21(DE3) growth is suppressed by triclosan with an MIC of 0.2 μg/mL. Similar experiments will reveal a suitable concentration for use with any triclosan-sensitive bacteria. Any growth medium to which triclosan has been added to a concentration sufficient to suppress the growth of untransformed E.coli is suitable for use in the invention as the medium for the growth and selection of transformed E.coli. Suppression of the growth of the bacteria includes killing of the bacteria. Particular complex media include, by way of non- limiting example, LB broth (G.Bertani, 1951), or Terrific broth (TB) (Tartoff and Hobbs. 1987) to which triclosan has been added to a concentration sufficient to suppress the growth of untransformed bacteria. By "complex media" is meant media wherein the exact composition and concentration of their components is not known. Complex media are typically derived from, for example, partially digested yeast, beef, soy, and/or additional proteins, and due to their rich array of nutrients are suitable for efficient growth of bacteria. Chemically defined media are also suitable for the use of the invention.

Transformation

The method for selection of transformed bacteria involves transforming triclosan-sensitive bacteria, with a vector carrying a functional FabV gene, as a selection marker. Transformation refers to the insertion of an exogenous polynucleotide into a host cell, which can be for example a competent cell, irrespective of the method used for insertion, for example, transformation by direct uptake, transfection, infection, and other methods known in the art are suitable. By way of non-limiting example, such methods for direct DNA introduction may include calcium phosphate co-precipitation, electroporation, lipofection, and microinjection. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid or an episome, or alternatively, may be integrated into the host genome. As used herein the term "transformed bacteria" denotes host bacteria that have been genetically engineered to produce one or more heterologous polypeptides under the control of one or more promoter-operators. As disclosed herein, a first heterologous polypeptide is the FabV protein (FabV enoyl-acyl carrier protein reductase) and a second heterologous polypeptide is a protein of interest. Such bacteria are referred to herein as "transformants".

The exogenous polynucleotide is preferably maintained as a non-integrated vector, for example, an episome, although in certain embodiments the vector may be integrated into the host genome.

Downstream uses of expressed proteins

The method of selection is suitable for the propagation of the nucleic acid of interest. Propagation of the nucleic acid of interest is achieved by replication of the expression vector, which results in an increase in the copy number of the nucleic acid of interest in the transformed bacterium. The method of selection is especially useful for the recombinant protein production, as demonstrated in Example 5.

The transformed bacterium can be used for production of the nucleic acid of interest, or for production of a protein of interest expressed by the nucleic acid of interest. The protein or nucleic acid can then be isolated. The invention also provides a method for preparing a pharmaceutical composition, comprising isolating the nucleic acid of interest or protein expressed by the nucleic acid of interest, and preparing a pharmaceutical composition comprising the isolated nucleic acid or protein. The term "pharmaceutical composition" according to this invention refers to protein isolated and purified according to this invention and suitable for in vivo administration, for example for use as a vaccine in immunising subjects against various diseases. These compositions will typically include the protein expressed from the nucleic acid of interest and any number of pharmaceutically acceptable excipients.

General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y. The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do no materially alter the basic and novel characteristics of the claimed composition, method or structure. The term "consisting of is generally taken to mean that the invention as claimed is limited to those elements specifically recited in the claim (and may include their equivalents, insofar as the doctrine of equivalents is applicable). The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. For example, "substantially free" from Y can be understood as a composition containing not more than 5% Y, not more than 4% Y, not more than 3% Y, not more than 2% Y, not more than 1% Y, or not more than 0.1% Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

As used herein, unless otherwise clear from context the term "or" is understood to be inclusive and can be used interchangeably with the term "and/or".

A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence. A host cell can be used, for example, for expression of a nucleic acid of interest or propagation of a plasmid vector or both.

All GenBank Accession numbers provided herein are incorporated by reference in the version available on the date of filing the instant application.

MODES OF CARRYING OUT THE INVENTION

Example 1 - Triclosan selection validation

In order to validate the triclosan selection method, a pET24 derived expression vector pFabVk-

GFP, was constructed, as shown in figure lb. The vector expresses the P. aeruginosa PAOl FabV gene under the control of a weak constitutive promoter (P3 promoter from ampC gene) and a reporter gene (Green fluorescent protein, GFP).

In order to confirm the complementation system, the pFabVk-GFP vector was transformed into E.coli of the BL21 (DE3) strain.

Figure 2a shows that E.coli transformed with the pFabVk-GFP vector were able to grow in the LB broth + triclosan (0.5 μg/mL) medium, whereas E.coli transformed with the pET24-GFP vector, which doesn't contain the FabV gene, were not able to grow in this medium.

Figure 2b shows a positive control in which the E.coli transformed with either the pFabVk-GFP or the pET24-GFP vector were both capable of growth in LB broth + kanamycin (5(^g/mL) medium, as both vectors contain a gene for kanamycin resistance.

Example 2 - Triclosan resistance optimal triclosan concentration

In order to establish the minimum inhibitory concentration, at which triclosan inhibits the growth of E.coli, the growth of E.coli transformed with either the pFabVk-GFP vector or the pET24- GFP vector was monitored at ODgoo over 8 hours. The results are shown in figure 3, and show that at low triclosan concentrations, some growth of E.coli transformed with the pET24-GFP vector was observed, but at high triclosan concentrations (>0^g/mL) growth of E.coli transformed with the pET24-GFP vector was not observed, whereas that of E.coli transformed with pFabVk-GFP was not affected.

Figure 4 shows the specific growth rate per hour, at different triclosan concentrations, of the E.coli that were either transformed with the pFabVk-GFP vector or the pET24-GFP vector. It shows that growth is maintained in the E.coli transformed with the pFabVk-GFP vector even at high triclosan concentrations. The growth rate of E.coli transformed with the pET24-GFP vector however decreases sharply as triclosan concentration increases to 0.2 ug/mL, then it levels off at a very low rate of growth as the triclosan concentration increases further. Figure 4 therefore indicates that a triclosan concentration of O^g/mL represents a minimum inhibitory concentration for the inhibition of E.coli growth.

Cultivation of the E.coli in this example was carried out at 37°C in 96 Deep Well plate in 1 mL of LB medium supplemented with different concentrations of triclosan or kanamycin at 50 mg/L.

Example 3 - Triclosan selection: Expression Results

Protein production rate during cultivation in standard conditions between E.coli transformed with either the pET24-GFP vector or with the pFabVk-GFP vector was compared. As shown in Figure 5, protein production was monitored by specific Fluorescence from three samples:

(a) E.coli BL21(DE3) transformed with the pET24-GFP vector, grown in LB broth.

(b) E.coli BL21(DE3) transformed with the pET24-GFP vector, grown in LB broth + kanamycin (50mg/L). (c) E.coli BL21(DE3) transformed with the pFabVk-GFP vector, grown in LB broth + triclosan 0.2mg/L.

Figure 5 demonstrates that 20 hours after the induction of the GFP gene, fluorescence in the sample where selection had occurred based on the FabV selection method (c) was significantly higher than in the sample where no selection of transformed E.coli had occurred (a).

Cultivation of the three samples was carried out at 37°C in 96 Deep Well plate with 1 mL LB or LB + triclosan 0.2 mg/L (LBT) or LB + kanamycin 50 mg/L (LBK). Induction of GFP was performed at OD 0.6 with IPTG ImM.

Example 4 - Expression results

Protein production rate during cultivation in high cell density conditions between E.coli BL21(DE3) transformed with either the pET24-GFP vector or with the pFabVk-GFP vector was compared. As shown in Figure 6, protein production was monitored by specific Fluorescence from three samples:

(a) E.coli BL21(DE3) transformed with the pET24-GFP vector, grown in EnBase®.

(b) E.coli BL21(DE3) transformed with the pET24-GFP vector, grown in EnBase® + kanamycin (50mg/L).

(c) E.coli BL21(DE3) transformed with the pFabVk-GFP vector, grown in EnBase® + triclosan 0.2mg/L.

Figure 6 demonstrates that 25 hours after the induction of the GFP gene, fluorescence in the sample where selection had occurred based on the FabV selection method (c) was significantly higher than in the sample where no selection of transformed E.coli had occurred (a). Protein production in the sample where selection had occurred based on the FabV selection method (c) was comparable to protein production in the antibiotic-based selection method (b).

Cultivation of the three samples was carried out at 30°C in 96 DeepWell plate with 1 mL EnBase® or EnBase® + triclosan 0,2 mg/L (LBT) or EnBase® + kanamycin 50 mg/L (LBK). Induction was performed after 16 hours of cultivation with IPTG.

Example 5: Protein production

E.coli BL21(DE3)/ pFabVk-GFP was cultivated for repeated cycles in LB + triclosan 0.5 μg/mL (LBT). Additionally BL21(DE3)/pET24-GFP was cultivated for repeated cycles in LB without antibiotic (LB). At different intervals, aliquots of the cultures were stored and re-inoculated to analyze recombinant protein expression over 15 cycles of cultivation. Each cycle of cultivation corresponds to 1 : 100 dilution of the culture in LB or LBT medium. Cultivation was carried out at 37°C in 20 mL LB medium. In the FabV-selection method, triclosan was added at final concentration of 0.2 μg/mL. At generation 0, kanamycin was added to final concentration of 50 μg/mL. NB: The pFabVk-GFP also contains the gene for kanamycin resistance. In the generation "zero" both clones (i.e. BL21(DE3) transformed with pET24-GFP or pFabVk-GFP) were grown in the presence of 5(^g/ml kanamycin to ensure the same starting point for later, following cycles of growth which were performed in the absence of antibiotic selection.

Figure 7 shows protein production from BL21(DE3)/ pFabVk-GFP in LBT and BL21(DE3)/pET24-GFP in LB. Protein production is maintained at high levels in E.coli transformed with the FabV gene and grown in LBT over 15 cultivation cycles. However in E.coli transformed with pET24-GFP and grown in LB, and therefore not subject to selection, protein production decreased to negligible levels after 15 cultivation cycles.

Example 6: Plasmid maintenance

The plasmid copy number (PCN) was evaluated in the triclosan selection method. The comparative CT method (AACT), which assesses relative target quantity in relation to a calibrator sample with known a known PCN (Skulj et al, 2008), was used to assess PCN in the samples. Table 1 shows three test conditions:

(a) LBK: BL21(DE3)/pFabVk-GFP cultivated in LB + kanamycin. (LBK has not undergone any cycles of dilution, and therefore represents the generation zero positive control.) LBT 20G: BL21(DE3)/pFabVk-GFP after 20 cycles of dilution in LB+Triclosan.

(b) LB 5G: BL21 (DE3)/pFabVk-GFP after 5 cycles of dilution in LB without selection.

In all three samples, cultivation was carried out at 37°C in 20 mL LB medium. Induction was performed at OD600nm = 0.6 with IPTG lmM.

In each condition, samples were either withdrawn before induction (NI), 4 hours after induction (4h) or 18 hours after induction (o/n).

Table 1 shows that a high plasmid copy number is maintained with triclosan selection during recombinant protein production (LBT20G). The PCN for the FabV selection sample after 20 cycles of cultivation and 4 hours after induction was 182 compared to 19 in the sample without selection (LB5G). Plasmid copy number was also higher in the LBT20G sample than that exhibited by the antibiotic-based selection sample (LBK).

Table 1 - Plasmid copy number (PCN)

It will be understood that all features referred to in one embodiment or aspect described above can be applied mutatis mutandis to other embodiments and aspects.

SUMMARY OF SEQUENCE IDENTIFIERS

SEQ ID NO: l - Pseudomonas aeruginosa PAOl PA2950 trans-2-enoyl-CoA reductase (DNA);

SEQ ID NO:2 - Pseudomonas aeruginosa PAOl trans-2-enoyl-CoA reductase (Amino acid);

SEQ ID NO: 3 - pFabV (empty vector with fabV gene and multiple cloning site);

SEQ ID NO:4 - pFabVkan (empty vector with fabV gene, multiple cloning site and kanamycin resistance gene);

SEQ ID NO:5 - Green Fluorescent Protein (GFP);

SEQ ID NO: 6 - pFabV-gfp (pFabV containing gfp gene and kanamycin resistance gene);

SEQ ID NO: 7 - Plasmid pET24b+;

SEQ ID NO: 8 - pET24-gfp (pET24 vector sequence with GFP gene sequence);

References

[I] Levy, S.B., J.antimicrob. Chemother. 49 (2002) 25-32

[2] Heath et al.,J Biol Chem 1999, 274: 11110-4.

[3] Zhu et al., Antimicrobial agents and chemotherapy, 2010, 689-698

[4] Russell Lancet Infect Dis 2003, 3:794-803.

[5] G.Bertani, J. Bacteriol. 1951, 62(3):293.

[6] Tartoff and Hobbs. 1987. Bethesda Research Laboratories Focus 9: 12.

[7] Skulj et al, Microbial Cell Factories 2008, 7-6

[8] Datsenko and Wanner, 2000, PNAS 97(12): 6640-5

[9] Hirsbeck et al, 2012, Structure. 20(1): 89-100.

[10] www.epa.gov/oppsrrdl/REDs/factsheets/triclosan_fs.htm

[I I] Massengo-Tiasse et al, 2008. J. Biol. Chem. 283: 1308-1316.

[12] Goh, S. & Good, L., 2008, BMC biotechnology 8:61.

[13] Beuth; 1998. Empfindlichkeitsprufung von mikrobiellen Krankheitserregern gegen Chemotherapeutika Teil 8: Mikrodilution; Allgemeine methodenspezifische Anforderungen (DIN 58940-8)

All references, publications, and patents cited in the present application are herein incorporated by reference in their entireties.

Claims

1. A method for selection of transformed bacteria comprising a nucleic vector which comprises a nucleic acid sequence of interest, which method comprises the steps of;

i) transforming triclosan-sensitive bacteria with a vector comprising (a) as a selection marker a nucleic acid sequence encoding a functional FabV protein; and (b) the nucleic acid sequence of interest, which does not encode a functional FabV protein, to give transformed bacteria, and;

ii) growing the transformed bacteria in a growth medium containing triclosan at a concentration sufficient to suppress the growth of triclosan-sensitive bacteria; and iii) selecting one or more bacteria that are able to grow in the growth medium.

2. The method of claim 1, wherein the growth medium is a complex medium comprising triclosan at a concentration sufficient to suppress the growth of triclosan-sensitive bacteria.

3. The method of claim 1 or claim 2, wherein the growth medium contains triclosan in a concentration equal to or greater than 0.2 μg/mL.

4. The method of any preceding claim, wherein the triclosan-sensitive bacteria is an E.coli strain.

5. An expression vector for complementing triclosan-sensitive bacteria, comprising (a) as a selectable marker a nucleic acid sequence encoding a functional FabV protein; and (b) a nucleic acid sequence of interest which does not encode a functional FabV protein.

6. The expression vector of any of claim 5 wherein the FabV encoding sequence is operably linked to the P3 promoter from the ampC gene.

7. The expression vector of any claim 5 or claim 6 wherein the vector comprises an Fl, ColEl or Ml 3 origin of replication.

8. The expression vector of any one of claims 5 to 7 wherein the nucleic acid of interest encodes a polypeptide of interest.

9. Use of the vector of any one of claims 5 to 8 in a method of selection of transformed bacteria.

10. A bacterium containing the expression vector of any one of claims 5 to 8, wherein the FabV encoding sequence of the vector confers triclosan resistance to the bacterium, which would otherwise be sensitive to triclosan.

11. A method of producing a protein of interest, comprising a step of growing the transformed bacterium of claim 10 under conditions such that (a) the growth of triclosan- sensitive bacteria is suppressed, and (b) the protein of interest is produced.

12. A method of producing a nucleic acid of interest, comprising a step of growing the transformed bacterium of claim 10 under conditions such that (a) the growth of triclosan- sensitive bacteria is suppressed, and (b) the nucleic acid of interest is propagated.

13. A method for preparing a pharmaceutical composition, comprising purifying a protein produced by the method of claim 11, and preparing a pharmaceutical composition comprising the purified protein.

14. A method for preparing a pharmaceutical composition, comprising purifying the nucleic acid of interest produced in the method of claim 12, and preparing a pharmaceutical composition comprising the purified nucleic acid of interest.