Biosynthetic methods

Abstract

The present invention generally relates to methods for producing products using cells, and more particularly to the field of industrial biotechnology or industrial microbiology in which living cells are used to produce useful chemical substances and products, such as biological products such as proteins. The present invention relates to a method for producing products using cells, comprising the steps of: excising one or more nucleic acid sequences including a target gene and an origin of replication from a chromosome within a cell; forming an extrachromosomal DNA molecule including the excised target gene and the excised origin of replication; and inducing the expression of the target gene from the extrachromosomal DNA molecule. The present invention also relates to cells, polynucleotide vectors, and their use for producing products.

Description

[Technical Field]

[0001] The present invention generally relates to methods for producing products using cells, and more particularly to the field of industrial biotechnology or industrial microbiology in which living cells are used to produce useful chemical substances and products, such as biological products such as proteins. Recombinant proteins are an important group of products used in many applications, including food processing, therapeutics, diagnostics, and chemical synthesis. For practical reasons, recombinant proteins need to be synthesized by living cells. Depending on the properties of the product, both prokaryotic and eukaryotic cells are used for this purpose. Escherichia coli (E. coli), a Gram-negative bacterium, is one of the most widely used protein-producing hosts, and many commercially available recombinant proteins are produced in E. coli.

[0002] A typical attempt at protein production in E. coli begins with cloning the target gene (GOI (gene-of-interest)) into a plasmid. Plasmids are autonomously replicating DNA molecules that can exist in multiple copies in bacteria (and some eukaryotes). This is beneficial in protein production. The presence of multiple copies of a GOI within a cell inherently increases the expression level of the target protein (POI (protein-of-interest)). Therefore, plasmid-based protein expression systems have a clear advantage over methods in which the GOI exists as a single copy within the genome.

[0003] To prevent plasmid loss during the production process, it is necessary to selectively select for plasmids. The most common method for this is the use of antibiotics. In this setup, the plasmids possess antibiotic resistance genes, and antibiotics are added to the growth medium. As a result, only cells containing the plasmids are able to grow.

[0004] The use of antibiotics in protein production presents several challenges. Some antibiotics are degraded or inactivated, requiring replenishment during the culture period to maintain efficient selection. In some cases, antibiotics must be removed during the protein purification process, adding further purification steps that can increase costs and reduce protein yield. Antibiotic removal is crucial when the protein is used to prepare human food (food enzymes) or as a pharmaceutical (therapeutic proteins). Humans can be allergic to certain antibiotics, and unintentional use of these antibiotics can damage the normal microbiome. Furthermore, the final product must not contain antibiotic resistance genes that could spread through horizontal gene transfer. These requirements have led to the establishment of strict quality control mechanisms by regulatory authorities and encouragement to discontinue the use of antibiotics in protein production. Many protein production companies wish to completely eliminate the use of antibiotics in large-scale production.

[0005] Several alternative selection mechanisms have been developed. One group of methods utilizes episomal complementation of trophoblastic strains to stabilize plasmids. In this case, genes encoding essential metabolic functions are inactivated within the genome and reintroduced onto the plasmid. This makes the presence of the plasmid essential for growth if metabolites cannot be obtained by other means, and the GOI is maintained on the same plasmid. Patent EP0284126B1 refers to genes such as leu, his, and trp as trophoblastic selection markers. Patent US8465946B2 discloses a selection system using the glycerol-3-phosphate dehydrogenase gene as a trophoblastic marker, and patent EP0185512B1 discloses the dal gene as a trophoblastic marker.

[0006] However, the use of such nutritional requirement markers in industrial fermentation is difficult because industrial fermentation media contain almost all the necessary substances (such as amino acids). Industrial media contain undefined complex mixtures such as corn steep liquor and yeast extract, and cells can compensate for their inability to synthesize certain metabolites by absorbing those metabolites from the culture medium. The use of chemically defined media consisting only of pure components is, in most cases, very expensive.

[0007] Another known method involves using a toxin-antitoxin pair to select plasmids without the use of antibiotics. The toxin gene is typically integrated into the host's chromosomal DNA, while the antitoxin gene is carried on a plasmid. When both the toxin and antitoxin are expressed within a cell, the binding of the antitoxin neutralizes the toxin, allowing the cell to proliferate. When the plasmid is lost from the cell, the antitoxin level decreases, the toxin escapes neutralization, and cell proliferation is inhibited. Such systems are described, for example, in US patents 8470580B2 and 10718001B2, both of which utilize a CcdA / CcdB toxin-antitoxin system. A drawback of such systems is their cumbersome handling. This is because cells expressing the toxin must always retain the plasmid expressing the antitoxin; therefore, when introducing a new plasmid with a GOI, the existing plasmid must be removed. Furthermore, the strength of selection can vary depending on the expression levels of the toxin and antitoxin.

[0008] Another selectable system is an RNA-selectable marker, in which RNA generated from a plasmid suppresses the expression of a toxic gene encoded in the genome. An example of such a system is described in US Patent No. 10144935B2.

[0009] As an alternative to plasmid-based expression, GOIs can be incorporated into the genome. In this case, the entire setup is very stable and does not require selection. The disadvantage of this method is that, because only a single copy of the GOI exists, the expression level is lower compared to multicopy plasmids. It is possible to amplify the genes incorporated into the chromosome [1][2], resulting in the formation of a tandem array of GOIs. However, such setups are unstable and often revert to a single-copy state [3].

[0010] Patent application EP0284126B1 discloses a method for multicopy insertion in which an essential gene is located between two copies of a GOI. Patent application WO1996023073A1 describes a method for randomly inserting a GOI into a bacterial genome along with a removable marker, and then removing the marker gene (antibiotic resistance gene). By repeating this procedure, it is possible to insert multiple copies of a GOI. However, if the goal is to achieve a copy number similar to that of a plasmid (>20), this one-to-one insertion method is extremely time-consuming.

[0011] Several variations exist of the above method, many of which are reviewed in [4] and [5].

[0012] Modification of GOI copy number in living cells has been described using a plasmid with two origins of replication [6]. During plasmid maintenance, only the low-copy-number origin of replication (oriS) is active, and the plasmid is present in small numbers per cell. Activation of the high-copy-number oriV increases the plasmid copy number. This has been used to increase the yield of the plasmid [6] or the protein encoded by the plasmid [7]. However, GOI is always present on the plasmid, and selection for plasmid maintenance requires the contingent presence of antibiotics. Other systems requiring the contingent presence of antibiotics have also been described in the art [8][9].

[0013] Therefore, there is a need for methods to express target genes at high levels in host cells without using selection markers such as antibiotic resistance.

[0014] This specification describes a novel expression system that requires only a single insertion of a GOI at a predetermined location on the genome, requires no selection (antibiotics or anything else) during the propagation and production process, and achieves high levels of protein expression comparable to, or even exceeding, those of multicopy plasmids. The solution provided herein is best suited for use in a two-stage bioproduction process. In a two-stage bioprocess, the process is divided into two stages. First, cells proliferate at maximum rate without (significant) production, and once sufficient biomass has accumulated, product production is induced. During the proliferation stage, the GOI is incorporated (i.e., present) in the genome as a single copy. Such a configuration is highly stable and requires no selection whatsoever. Once the desired level of biomass has accumulated, site-directed recombinase is induced within the cell. The GOI, along with its surrounding sequence, is excised from the genome, circularized, and forms a fully functional plasmid. This plasmid begins to replicate, and its copy number increases. Importantly, no selection or antibiotic resistance genes are required. This is because plasmid formation occurs late in the proliferation stage, and there is not enough cell division remaining for the plasmid to be lost from the cell. Once induced, the product, i.e., the target protein (POI), is expressed at high levels, comparable to or exceeding expression by conventional expression plasmids.

[0015] Therefore, in one aspect, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. The present invention provides a method comprising the step of forming an extrachromosomal DNA molecule containing the excised target gene and an excised origin of replication (or the excised origin of replication).

[0016] In an embodiment, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication (or the excised origin of replication), The present invention provides a method comprising the step of inducing the expression of the target gene from the extrachromosomal DNA molecule described above.

[0017] In one embodiment, the expression of the target gene from an extrachromosomal DNA molecule is induced by the formation of the extrachromosomal DNA molecule.

[0018] In one embodiment, the expression of the target gene from an extrachromosomal DNA molecule is induced simultaneously with or after the formation of the extrachromosomal DNA molecule.

[0019] The process of inducing the expression of a target gene from an extrachromosomal DNA molecule typically refers to an event or action that induces the expression (or increased expression) of the target gene when the target gene is part of an extrachromosomal DNA molecule. Therefore, the process includes the setup of administering or introducing an active substance (e.g., a chemical inducer) or condition (e.g., a temperature change) that leads to the induction of the target gene's expression, which is performed before, simultaneously with, or after the excision and formation processes. Thus, actions that lead to the induction of the target gene's expression (e.g., adding a chemical inducer to, for example, cell culture medium, or changing the temperature of the cell culture medium) may be performed even before the excision and formation processes.

[0020] This can be done, for example, in a setup where the expression of the target gene is impossible when the target gene is part of a chromosome (e.g., because the target gene is not operably linked to a promoter in its chromosomal arrangement), but possible when the target gene is part of an extrachromosomal molecule (e.g., because the target gene is operably linked to a promoter by the formation of an extrachromosomal DNA molecule). In such a setup, an action leading to the induction of the expression of the target gene may be performed before the excision step and the formation step are carried out. In that case, the induction of the expression of the target gene from the extrachromosomal DNA molecule may occur simultaneously with, or after (e.g., a little later or immediately after) the formation of the extrachromosomal DNA molecule. Also, simultaneously with or after the formation of the extrachromosomal DNA molecule (e.g., immediately after the extrachromosomal DNA molecule is formed, a little later or even later after the extrachromosomal DNA molecule is formed, such as after the extrachromosomal DNA molecule is replicated), it is also possible to induce the expression of the target gene from the extrachromosomal DNA molecule by administering or introducing an agent (e.g., a chemical inducer) or a condition (e.g., a temperature change). In such a case, the induction of the expression of the target gene from the extrachromosomal DNA molecule may occur upon the administration or introduction of the agent or condition.

[0021] The principle described in relation to the step of "inducing the expression of the target gene from the extrachromosomal DNA molecule" above can be applied, with the necessary modifications, to the step of inducing the replication of the extrachromosomal DNA molecule or any other induction step in the methods described in this specification.

[0022] Also, in other embodiments described in more detail elsewhere in this specification, for example, by causing an operative linkage to occur between an appropriate promoter and the target gene during the formation of the extrachromosomal DNA molecule, it is also possible to induce the expression from the extrachromosomal DNA molecule by the formation of the extrachromosomal DNA molecule.

[0023] Each step of the method of the present invention can be carried out in any suitable order. Preferably, each step of the method of the present invention is carried out in the order listed.

[0024] All of the excision, formation, replication, and expression steps described herein can be performed intracellularly within the same cell.

[0025] The term "origin of replication" is a technical term understood by those skilled in the art. Generally, it refers to any sequence, such as a nucleotide sequence, to which DNA replication can be initiated by the binding of appropriate proteins and enzymes that are part of the replication mechanism or required for the replication process. Origins of replication in a particular cell or cell line (e.g., bacterial strain) may be referred to by specific names in the art. For example, the origin of replication present on the chromosome of *E. coli* is called oriC.

[0026] The extrachromosomal DNA molecules described herein are typically double-stranded DNA molecules. Extrachromosomal DNA molecules are formed from nucleic acid sequences excised from chromosomes. An extrachromosomal DNA molecule contains a target gene and an origin of replication. The origin of replication, of course, has the function of enabling the replication of the extrachromosomal DNA molecule within the cell. The replication mechanism, which works in cooperation with the origin of replication to cause the replication of the extrachromosomal DNA molecule, may be located at any suitable location within the cell. For example, it may be located on the excised extrachromosomal DNA molecule (or on another extrachromosomal DNA molecule), on the chromosome, or a combination of these (e.g., both on the excised extrachromosomal DNA molecule and on the chromosome). The target gene is positioned on the extrachromosomal DNA molecule to promote or enable its expression. Therefore, the function of the extrachromosomal DNA molecule is to have the ability to replicate (the ability to increase the copy number) and the ability to express the target gene, thereby enabling high expression of the target gene. Extrachromosomal DNA molecules may be linear or circular (or circularized). The extrachromosomal DNA molecule may be a plasmid, for example, a circular or linear plasmid, but more preferably a circular plasmid.

[0027] The extrachromosomal DNA molecule is formed from one or more nucleic acid sequences that are excised from the chromosome in the method of the present invention.

[0028] The excision step may include excising multiple nucleic acid sequences from a chromosome, which are then joined together (e.g., by ligation using DNA ligase) to form an extrachromosomal DNA molecule. These multiple nucleic acid sequences together constitute the target gene and the origin of replication. Therefore, the target gene and the origin of replication may be located on (or within) different such nucleic acid sequences.

[0029] Therefore, in scenarios where the excision process involves the excision of multiple nucleic acid sequences from a chromosome, the excision of an origin of replication from the chromosome (or excision event, excision reaction, excision process) may differ from the excision of the target gene from the chromosome (or excision event, excision reaction, excision process). In this scenario, the excised nucleic acid sequence containing the target gene can then be joined (e.g., by the action of a DNA ligase) to an origin of replication (or an excised nucleic acid sequence containing that origin of replication) (and, if necessary, other excised nucleic acid sequences (if three or more nucleic acid sequences are excised)) to form an extrachromosomal DNA molecule.

[0030] Alternatively, more preferably, the one or more nucleic acid sequences are a single nucleic acid sequence. Here, the excision step includes excising a single nucleic acid sequence. For example, the excision step may consist of excising a single nucleic acid sequence from a chromosome, and the extrachromosomal DNA molecule may consist of the single nucleic acid sequence, i.e., the nucleic acid sequence excised alone. Thus, in such embodiments, the target gene is excised as part of the same DNA molecule as a certain origin of replication (or said origin of replication). Another way of looking at it is that the target gene is excised from a chromosome by the same excision (or excision event, excision reaction, excision process) as the excision of a certain origin of replication (or said origin of replication). Yet another way of looking at it is that in the excision step, the target gene and the origin of replication are excised as a single (or one, i.e., only one) DNA molecule. Thus, as a result of the excision step, the target gene and the origin of replication exist outside the chromosome (e.g., form an extrachromosomal DNA molecule) as part of the same (or single, or one, i.e., only one) DNA molecule. In such embodiments where the target gene and the origin of replication are excised as a single DNA molecule (or a single nucleic acid sequence), the target gene and the origin of replication may be located in close proximity to each other on the chromosome, for example, so that these components can be easily excised together (e.g., in a single excision event (such as a single recombination event)).

[0031] The excision and formation steps of the method of the present invention may be performed sequentially. For example, after the excision step, before the formation of an extrachromosomal DNA molecule, further modification of the excised DNA molecule may be desired or required. For example, one or more nucleic acid sequences excised in the excision step may be excised as one or more linear DNA molecules, in which case the method (e.g., the formation step of the method) may include ligating one or more ends of the excised DNA molecules, for example (in embodiments in which multiple DNA sequences are excised from a chromosome) to join (i.e., ligate) multiple excised DNA molecules and / or to circularize the excised DNA sequences. Also, for example, if "one or more nucleic acid sequences" as referred herein consists of a single (or one, i.e., only one) DNA sequence, the method (or the formation step of the method) may include ligating both ends of the excised DNA sequence, for example to circularize the excised DNA sequence. In such examples, the above-mentioned circularization (or circularization event, circularization reaction, circularization process) can be considered to form an "extrachromosomal DNA molecule" (e.g., a single extrachromosomal DNA molecule) as referred herein, and therefore the excision and formation steps can be considered to occur sequentially. Circularization of nucleic acid sequences can be achieved by standard methods known in the art, such as using DNA ligase.

[0032] Naturally, in embodiments where the excision and formation processes are expected to be performed sequentially, the excision process is intended to be performed before the formation process. Alternatively, more preferably, the excision step and the formation step of the method of the present invention are performed simultaneously.

[0033] For example, this could apply if the excision process involves excising a target gene along with a replication origin (or as part of the same molecule), and no further modifications are made to the excised DNA molecule thereafter. In such cases, the excised DNA molecule can be considered an extrachromosomal DNA molecule, and therefore, the excision and formation processes can be considered to occur simultaneously.

[0034] In a more preferred embodiment, the excision and formation steps may be performed together. For example, there may be a single step, event, or reaction, for example, a single recombination step (or a single recombination event or reaction). Thus, in another embodiment, the present invention provides a method for producing a product by cells, comprising the step of recombining a nucleic acid sequence derived from a chromosome within the cell to form an extrachromosomal DNA molecule, wherein the nucleic acid sequence (or the extrachromosomal DNA molecule) includes a target gene and an origin of replication. Other aspects and embodiments of the present invention described herein may be applied to this embodiment of the invention as appropriate, with necessary modifications. Thus, for example, references to “excision and formation” are expressed as “recombination” in this context, and references to “excised” nucleic acid sequences are expressed as “recombined” nucleic acid sequences in this context.

[0035] In an embodiment, the present invention is a method for producing a product using cells, A step of forming an extrachromosomal DNA molecule by rearranging nucleic acid sequences derived from chromosomes within the above-mentioned cell, wherein the nucleic acid sequence (or the extrachromosomal DNA molecule) includes a target gene and an origin of replication, The present invention provides a method comprising the step of inducing the expression of the target gene from the extrachromosomal DNA molecule described above.

[0036] In this specification, the process of excising nucleic acid sequences from chromosomes to form extrachromosomal DNA molecules (in some preferred embodiments, a recombination step or event) may also be referred to as "popping" or "popping out."

[0037] The excision and reshaping steps can be carried out using standard genome editing technologies in the art. For example, a CRISPR / Cas system or recombinase can be used, for example, site-directed recombination can be used, for example, a serine recombinase or tyrosine recombinase system can be used.

[0038] In another embodiment, the present invention relates to a method for producing a product using a cell comprising a chromosome containing a target gene and an origin of replication, The present invention provides a method comprising the step of excising one or more nucleic acid sequences containing a target gene and an origin of replication from the above-mentioned chromosome to form an extrachromosomal DNA molecule (or, in order to form an extrachromosomal DNA molecule, excising one or more nucleic acid sequences containing a target gene and an origin of replication from the above-mentioned chromosome), wherein the extrachromosomal DNA molecule contains (or consists of) the excised target gene and the excised origin of replication (or the excised origin of replication).

[0039] In embodiments, the present invention relates to a method for producing a product using a cell comprising a chromosome containing a target gene and a replication origin, A step of excising one or more nucleic acid sequences containing a target gene and an origin of replication from the above chromosome to form an extrachromosomal DNA molecule (or, in order to form an extrachromosomal DNA molecule, excising one or more nucleic acid sequences containing a target gene and an origin of replication from the above chromosome), wherein the extrachromosomal DNA molecule contains (or consists of) the excised target gene and the excised origin of replication (or the excised origin of replication), The present invention provides a method comprising the step of inducing the expression of the target gene from the extrachromosomal DNA molecule described above.

[0040] In another aspect, the present invention relates to a method for producing a product using cells, The process involves excising the target gene and the origin of replication from the chromosome within the cell, The present invention provides a method comprising the step of forming an extrachromosomal DNA molecule containing the excised target gene (or the excised origin of replication) within the cell.

[0041] In an embodiment, the present invention is a method for producing a product using cells, The process involves excising the target gene and the origin of replication from the chromosome within the cell, A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication (or the excised origin of replication) within the cell, The present invention provides a method comprising the step of inducing the expression of the target gene from the extrachromosomal DNA molecule described above.

[0042] Other aspects and embodiments of the present invention described herein may be applied to these aspects of the invention as appropriate, with necessary modifications.

[0043] The type of origin of replication used for extrachromosomal DNA molecules is not particularly limited. For example, the origin of replication may be an inducible origin of replication, a non-inducible (or active, constitutive, or constitutive) origin of replication, or an origin of replication that cannot be induced artificially or by artificial means.

[0044] In embodiments, the origin of replication (and / or the second origin of replication in related embodiments as defined herein) is non-inducible and preferably an origin of replication on a chromosome, i.e., oriC, and more preferably an oriC of Escherichia coli.

[0045] In embodiments, the origin of replication (and / or the second origin of replication in the relevant embodiments defined herein) is inducible and preferably an origin of replication or oriV on a plasmid, more preferably an origin of replication or oriV on plasmids RK2, pBR322, pMB1, ColE1, R6K, p15A, pSC101, or pUC.

[0046] In this art, in many plasmids, the oriV (i.e., "origin of vegetative replication") often contains or consists of direct repeats or iteron DNA sequences, which interact with the Rep protein during replication initiation to form an initial complex.

[0047] In some embodiments, the replication origin may be stringent or relaxed.

[0048] In embodiments, the origin of replication (or the activity of the origin of replication) is inducible, activatable, controllable, or under the control of the activator. It is advantageous to use an inducible (or activatable, controllable, etc.) origin of replication because it allows control over the timing and level (or copy number) of replication of the extrachromosomal DNA molecule (i.e., the extrachromosomal DNA containing the target gene and the origin of replication).

[0049] In embodiments, the origin of replication is an origin that is induced or activated (or induced only or activated only) during the formation of an extrachromosomal DNA molecule. Alternatively, the origin of replication is inactive when it is in its initial location (e.g., on a chromosome) (or before the formation of an extrachromosomal DNA molecule), but becomes active when it becomes part of an extrachromosomal DNA molecule.

[0050] The origin of replication may be under the control of (or depend on the presence or level of) an exogenous or heterologous active agent (or inducer). The origin of replication may be induced by the administration or inoculation of an active agent (or inducer). For example, it may be induced by, for example, a human operator manually administering or inoculating cells, cell populations, or cell cultures with an active agent (or inducer). Alternatively, the origin of replication may be induced by the formation of extrachromosomal DNA molecules.

[0051] The biochemical mechanisms that make origins of replication inducible (or repressible or controllable) are not particularly limited. For example, the activator (or inducer) that induces the activation of an origin of replication may be constitutively expressed or inductively expressed. This can be achieved, for example, by operably linking the gene encoding the activator to a promoter. This promoter may be an inducible promoter or a constitutive promoter. The promoter may be operably linked to the activator by the formation of an extrachromosomal DNA molecule (or through its formation, via its formation, at the time of formation, or as a result of its formation).

[0052] For example, an inducible origin of replication may be an oriV (originally derived from the RK2 plasmid) under the control of the protein TrfA. The oriV requires TrfA for activation (i.e., initiation of replication), and if TrfA is absent (or not expressed), the oriV is inactive (i.e., does not initiate replication). In embodiments, the origin of replication is an endogenous or homologous origin of replication, or an origin of replication on a chromosome, or an origin of replication on a native or wild-type chromosome, or an origin of replication on an endogenous or homologous chromosome.

[0053] In embodiments, the origin of replication is a chromosome-based (or naturally chromosome-based, or native chromosome-based) origin of replication (or chromosome-derived, chromosome-origin, or naturally or native chromosome-based) origin of replication. This means that the origin of replication is naturally (or originally, endogenously, or normally) located on a chromosome (regardless of its intracellular location before, during, or after the implementation of the method of the present invention). For example, the origin of replication may be oriC, for example, oriC derived from E. coli. Thus, such an origin of replication is typically native or endogenous to the cell type used to produce the product in the method of the present invention (or is the wild-type origin of replication for that cell type).

[0054] In embodiments, the origin of replication is an extrachromosomal (or non-chromosomal) (or naturally extrachromosomal (or non-chromosomal)) origin of replication (or an extrachromosomal origin, of extrachromosomal origin, or naturally extrachromosomal origin of replication), preferably a plasmid-derived or vector-derived (or a plasmid-derived, vector-derived, or naturally plasmid- or vector-derived) origin of replication (regardless of its intracellular location before, during, or after the method of the present invention). For example, the origin of replication may be an oriV, for example, an oriV derived from the RK2 plasmid.

[0055] In embodiments, the origin of replication is an origin of replication whose normal or natural location is in a cell different from the cell used in the method. For example, the origin of replication may be exogenous, heterologous, or nonnative, or it may be an extrachromosomal (or extrachromosomally derived) origin of replication (e.g., a non-chromosomal origin of replication or an origin of replication derived from a plasmid or vector), or it may be an exogenous or heterologous extrachromosomal (e.g., on a plasmid or vector) origin of replication. For example, (as already mentioned above,) the origin of replication may be an oriV, for example, an oriV derived from the RK2 plasmid. Thus, a heterologous origin of replication is incorporated into the chromosome.

[0056] In embodiments, the extrachromosomal DNA molecule (i.e., the extrachromosomal DNA molecule formed in the formation step of the method of the present invention) includes two or more origins of replication. For example, it may include two, three, four, or five origins of replication, preferably two. For example, the extrachromosomal DNA molecule may include an origin of replication on a chromosome (or naturally on a chromosome) and an origin of replication outside of a chromosome (or naturally outside of a chromosome, or naturally non-chromosomal).

[0057] One example of a POP system designed by the present inventors is POP2 (an example is shown in Figure 1). In the case of POP2, the chromosome contains two origins of replication (oriV and oriC) before the excision and formation processes (before popping). Then, during the popping process, oriV is excised from the chromosome as a single unit together with the target gene, forming a plasmid.

[0058] In one embodiment, the origin of replication and the target gene, which will later become part of an extrachromosomal DNA molecule, are excised from the chromosome as part of the same nucleic acid sequence (or the same DNA molecule).

[0059] In embodiments, nucleic acid sequences (i.e., nucleic acid sequences for forming extrachromosomal DNA molecules) are excised from chromosomes by site-directed recombination. Site-directed recombination requires the activity of site-directed recombinases at the site-directed recombination sites. Therefore, to achieve excision by site-directed recombination, the target gene (and optionally, preferably, the origin of replication) may be flanked by site-directed recombination sites. Thus, cells may contain one or more site-directed or specific site-directed recombinases (or one or more genes encoding such site-directed recombinases). Therefore, in embodiments, the nucleic acid sequences are flanked by site-directed recombination sites.

[0060] In this embodiment, the excision step and the formation step are performed simultaneously.

[0061] In one embodiment, the excision step and the formation step are performed simultaneously by site-specific recombination.

[0062] In embodiments, the origin of replication for an extrachromosomal DNA molecule is an extrachromosomal (i.e., naturally extrachromosomal or non-chromosomal) origin of replication, a non-native origin of replication, or an artificially induced origin of replication, more preferably an origin of replication on a plasmid such as an oriV, and even more preferably an oriV derived from an RK2 plasmid.

[0063] If the origin of replication is inducible, the cell may contain genes (or genes encoding regulatory agents, or regulatory agents) for controlling the activity (or induction) of the origin of replication. Such genes may be located on the chromosome (or on a given chromosome) or outside the chromosome.

[0064] The genes for controlling the activity of the origin of replication may be located on the chromosome (or on the chromosome (or a certain chromosome)) or extrachromosomally (for example, after the excision or formation step, or throughout the entire method). Thus, in embodiments, the origin of replication (or the activity of the origin of replication) on an extrachromosomal DNA molecule is controlled by genes located on the chromosome (i.e., on the chromosome (or a certain chromosome)) or extrachromosomally.

[0065] The genes for controlling the activity of the origin of replication may be located on a chromosome throughout the entire method, or may remain on that chromosome (or a certain chromosome) throughout the entire method. Therefore, in embodiments, the origin of replication (or the activity of the origin of replication) is controlled by genes located on that chromosome (or a certain chromosome).

[0066] The gene for controlling the activity of the origin of replication (or a particular origin of replication) may be located outside the chromosome throughout the entire method, or may remain outside the chromosome (or outside the chromosome) throughout the entire method. Therefore, in embodiments, the origin of replication (or a particular origin of replication) (or the activity of the origin of replication (or a particular origin of replication)) is controlled by a gene located outside the chromosome (or outside the chromosome).

[0067] Genes that control the activity of origins of replication can be excised from the chromosome as part of the excision process. More preferably, such genes are excised as part of the same nucleic acid sequence as the target gene (and optionally, preferably, the origin of replication (or a certain origin of replication)).

[0068] In one embodiment, the one or more nucleic acid sequences include (or are surrounded by) site-specific recombination sites.

[0069] In the embodiment, the nucleic acid sequence includes genes for controlling the activity of the origin of replication.

[0070] The product production method of the present invention can be further improved by combining it with Switcher Technology, first disclosed in Japanese Patent Application WO2022117827A1. Specifically, this involves inactivating origins of replication located within chromosomes, which are preferably performed by excising the origins of replication from the chromosomes. In cells having only a single chromosome, inactivating origins of replication located within the chromosome is advantageous in that it stops cell division and proliferation while keeping the cell metabolically active. The use of Switcher Technology is also advantageous in the present invention in that it reduces the extent to which copies of extrachromosomal DNA molecules are lost from the cell by stopping cell division.

[0071] Accordingly, in embodiments, the method of the present invention includes inactivating origins of replication present within a chromosome. In embodiments, as a result of the method, the chromosome lacks (or has no functional origins of replication, or does not have functional origins of replication). This may be achieved by excising endogenous origins of replication on the chromosome, or by mutating endogenous origins of replication on the chromosome, for example, so that the origins of replication become non-functional. This is advantageous because it may result in increased protein production. Accordingly, in embodiments, the method includes a step of inactivating endogenous (or native) origins of replication (or multiple origins of replication) present within a chromosome, preferably, as a result of the method, the chromosome lacks functional origins of replication.

[0072] Therefore, in embodiments of the method of the present invention, as a result of the method, the chromosomes lack (or do not contain or contain) functional origins of replication.

[0073] Accordingly, in embodiments, the method of the present invention comprises excising a second origin of replication from the chromosome, and preferably the extrachromosomal DNA molecule includes the excised second origin of replication.

[0074] In the embodiment, the nucleic acid sequence (excised from the chromosome) includes the target gene and two origins of replication, and the extrachromosomal DNA molecule includes the target gene and one or both origins of replication.

[0075] In this embodiment, the nucleic acid sequence (excised from the chromosome) includes the target gene and two origins of replication, and the extrachromosomal DNA molecule includes one of the origins of replication but not the second origin.

[0076] In embodiments, endogenous (or native) chromosomal origins (or multiple origins) are excised from the chromosome as part of the excision process (e.g., as part of the same nucleic acid sequence as the target gene), and these endogenous and / or chromosomal origins form part of an extrachromosomal DNA molecule. Thus, in such embodiments, endogenous and / or chromosomal origins are transferred from the chromosome and form part of an extrachromosomal DNA molecule.

[0077] In embodiments, one or more nucleic acid sequences (i.e., nucleic acid sequences excised from a chromosome as described herein) include two origins of replication. In such embodiments, one of the origins of replication (preferably a non-native, heterologous, artificially induceable, or plasmid-based origin) may be excised to form part of an extrachromosomal DNA molecule, while the other origin (preferably a native, chromosomal, or endogenous origin) does not form part of an extrachromosomal DNA molecule. Alternatively, both origins of replication may form part of an extrachromosomal DNA molecule.

[0078] Alternatively, in embodiments, the method includes irreversibly inactivating the origins of replication (or all origins of replication) present in the chromosome (or multiple chromosomes) of the cell, or excising the origins of replication (or all origins of replication) from the chromosome (or multiple chromosomes) of the cell. For example, if the chromosome of the cell (modified for the present invention) contains one origin of replication (or two origins of replication), the method includes excising the origin of replication (or both origins of replication) from the chromosome.

[0079] By combining the two technologies, three designs were obtained: POP1, POP3 (including POP3A and POP3B), and POP4. General schematic diagrams of these designs are shown in the drawings and will be described in more detail in the examples. Thus, the POP2 system differs from POP1, POP3, and POP4 in that, while in POP1, POP3, and POP4 the origin of replication (endogenous origin of replication) (e.g., oriC) is removed from the chromosome as part of the method, this removal does not occur in the POP2 design. In other words, in the POP2 design, native, endogenous, or wild-type origins of replication on the chromosome remain on the chromosome and are not removed or irreversibly inactivated, for example.

[0080] We also designed POP5, POP6, and POP7. These are the same as POP1, but the trfA gene and RK2 oriV are replaced with pMB1 type origins. These were created to confirm that the concept of POP is not limited to plasmid types whose replication depends on the Rep protein.

[0081] We also designed POP8, a system that does not require chemical inducers. In particular, it does not require the addition of chemical inducers to induce the expression of the target gene from the plasmid, because the expression of the target gene is induced by plasmid formation.

[0082] In the case of POP2, the chromosome before popping contains two origins of replication (oriV and oriC). During the popping process, oriV is excised from the chromosome as a single unit along with the target gene, forming a plasmid. Meanwhile, the other origin of replication (oriC) remains on the chromosome without being excised.

[0083] Therefore, in the embodiment, one or more nucleic acid sequences excised from the chromosome include a target gene and two origins of replication, and the extrachromosomal DNA molecule includes the excised target gene and the excised origins of replication (or one or at least one of the excised origins of replication). More preferably, the extrachromosomal DNA molecule includes the excised target gene and one of the excised origins of replication, but does not include the other excised origin of replication. Preferably, the origin of replication consists of one non-native (or heterologous) or inducible (or artificially inducible) origin of replication (preferably an origin of replication on a plasmid, more preferably oriV, even more preferably oriV derived from an RK2 plasmid) and one native, or wild-type, or non-inducible (or not artificially inducible) origin of replication (preferably an origin of replication on a chromosome, more preferably oriC, even more preferably oriC from Escherichia coli). Preferably, the origin of replication that forms part of the extrachromosomal DNA molecule is a non-native (or heterologous, etc.) origin of replication, and / or the origin of replication that does not form part of the extrachromosomal DNA molecule is a native (or wild-type, etc.) origin of replication.

[0084] In POP1 and POP8, the chromosome contains two origins of replication (oriV and oriC) before popping. During the popping process, both origins of replication are excised from the chromosome as a single unit along with the target gene, forming a plasmid. The difference between POP1 and POP8 is that in POP1, the target gene is under the control of an inductive promoter (both before and after popping), whereas in POP8, the target gene is operablely linked to a constitutive promoter by popping (i.e., by plasmid formation).

[0085] Therefore, in the embodiment, one or more nucleic acid sequences excised from the chromosome include the target gene and two origins of replication, and the extrachromosomal DNA molecule includes the excised target gene and the two excised origins of replication. Preferably, the origins of replication consist of one non-native (or heterologous) or inducible (or artificially inducible) origin of replication (preferably an origin of replication on a plasmid, more preferably oriV, even more preferably oriV derived from an RK2 plasmid) and one native, or wild-type, or non-inducible (or not artificially inducible) origin of replication (preferably an origin of replication on a chromosome (or on a native chromosome, or on a wild-type chromosome), more preferably oriC, even more preferably oriC from Escherichia coli).

[0086] In POP5, POP6, and POP7, the chromosome before popping contains two origins of replication (oriC and pMB1 type origins). During the popping process, both origins of replication are excised from the chromosome as a single unit along with the target gene, forming a plasmid.

[0087] In the case of POP4, the chromosome before popping contains one origin of replication (oriC). During the popping process, this origin of replication, along with the target gene, is excised from the chromosome as a single unit, forming a plasmid.

[0088] Therefore, in embodiments, one or more nucleic acid sequences (excised from the chromosome) include a target gene and an origin of replication, and the extrachromosomal DNA molecule includes the excised target gene and the excised origin of replication. Preferably, the origin of replication (or the excised origin of replication) is a native or non-inducible (or not artificially inducible) origin of replication (preferably an origin of replication on a chromosome (or on a native chromosome or on a wild-type chromosome), more preferably oriC, even more preferably oriC of E. coli). Alternatively, the origin of replication is a non-native (or heterologous) or inducible (or artificially inducible) origin of replication (preferably an origin of replication on a plasmid, more preferably oriV, even more preferably oriV derived from an RK2 plasmid).

[0089] In POP3A and POP3B, the chromosome before popping contains two origins of replication (oriV and oriC). During the popping process, oriV is excised from the chromosome as a single unit along with the target gene, forming a plasmid. Furthermore, oriC is excised as a separate molecule. This molecule does not form part of the plasmid (extrachromosomal DNA molecule) containing the excised target gene. Therefore, in POP3, the excision of oriC and the formation of the POP plasmid are two separate events.

[0090] Accordingly, in embodiments of the method of the present invention, one or more nucleic acid sequences (excised from the chromosome) include a target gene and an origin of replication, and the method includes the step of irreversibly inactivating all origins of replication present in the chromosome (or all chromosomes in the cell). Preferably, the origin of replication is a native or non-inducible (or not artificially inducible) origin of replication (preferably an origin of replication on a chromosome (or on a native chromosome or on a wild-type chromosome), more preferably oriC, even more preferably oriC of E. coli). Alternatively, the origin of replication is a non-native (or heterologous) or inducible (or artificially inducible) origin of replication (preferably an origin of replication on a plasmid, more preferably oriV, even more preferably oriV derived from an RK2 plasmid).

[0091] Alternatively, in embodiments of the method of the present invention, one or more nucleic acid sequences (excised from the chromosome) include a target gene and two origins of replication, and the extrachromosomal DNA molecule includes the target gene and one of the origins of replication (or one or at least one of the two origins of replication).

[0092] Alternatively, in an embodiment of the method of the present invention, one or more nucleic acid sequences (excised from the chromosome) include a target gene and two origins of replication, and the extrachromosomal DNA molecule includes the target gene and one of the origins of replication, but does not include the other origin of replication.

[0093] One advantage of the present invention is that it enables high-copy-number expression of a target gene within a cell without the need for a selectable marker (selection marker) present within the extrachromosomal DNA molecule. While we do not wish to be constrained by theory, it is understood that a selectable gene or antibiotic resistance gene is unnecessary because the formation of the extrachromosomal DNA molecule occurs in the later stages of proliferation, and there are not enough cell divisions remaining to cause the loss of the extrachromosomal DNA molecule from the cell. The reduction of extrachromosomal DNA molecule loss from the cell can also be achieved, or further improved, when switcher technology is used, i.e., when the origin of replication on the chromosome is inactivated by this method (e.g., the excision and formation steps) so that the cell no longer has a functional origin of replication within its chromosome.

[0094] Therefore, in the preferred method of the present invention, selectable markers such as antibiotic resistance genes are unnecessary or not used.

[0095] Selection markers are typically nucleotide sequences encoding proteins that confer selective resistance to expressed cells. Selection markers are typically antibiotic resistance genes. Chloramphenicol resistance genes, ampicillin resistance genes, and kanamycin resistance genes are typical selection markers. Other selection markers include genes that confer the ability to utilize artificial nitrogen sources or genes that confer the ability to use artificial carbon sources. Therefore, in embodiments, the nucleic acid sequences and / or extrachromosomal DNA molecules excised from the chromosomes do not contain (or do not have) these components.

[0096] Therefore, in the embodiment, the one or more nucleic acid sequences do not include a selectable marker.

[0097] Therefore, in the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) a selection marker (or does not contain (or has not contain or have) a selection marker or selectable marker).

[0098] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) a selection marker, which is a nucleotide sequence encoding a protein that confers selective resistance to the expressed cell.

[0099] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) an antibiotic resistance gene.

[0100] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) a chloramphenicol resistance gene, an ampicillin resistance gene, and / or a kanamycin resistance gene.

[0101] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) a gene that confers the ability to utilize artificial nutrients.

[0102] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) genes that confer the ability to utilize an artificial nitrogen source and / or genes that confer the ability to use an artificial carbon source.

[0103] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not contain or have) the dnaA gene, or does not contain (or does not contain or have) the dnaA gene derived from Bacillus subtilis, or does not contain (or does not contain or have) the origin of replication of Bacillus subtilis.

[0104] In the embodiment, the nucleic acid sequence and / or extrachromosomal DNA molecule excised from the chromosome does not contain (or does not have) toxin markers, antitoxin markers, and / or nutritional requirement markers.

[0105] Therefore, the exemplary cell may include a chromosome containing a target gene and an origin of replication, flanked by a recombination site (e.g., a site-specific recombination site) before the excision and formation steps are performed.

[0106] In embodiments, one or more nucleic acid sequences as defined herein (i.e., one or more nucleic acid sequences excised from the chromosome) are (or each of them) flanked by site-specific recombination sites (e.g., site-specific recognition and cleavage sites) for recognition (or recognition and cleavage) by site-specific recombinase. In the case of multiple nucleic acid sequences, the pairs of site-specific recombination sites may be the same or different. In the case of multiple nucleic acid sequences, the pairs of site-specific recombination sites may be appropriately complementary so that, after excision, the multiple excised nucleic acid sequences can be joined (or ligated) to form the extrachromosomal DNA molecule described herein.

[0107] Subsequently, an appropriate site-specific recombinase can bind to and cleave the site in order to promote or enable DNA recombination.

[0108] In embodiments, one or more nucleic acid sequences (to be excised from the chromosome) are (or each of them) flanked by site-specific recombination sites, each site being recognizable by one or more site-specific recombinase enzymes. For example, if one nucleic acid sequence is excised in the excision step of the present invention, there may be two site-specific recombination sites. One may be located on the 5' side (or 5' end, or one end) of the nucleic acid sequence, and the other may be located on the 3' side (or 3' end, or the other end) of the nucleic acid sequence. If multiple nucleic acid sequences are excised in the excision step of the present invention, there may be multiple pairs of site-specific recombination sites. For example, there may be one pair for each nucleic acid sequence, and each pair may be arranged, for example, to flank the nucleic acid sequence.

[0109] In embodiments, the site-specific recombination sites (or sites included in each pair of site-specific recombination sites) have different sequences, for example, the attB site and the attP site. Alternatively, the site-specific recombination sites (or sites included in each pair of site-specific recombination sites) have the same sequence (i.e., the site-specific recombination sites are identical), for example, the loxP site. Appropriate site-specific recombination sites are selected depending on the recombinase enzyme used and can be easily derived by those skilled in the art.

[0110] As used herein, the term “recombinase” encompasses any site-specific enzyme capable of performing the excision and / or formation steps of the present invention. Such recombinases may be capable of DNA manipulation by excision, insertion, inversion, or translocation. The term “recombinase” also encompasses integrases, invertases, and resolvers, in addition to recombinases.

[0111] As used herein, the terms “site-specific recombinase” (or site-specific integrase, site-specific invertase, site-specific resolverase) mean an enzyme that catalyzes DNA exchange reactions between target site sequences specific to the recombinase. Accordingly, site-specific recombinases (and site-specific recombinase sites) as used herein can facilitate the excision and / or formation steps of the present invention.

[0112] The relative orientation of site-specific recombination sites, such as a pair of site-specific recombination sites, determines the outcome of a recombination event. The term “orientation” (or direction, orientation) of site-specific recombination sites as used herein is commonly understood in the art and refers to the direction in which site-specific recombinase sites are positioned within a polynucleotide. For example, if site-specific recombination sites on the same polynucleotide (e.g., a pair of site-specific recombination sites) have opposite orientations, inversion of the flanking sequence within the polynucleotide may occur (e.g., when using a pair of loxP sites (in the Cre-lox system) or when using a pair consisting of attP and attB sites (e.g., as seen in the serine recombinase system)). Conversely, if two site-specific recombination sites have the same orientation (or parallel orientation), excision of the flanking sequence from the polynucleotide may occur.

[0113] Therefore, in embodiments, site-specific recombination sites (preferably serine site-specific recombination sites) are positioned to flank (i.e., on both sides or at both ends) an intrachromosomal nucleic acid sequence (i.e., the nucleic acid sequence to be excised) by site-specific recombination (e.g., catalyzed by an appropriate enzyme) (and, if necessary, form or simultaneously form an extrachromosomal DNA molecule). Preferably, the site-specific recombination sites are positioned in the same orientation within the chromosome.

[0114] In embodiments, the site-specific recombination site includes a site-specific recombination site for recognition by serine recombinase. Preferably, the serine recombinase is γδ, Bxb1, φC31, or TP901. However, many different serine recombinases have been described and experimentally identified in the art, and more than 4000 new ones have been predicted by bioinformatics (Yang et al., 2014)

[12] . Any of these may be used.

[0115] In one embodiment, the site-specific recombination site includes a site-specific recombination site for recognition by serine integrase.

[0116] The mechanism of recombination by serine site-specific integrases (a subfamily of serine site-specific recombinases) is generally understood in the art. Serine site-specific integrases catalyze the recombination of attP and attB sites, generating attL and attR sites. The outcome of the recombination of attP and attB sites depends on the orientation of the site-specific recombination sites. When attP and attB sites have the same orientation and are located on the same polynucleotide, the sequence sandwiched between them is excised from the polynucleotide, leaving either an attR or attL site in the remaining polynucleotide.

[0117] In embodiments, the site-specific recombination site includes a site-specific recombination site for recognition by tyrosine recombinase, preferably Cre, Dre, Flp, KD, B2, or B3.

[0118] Suitable site-specific recombinase technologies for use in the present invention include, for example, serine site-specific recombination, tyrosine site-specific recombination, Cre-Lox recombination, FLP-FRT recombination, and homologous recombination.

[0119] In one embodiment, the site-specific recombinant site comprises or consists of a pair of site-specific recombinant sites.

[0120] As used herein, the term "pair" of sites refers to two sites recognized by the same site-specific recombinase enzyme, and both sites must be present for a recombination event to occur. Examples of a pair of site-specific recombination sites include an attP site and an attB site (e.g., for recognition by serine recombinase / integrase), or two loxP sites (e.g., for recognition by Cre recombinase).

[0121] Other examples of site-specific recombination site pairs are known in the art. Therefore, other examples of site-specific recombination site pairs include two FRT sites (e.g., for recognition by Flp recombinase), two rox sites (e.g., for recognition by Dre recombinase), two KD recombinase target (KDRT) sites (e.g., for recognition by KD recombinase), two B2 recombinase target (B2RT) sites (for recognition by B2 recombinase), and two B3 recombinase target (B3RT) sites (for recognition by B3 recombinase).

[0122] Therefore, the "pair" of sites described herein may be two different sites (having two different sequences) or two identical sites (having the same sequence), depending on the recombinase enzyme used.

[0123] Modified cells containing nucleic acid sequences to be excised from chromosomes according to the present invention may unintentionally undergo changes that render the nucleic acid sequences unexcisable. Loss of excisability may be caused, for example, by mutations in one or more site-specific recombination sites, or by mutations in one or more genes encoding site-specific recombinases specific to those sites.

[0124] Therefore, it may be beneficial to prepare cells in which the nucleic acid sequence to be excised from the chromosome (or, in the case of multiple nucleic acid sequences, each nucleic acid sequence) is flanked by two, three, or four pairs (or at least, two, three, or four pairs) of site-specific recombination sites, preferably two pairs of site-specific recombination sites. While we do not wish to be bound by theory, the probability that such cells lose their ability to excise nucleic acid sequences from chromosomes is reduced compared to cells in which the nucleic acid sequence is flanked by only one site-specific recombination site, or contains only one site-specific recombination site.

[0125] Therefore, in the embodiment, the site-specific recombination site includes or consists of two or more pairs, preferably two pairs of site-specific recombination sites.

[0126] Alternatively, site-specific recombination sites may consist of three, four, five, or six pairs of site-specific recombination sites.

[0127] If the nucleic acid sequence to be excised from a chromosome (or each nucleic acid sequence) is flanked by two or more pairs of site-specific recombination sites, these pairs of site-specific recombination sites may be arranged in any order along the chromosome, provided that the order allows for successful recombination.

[0128] In the embodiment, when two pairs of site-specific recombination sites are present, one pair of site-specific recombination sites may be nested inside the other pair. That is, one pair of recombination sites is sandwiched between the other pair of recombination sites. Therefore, for the two pairs of recombination sites A and B, these sites are arranged along the polynucleotide in an ABBA order or a BAAB order.

[0129] Alternatively, the site-specific recombination sites within each pair are arranged alternately along the polynucleotide so that no pair is nested inside the other. In other words, for the two pairs of recombination sites A and B, these sites are arranged in the polynucleotide in an ABAB order or a BABA order.

[0130] Each component of two or more recombination sites is spatially arranged to enable recombination. Therefore, in one preferred embodiment, a suitable number of nucleotides are arranged to separate adjacent A and B sites (i.e., adjacent sites belonging to each pair). In an alternative preferred embodiment, there are no nucleotides separating adjacent A and B sites (i.e., adjacent sites belonging to each pair). In other words, adjacent A and B sites (i.e., adjacent sites belonging to each pair) form a single continuous sequence.

[0131] The embodiments described in the above paragraphs are also applicable to further pairs of site-specific recombination sites, for example, three site-specific recombination sites, with necessary modifications.

[0132] In embodiments, the cells of the present invention further include a gene encoding a site-specific recombinase.

[0133] The excision and formation steps can be facilitated by site-specific recombinase (or site-specific recombinase activity). This can be achieved by using cells in which the chromosome has been modified so that the nucleic acid sequence to be excised is flanked by site-specific recombination sites. However, the excision method is not limited to this, and other genome editing technologies can also be used to excise the nucleic acid sequences described herein. For example, tyrosine recombinase or CRISPR / Cas technology can be used.

[0134] In embodiments, the gene encoding the site-specific recombinase is operably ligated to a promoter. For example, in the POP plasmids (POP1-POP8) illustrated in the examples, the site-specific recombinases are φC31 integrase and Bxb1, which are under the control of the mutant λPR(T41C) promoter and the λPL promoter, respectively.

[0135] Site-specific recombinases are appropriately selected to act with the chosen site-specific recombination sites. Therefore, in embodiments where two or more recombination sites are used, the cell (or intracellular chromosome) may further contain, as appropriate, genes encoding site-specific recombinases corresponding to each pair of recombination sites, for example, two or more recombinases, for example, three, four, five, or six recombinase genes. Furthermore, the genes encoding each site-specific recombinase enzyme may be operably linked to the same or different promoters. In addition, the genes encoding each site-specific recombinase enzyme may reside on the same or different nucleic acid sequences. For example, they may reside on the same or different chromosomes, or on the same or different extrachromosomal DNA molecules (e.g., plasmids or constructs). Alternatively, one or more of the genes may reside on a chromosome, while the others reside on extrachromosomal DNA molecules. Preferably, the genes are located on a chromosome (or the same chromosome).

[0136] It will be understood that embodiments of promoters and induction systems described herein (e.g., those relating to site-specific recombinases) may be used, with necessary modifications, to characterize the control of induction (or expression) of the target genes described herein, either as alternatives or additionally.

[0137] As used herein, the term “promoter” has the meaning recognized in the art and is generally understood to mean a DNA sequence to which a protein binds in order to control, initiate, and / or inhibit the transcription of the DNA sequence downstream of that promoter. Where it is stated that a gene encoding a recombinase enzyme (or target gene) is “operably ligated” to a promoter, it means that the promoter is in the correct functional position and / or orientation relative to that gene in order to control its transcription.

[0138] In embodiments, promoters (e.g., promoters for inducing excision and / or formation steps, and / or promoters for target genes, and / or promoters for site-specific recombinases, and / or promoters for agents that control the origin of replication) are sensitive to internal stimuli or internal conditions. For example, the promoters include the PCP_2836 promoter (Ma et al., 2018, Microbial Cell Factories, BioMed Central Ltd., 17(1), p.185. doi:10.1186 / s12934-018-1031-7.), the phoPR promoter (Paul et al., 2004, Journal of Bacteriology, American Society for Microbiology Journals, 186(13), pp.4262-4275. doi:10.1128 / JB.186.13.4262-4275.2004), and the P170 promoter (Madsen et al., 1999, Molecular Microbiology, Blackwell Publishing Ltd., 32(1), pp.75-87. The promoter may be induced when the cell culture enters a specific growth stage, as in the case of doi:10.1046 / j.1365-2958.1999.01326.x). Preferably, the promoter may be induced when the cell culture enters the exponential growth phase or during the exponential growth phase. Other preferred conditions include self-inducing media (e.g., those described in Studier et al., 2005 (Protein Expression and Purification, 2005, 41:207-234)) and self-inducing systems (e.g., SILEX described in Briand et al., 2016 (Scientific Reports, 6:33037)).

[0139] Promoter sensitive to internal conditions (e.g., promoters for inducing excision and / or formation processes, as well as promoters for target genes, as well as promoters for site-specific recombinases, and / or promoters for agents that control the origin of replication) may be preferred under conditions in industrial applications because these promoters do not require user intervention (e.g., administration of chemical inducers) for induction.

[0140] In embodiments, the promoter (e.g., a promoter for inducing excision and / or formation steps, as well as a promoter for a target gene, as well as a promoter for a site-specific recombinase, and / or a promoter for an active agent that controls the origin of replication) is sensitive to one or more external stimuli, and is, for example, an inducible promoter. Therefore, the promoter used may be selected based on one or more external stimuli that the user wishes to use to initiate excision and / or formation.

[0141] In some such embodiments, promoters (e.g., promoters for inducing excision and / or formation steps, and / or promoters for target genes, and / or promoters for site-specific recombinases, and / or promoters for agents that control the origin of replication) are temperature-sensitive, pH-sensitive, photosensitive, or chemically sensitive. Promoters with these and other properties, e.g., other inducible properties, are well known and described in the Art, and suitable promoters for use in the present invention (e.g., promoters for inducing excision and / or formation steps, and / or promoters for target genes, and / or promoters for site-specific recombinases, and / or promoters for agents that control the origin of replication) can be easily selected.

[0142] For example, other types of gene expression control methods that can be used for the same purpose (e.g., induction of excision and / or formation processes, and / or induction of the expression of a target gene, and / or induction of the expression of site-directed recombinases, and / or induction of agents that control the origin of replication) include induction by inducers (e.g., IPTG, arabinose, homoserine lactones, anhydrotetracycline) (Marschall, Sagmeister and Herwig, 2017)

[16] , (Lutz and Bujard, 1997)

[15] , (Cox, Surette, and Elowitz, 2007)

[17] , targeted proteolysis (Cameron and Collins, 2014)

[18] , and various modifications of CRISPR / Cas9 (Qi et al., 2013)

[19] , but are not limited to these. Those skilled in the art will understand how to use such other types of gene expression control methods. For example, in the case of chemically induced promoters, an appropriate chemical substance is added to the growth medium and taken up into the cell via diffusion or facilitated transport. Within the cell, the chemical substance binds to its target transcription factor, thereby activating transcription of that transcription factor from its target promoter. If a transcription repressor controls expression from a promoter and is conferred sensitivity to proteolysis, then proteolysis of the transcription repressor can induce expression from the promoter. Catalytically inactive CRISPR / Cas9 can be targeted to promoters by expressing appropriate guide RNA within the cell, resulting in activation or repression of the promoter depending on the precise configuration.

[0143] Therefore, in embodiments, promoters (e.g., promoters for inducing excision and / or formation steps, and / or promoters for target genes, and / or promoters for site-directed recombinases, and / or promoters for active agents that control the origin of replication) are chemically sensitive, and suitable promoters are readily available in the art. For example, preferred promoters are those that can be induced using isopropyl β-d-1-thiogalactopyranoside (IPTG), arabinose, homoserine lactones, or anhydrotetracycline. For example, the above promoters are promoters derived from the lac operon, ara operon, tet operon, or lux operon, such as the lac promoter (lac p), araBAD promoter, ppuI promoter, and tet promoter (P tet It is a promoter, or a luxI promoter.

[0144] For example, the expression of a gene encoding a site-directed recombinase (and / or the expression of a target gene, and / or the expression of a site-directed recombinase, and / or the expression of an agent that controls the origin of replication) may be controlled under a lac operon expression system. The expression of the gene (and / or target gene) can be induced by IPTG or allolactose. For example, the gene (and / or target gene) may be located on a polynucleotide sequence or construct (e.g., on a chromosome, or on an extrachromosomal DNA molecule or plasmid), with a lac promoter and a lac operator located upstream of the gene, and the polynucleotide sequence may further include a lac repressor (lacI). Polynucleotide sequences and vectors suitable for controlling a target gene under this system are known in the art.

[0145] Alternatively, the expression of a gene encoding a site-directed recombinase (and / or the expression of a target gene, and / or the expression of a site-directed recombinase, and / or the expression of an agent that controls the origin of replication) may be controlled under an ara operon expression system. The expression of the gene (and / or target gene) can be induced by arabinose. For example, the gene (and / or target gene) may be located on a polynucleotide sequence or construct (e.g., on a chromosome or on an extrachromosomal DNA molecule), and an araBAD promoter may be located upstream of the gene (and / or target gene), and the polynucleotide sequence or construct may also include other genetic elements necessary for gene regulation using the ara operon. The genetic elements of the ara operon necessary for gene regulation are known to those skilled in the art and are shown above.

[0146] Alternatively, more preferably, the expression of a gene encoding a site-directed recombinase (and / or the expression of a target gene, and / or the expression of a site-directed recombinase, and / or the expression of an agent that controls the origin of replication) may be controlled under a lux operon expression system. The expression of the gene (and / or target gene) can be induced by acylhomoserine lactones (or acylhomoserine lactones). For example, the gene (and / or target gene) may be located on a polynucleotide sequence or construct (e.g., on a chromosome or on an extrachromosomal DNA molecule), the luxI promoter may be located upstream of the gene (or operably linked to the gene), and the polynucleotide sequence or construct may also include the luxR gene, preferably in a configuration in which the luxR gene is constitutively expressed.

[0147] Alternatively, the cell may contain a gene encoding a site-directed recombinase, the expression of which can be controlled under a Tet-Off or Tet-On expression system. The expression of this gene can be induced by tetracycline (or anhydrotetracycline or doxycycline). For example, the cell may contain (i) a gene located on a plasmid, the tetracycline (or anhydrotetracycline or doxycycline)-dependent promoter located upstream of the gene, and (ii) a tTA expression plasmid or an rtTA expression plasmid. Plasmids suitable for controlling a target gene under this system are known in the art.

[0148] In some embodiments, the expression of a gene encoding a site-directed recombinase (and / or the expression of a target gene) may be induced or controlled by target protein degradation.

[0149] In embodiments, the expression of a gene encoding a site-directed recombinase (and / or the expression of a target gene) is induced or controlled by CRISPR interference (CRISPRi).

[0150] In a preferred embodiment, the promoter (e.g., a promoter for inducing excision and / or formation steps, and / or a promoter for a target gene, and / or a promoter for a site-directed recombinase, and / or a promoter for an active agent that controls the origin of replication) is temperature-sensitive.

[0151] Any suitable temperature-sensitive promoter can be used, and there are many examples of such promoters in the art.

[0152] The temperature-sensitive promoter used in the present invention can be selected to be activated (and / or exhibit maximum activity level) at any preferred temperature. Preferably, the promoter may be a temperature-sensitive promoter that is activated (and / or exhibits maximum activity level) at temperatures in the range of 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C, 50-55°C, or 55-60°C, or above 60°C. Preferably, the promoter may be a promoter that is activated (and / or exhibits maximum activity level) at about 37°C.

[0153] Accordingly, in embodiments, the temperature-sensitive promoters used in the present invention (e.g., promoters for inducing excision and / or formation steps, and / or promoters for target genes) can be induced by changing (i.e., increasing or decreasing) the temperature of the cells (or cell culture) to a temperature (or temperature range) in which the temperature-sensitive promoter is activated (and / or exhibits its maximum activity level). Thus, to induce excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules (and / or, in some cases, expression of target genes), the temperature can be changed to a range of 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C, 50-55°C, or 55-60°C, or above 60°C.

[0154] The temperature-sensitive promoter used may be activated at higher temperatures, in which case it may be necessary to initially set the cell (or cell culture) temperature lower (e.g., 30°C or below 30°C) and then increase it (e.g., to 37°C or above 37°C) to induce activation and, consequently, to induce excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules (and / or, in some cases, expression of the target gene). Conversely, the temperature-sensitive promoter used may be activated at lower temperatures, in which case it may be necessary to initially set the cell (or cell culture) temperature higher (e.g., 37°C or above 37°C) and then decrease it (e.g., to 30°C or below 30°C) to induce excision and formation (and / or, in some cases, expression of the target gene). Preferably, the promoter may be activated (and / or exhibit maximum activity level) at approximately 37°C (or above 37°C). Therefore, conveniently, to induce the excision and / or formation steps (and / or, optionally, the expression of the target gene) of the present invention, the temperature of the cells (or cell culture) may be initially set lower, for example, to about 30°C, e.g., 25°C to 33°C, and then increased thereafter (for example, to about 37°C, e.g., 36°C to 39°C, or a temperature higher than 37°C).

[0155] After raising or lowering the temperature of cells (or cell culture) to activate a temperature-sensitive promoter, the temperature may be maintained further, or it may be modified (lowered or raised) by, for example, a human operator. This modification is to provide the ideal or optimal temperature for the expression of the target gene, for example, if that temperature differs from the activation temperature (or activation temperature range) of the temperature-sensitive promoter. Maintaining the activation temperature after the activation of the temperature-sensitive promoter is not a requirement of the present invention. Therefore, after raising (or lowering) the temperature of cells (or cell culture) to match or exceed (or fall below) the activation temperature of the temperature-sensitive promoter, the temperature may also be lowered (or raised again). For example, it may be returned to the previous temperature or the starting temperature.

[0156] A preferred temperature-sensitive promoter for use in the present invention is a promoter that is controllable or modulated by a temperature-sensitive active substance (or temperature-sensitive repressor or temperature-sensitive activator), such as the phage-lambda cl857 repressor. In embodiments, the temperature-sensitive promoter is phage-lambda P R or P L promoter (or, P R or P L These promoters are modified versions of the promoter and are controlled by the lambda cl857 repressor. These promoters are activated (and / or exhibit maximum activity levels) at approximately 37°C (Jechlinger et al., 1999, FEMS Microbiology Letters. Wiley / Blackwell (10.1111), 173(2), pp.347-352. doi:10.1111 / j.1574-6968.1999.tb13524.x). Therefore, using these promoters, the excision and formation steps of the present invention (and / or, in some cases, the expression of the target gene) can be easily induced by changing the cell (or cell culture) temperature from approximately 30°C (e.g., 25°C-33°C) to approximately 37°C (e.g., 36°C-39°C, or a temperature higher than 37°C).

[0157] In embodiments, the functional elements used (or required) in the excision and / or formation steps of the present invention, for example, one or more or all of the functional elements (i.e., genetic elements), are located (or incorporated) on one or more episomes or plasmids or other suitable extrachromosomal DNA molecules present in the cell. For example, in embodiments, the genetic element (or gene) encoding site-specific recombinase is located (or incorporated) on one or more episomes or plasmids or one or more other suitable extrachromosomal elements present in the cell. Alternatively, the entire recombinase expression system may be located (or incorporated) on one or more episomes or plasmids (or other suitable elements). For example, the genetic element (or gene) encoding site-specific recombinase and the genetic element (or gene) encoding a recombinase regulatory component may be located (or incorporated) on one or more episomes or plasmids (or other suitable elements). The recombinase regulatory component is a temperature-sensitive promoter as defined herein (e.g., P R Or P L The promoter or a variant thereof may include, if appropriate, a genetic element (or gene) encoding a substance that promotes such sensitivity as defined herein. This could be, for example, a gene encoding a temperature-sensitive repressor, such as a gene encoding the cI857 repressor.

[0158] In an alternative, more preferred embodiment, the functional elements (e.g., one or more or all functional elements (i.e., genetic elements)) used (or required) in the excision step and / or formation step of the present invention are located (or integrated) on the genome, for example, on a chromosome (such as a bacterial chromosome). For example, in an embodiment, the genetic element (or gene) encoding a site-specific recombinase is located (or integrated) on the genome, for example, on a chromosome (such as a bacterial chromosome). Alternatively or more preferably, the entire recombinase expression system may be located (or integrated) on the genome. For example, the genetic element (or gene) encoding a site-specific recombinase and the genetic element (or gene) encoding a recombinase regulatory factor component may be located (or integrated) on the genome, for example, on a chromosome. The recombinase regulatory factor component may include a temperature-sensitive promoter as defined herein (e.g., P R or P L promoter or a variant thereof), and optionally, a genetic element (or gene) encoding an agent that promotes the sensitivity as defined herein, as appropriate. This may be, for example, a gene encoding a temperature-sensitive repressor, such as a gene encoding the cI857 repressor.

[0159] Alternatively, the genetic element (or gene) encoding a site-specific recombinase may be located on the genome, and the genetic element encoding the related recombinase regulatory factor component may be located on one or more episomes or plasmids, or vice versa.

[0160] In embodiments, one or more functional elements (i.e., genetic elements) used (or required) for irreversible inactivation of replication origins are codon-optimized. For example, a genetic element (or gene) encoding a site-directed recombinase may be codon-optimized. Alternatively, a genetic element (or gene) encoding a temperature-sensitive agent that promotes the sensitivity of a temperature-sensitive promoter (e.g., encoding a temperature-sensitive repressor, e.g., cl857) is codon-optimized when such a temperature-sensitive promoter system is used.

[0161] In embodiments, the product is a product produced intracellularly or that can be produced by a cell (or any product or any target product), for example, a product produced or that can be produced by a two-step bioprocess method in the art. Such a product is therefore a product that is desired to be prepared or produced in large quantities, measurable amounts, or significant amounts, for example, a desirable, useful, industrial, or commercial product, or a product of value for industry or research. Therefore, in embodiments, the product is a small molecule. Alternatively, the product may be a polypeptide, protein, or protein complex (or target protein), for example, an antibody, antigen, enzyme, growth factor, or cytokine. Preferably, the product is a pharmaceutical compound, pharmaceutical product, food additive, coloring compound, fragrance component, industrial chemical, fine chemical, or biofuel. Preferably, the biofuel is an alcohol, for example, butanol or isobutanol. Preferably, the product is encoded by a target gene, or the target gene encodes an enzyme or active substance for producing the product.

[0162] The method of the present invention is not limited to forming only a single extrachromosomal DNA molecule within a cell. For example, the method of the present invention may include multiple (e.g., two or more) target genes and multiple origins of replication for the excision and formation of multiple extrachromosomal DNA molecules (e.g., multiple plasmids) to express the multiple target genes. This can be achieved, for example, by using separate excision and formation mechanisms for the excision and formation of each extrachromosomal DNA molecule. For example, this can be achieved by using different serine recombinases for each nucleic acid sequence (or combination of nucleic acid sequences) required for the formation of each extrachromosomal DNA molecule (and further, by using cells containing chromosomes having different corresponding site-specific recombination sites).

[0163] It is well known to those skilled in the art that steps necessary to enable cells to produce a desired product, such as inducing or enabling the expression or regulation of genes required for product synthesis, or the expression of a target gene. For example, if the product is a small molecule, one or more enzymes capable of producing the product from a given substrate may be expressed. One or more such enzymes may be encoded by a target gene described herein. The substrate may be available uniformly or endogenously within the cell, for example, if the substrate is an intermediate in an intracellular metabolic pathway. Alternatively, the substrate may be supplied heterologously (e.g., by expressing heterologous genes within the cell) or exogenously (e.g., by administering it into a culture medium).

[0164] In the embodiment, the target gene encodes either a product or an enzyme necessary for producing the product.

[0165] The target gene may be homologous (endogenous) or heterologous. Preferably, the target gene is heterologous.

[0166] In embodiments, a gene (or target gene) is operably linked to a promoter. Such a promoter can control the expression of a product or enzyme encoded by the target gene (GOI), e.g., a homologous (endogenous) or heterologous product or enzyme. Thus, in some embodiments, such a promoter may be an endogenous promoter (e.g., a promoter native to the gene or cell), or a constitutive promoter to enable continuous or constitutive expression. Alternatively, such a promoter may be an inducible or heterologous promoter, which can be used, for example, to enable the induction of the production of a gene-encoded product within a cell at a desired time.

[0167] Suitable inducible promoters are well known and described in the art, and any of these can be used. For example, in embodiments, the promoter may be pH-sensitive, photosensitive, temperature-sensitive, or chemically sensitive. Further embodiments and details of such promoters and systems are described elsewhere in this specification (including embodiments described herein in relation to promoters for controlling site-specific recombinases, which, with necessary modifications, can be applied as embodiments of promoters for controlling target genes). Exemplary systems are also shown in the examples.

[0168] In other embodiments, gene (target gene) or promoter expression may be induced or controlled by targeted protein degradation or by other suitable techniques such as CRISPR interference (CRISPRi).

[0169] The concept of CRISPR interference (CRISPRi) is well known in the art, for example, as described by Lei et al. in 2013 (Cell 152, 1173-1183). In CRISPRi, a catalytically inactive Cas9 is targeted to a specific DNA sequence using an sgRNA with a specific sequence. This catalytically inactive Cas9 binds to the target DNA region (e.g., the promoter region), interfering with RNA polymerase binding and transcription elongation.

[0170] Therefore, in embodiments of cells in which the expression of a gene (target gene) or promoter is induced or controlled by CRISPR interference (CRISPRi), the cell includes a gene encoding catalytically inactive Cas9 and an sgRNA for targeting the catalytically inactive Cas9 to the gene or promoter in order to produce a desired product, thereby inhibiting the expression of the gene or promoter.

[0171] In the embodiment, the target gene and / or product are homologous. The term "homologous" is commonly understood in the art. Therefore, in the context of this invention, "homologous target gene" or "homologous product" refers to a target gene or product that is normally (i.e., naturally or natively) present in or produced by a cell (or cell line or cell system). Therefore, a cell may not need to be exposed to a heterologous substrate to have a homologous target gene or produce a homologous product. Thus, "homologous" may mean "endogenous."

[0172] In this embodiment, the target gene is a homologous target gene, and the target gene is located at a non-native position within the cell. If the product is homologous or endogenous, the cell may already be producing or capable of producing the product before excising the nucleic acid sequence from the chromosome and forming the extrachromosomal DNA molecule. In this case, the excision of the nucleic acid sequence from the chromosome and the formation of the extrachromosomal DNA molecule may be induced to increase the level of the product or to overexpress the product. For example, as described elsewhere in this specification, this may be to improve the cell's titer, yield, and / or productivity in the production of homologous or endogenous products, for example, compared to control cells.

[0173] Preferably, the product is not produced (or not produced in a significant amount, or not produced in a measurable amount) before the excision and formation steps are performed. Preferably, alternatively or additionally, the target gene is not expressed (or not expressed in a significant amount, or not expressed in a measurable amount) before the excision and formation steps are performed.

[0174] In an alternative, more preferred embodiment, the target gene and / or product is heterogeneous. The term "heterologous" is commonly understood in the art. Therefore, in the context of this invention, a "heterologous target gene" or "heterologous product" is a target gene or product that is not normally (i.e., naturally or natively) present in a cell (or cell line or cell system) or produced by it. Thus, for a cell to have a heterologous target gene or produce a heterologous product, it may need to be genetically modified or exposed to a heterologous substrate. Thus, "heterologous" can mean "exogenous." The term "heterologous" is in contrast to "homological" and "endogenous," and these terms are also commonly understood in the art.

[0175] Preferably, the product is not produced (or not produced in a significant amount, or not produced in a measurable amount) before the excision and formation steps are performed. Preferably, alternatively or additionally, the target gene is not expressed (or not expressed in a significant amount, or not expressed in a measurable amount) before the excision and formation steps are performed.

[0176] In embodiments, the nucleic acid sequence (excised from the chromosome) contains two or more (or more, e.g., two, three, four, or five) target genes, and / or the extrachromosomal DNA molecule contains two or more (or more, e.g., two, three, four, or five) target genes. The target genes may be identical or different.

[0177] The cells of the present invention (e.g., modified cells of the present invention) can be prepared using standard techniques in the art. For example, recombinase may be used, in which case homologous recombination may be used, and in which case the phage-lambda Red recombinase system may be used (see, for example, Progress in Biophysics and Molecular Biology 147 (2019), 33e46). Such homologous recombination using recombinase can be carried out, for example, using a vector containing the genetic element of interest. For example, a vector containing the target gene and an origin of replication (or two origins of replication) flanked by site-specific recombination sites. Here, the site-specific recombination sites themselves are flanked (i.e., nested) by nucleotide sequences homologous to the corresponding sequences in a chromosome (or said chromosome) of the target cell. The cells of the present invention (e.g., modified cells of the present invention) can also be prepared using non-homologous site-specific recombination. For example, a cargo sequence can be incorporated into the naturally occurring attB site in the E. coli chromosome using phage-lambda integrase.

[0178] Therefore, in one embodiment, the present invention provides a method for producing the cells of the present invention.

[0179] In embodiments, a method for producing cells of the present invention includes the step of inserting one or more polynucleotide vectors described herein into a chromosome (or said chromosome) of a cell. Preferably, the one or more polynucleotide vectors are inserted into a chromosome (or said chromosome) using a recombinase. For example, homologous recombination using, for example, the phage-lambda Red recombinase system, or non-homologous site-specific recombination using, for example, phage-lambda integrase may be used.

[0180] In the process of producing the cells of the present invention, a certain (or said) original (i.e., native, natural, wild-type, or endogenous) origin of replication in the starting bacterial cell may be replaced by a different origin of replication. For example, the inserted origin of replication may have a different sequence compared to a certain (or said) original (i.e., native, natural, wild-type, or endogenous) origin of replication in the starting bacterial cell (for example, the inserted origin of replication may originate from a different cell line or cell strain, or it may be an exogenous sequence). Or more preferably, a certain (or said) origin of replication is the same (i.e., the same native, natural, wild-type, or endogenous) origin of replication. That is, a polynucleotide fragment encoding the original origin of replication is replaced by another polynucleotide fragment encoding the same origin of replication via homologous recombination or other substitution process.

[0181] Similarly, in the product production methods and other embodiments of the present invention described herein, a replication origin (or such replication origin) may have a different sequence compared to a certain (or such) original (i.e., native, natural, wild-type, or endogenous) replication origin (for example, such replication origin may originate from a different cell line or cell type, or it may be an exogenous sequence).

[0182] As detailed below, the method of the present invention can advantageously provide an increase or improvement in gene copy number, gene expression, and / or product production. However, it should be noted that the method of the present invention may be useful even when the production level in the cells of the present invention is the same as that of cells of the prior art. This is because the method of the present invention does not require antibiotics, which facilitates product purification, waste management, etc., and therefore improves the efficiency and cost-effectiveness of the method.

[0183] In embodiments, the method of the present invention increases or improves the level, quantity, or concentration of a cellular product or product production (or product of production), preferably in terms of potency, yield, or productivity, or in terms of cell-per-cell potency, cell-per-cell yield, or cell-per-cell productivity. Preferably, the increase is a measurable or significant increase, for example, statistically or clinically significant. For example, cells of the present invention are preferred that can result in an increase of at least, or up to, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold in terms of the level of product production or product of production (preferably in terms of potency, yield, or productivity, or in terms of cell-per-cell potency, cell-per-cell yield, or cell-per-cell productivity), or in terms of the activity of one or more enzymes that produce the product or play an active role in the production of the product. Any suitable comparison can be used. For example, this could be an increase compared to the levels observed in the same type of cells when excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules are not induced (e.g., do not occur or are not possible), or an increase compared to the levels observed in control cells, control cell lines, or control strains, as described elsewhere.

[0184] As used herein, the term “control strain” (or “control cell”) may generally refer to a cell that does not have the extrachromosomal DNA molecules described herein, or a cell that does not have the ability to produce the extrachromosomal DNA molecules described herein. For example, a “control strain” may refer to a cell that differs from the cells of the present invention in that the control strain is unmodified, the control strain’s chromosomes are unmodified, or the control strain contains native, wild-type, or unmodified chromosomes (for example, a native, wild-type, or unmodified strain or cell). Alternatively, the terms “control cells,” “control cell line,” or “control strain” may refer to cells (e.g., cells of the present invention) when used under conditions in which the excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules are not induced / induced (e.g., cells of the present invention as defined herein). Alternatively, the terms “control cells,” “control cell line,” or “control strain” may refer to cells that express the product according to the method of the art. For example, control cells may be transformed with a conventional plasmid for the production of the product, where the conventional plasmid requires antibiotic selection to be retained within the cell. For example, the conventional plasmid may be the mRFP1-KanR plasmid used in the examples.

[0185] Suitable methods for measuring product production or the level of a product (preferably in terms of potency, yield, or productivity, or in terms of potency per cell, yield per cell, or productivity per cell) or the activity of the enzyme producing the product are well known to those skilled in the art. Accordingly, in some embodiments of the present invention, the method includes the steps of detecting or determining the amount or level (e.g., concentration) of a product produced by cells or a cell system (e.g., determining potency or yield), and further, if necessary, measuring the time required for the cells to produce the product to that amount or level (e.g., concentration).

[0186] Accordingly, typically, cells (or cell lines or cell systems) used in the present invention or the methods of the present invention exhibit higher (in some cases significantly higher) product production or levels of product (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell) compared to the control cells described above, after the formation of extrachromosomal DNA molecules. Where appropriate, the level of product production can be readily measured or determined by methods known in the art. Accordingly, cells (or cell lines or cell systems) that can exhibit higher (increased) or significantly higher (increased) product production or levels of product (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell) compared to a control, for example, when evaluated in vitro, constitute yet another aspect of the present invention.

[0187] Products produced by cells may be secreted into the culture medium (or supernatant) or retained within the cell. Therefore, the level of production or product can be measured, as appropriate, in terms of the level of product present in the culture medium (or supernatant), the level of product retained within the cell (e.g., this can be measured after standard protein extraction and purification), or both, depending on the product in question.

[0188] Another way of looking at it, cells (or cell lines or cell lines) that, when evaluated in vitro, for example, in the cell culture medium (or supernatant) and / or within cells, exhibit higher (increased) or significantly higher (increased) product production or levels of product (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell) when the formation of extrachromosomal DNA molecules is induced, compared to control cells, constitute yet another aspect of the present invention. For example, cells (or cell lines or cell lines) that, when evaluated in vivo, can exhibit higher (increased) or significantly higher (increased) product production or levels of product in a subject when induced (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell), for example, cells (or cell lines or cell lines) that can exhibit higher (increased) product production or local levels of product in a subject when excision and formation are induced, constitute yet another aspect of the present invention, particularly when such an effect is observed when such cells are administered to a subject.

[0189] The cells of the present invention can be administered to a target by any appropriate method commonly used in therapies such as cell-based therapies or in the treatment of indicated diseases. Such administration methods include, but are not limited to, oral administration, sublingual administration, transdermal administration, cutaneous administration, rectal administration, nasal administration, vaginal administration, or ocular administration, or inhalation administration, buccal administration, or injection administration. Furthermore, the cells or compositions of the present invention can be formulated for parenteral administration, for example, by injection or continuous infusion. The route of administration may be any route that effectively delivers the cells to the desired site without harming the recipient. Preferably, the above-mentioned higher level or increase in the cell (of product production, or of the activity of the product, or of the enzyme producing the product) is a measurable or significant increase, for example, statistically or clinically significant. For example, cells (or cell lines or cell lines) that can produce an increase of at least, or up to, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, or more compared to the control level, as described above, in terms of the level of the product, product production, or production product (preferably in terms of titer, yield, or productivity, or in terms of titer, yield, or productivity per cell), for example, at the local level or in vitro level of product production or production product, or at the level of activity of the enzyme producing the product (for example, when evaluated in vitro), compared to the control level. In other words, cells are preferred that can result in an increase of at least, or up to, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 times compared to the control level, as described above, in terms of the level of the product, product production, or product of production (preferably in terms of titer, yield, or productivity, or in terms of titer, yield, or productivity per cell), for example, at the local or in vitro level of product production or product of production, or at the level of activity of the enzyme producing the product. The in vitro level can be readily measured, for example, in a cell culture, as described elsewhere herein. Preferably, such an increase is an increase in the extracellular (e.g., secreted) level of the product and / or at the intracellular level (preferably in terms of titer or yield), or an increase in the activity of the enzyme producing the product, which is measured, for example, in vitro, for example, in the culture medium (or supernatant) or intracellularly of a cell culture.

[0190] Suitable methods for measuring the product, product production, or the level of the product (preferably in terms of potency, yield, or productivity, or in terms of potency per cell, yield per cell, or productivity per cell), or the activity of the enzyme producing the product are known to those skilled in the art, and any of these can be used.

[0191] In embodiments, the methods of the present invention increase the level of a cellular product, product production, or product (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell). Preferably, the increase is a measurable or significant increase, for example, statistically or clinically significant. For example, the excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules, when used in the methods of the present invention, result in the multiplier or rate of increase defined above in product production or the level of product (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell), or in the activity of the enzyme producing the product. Any suitable comparison can be used. Examples include an increase in the level (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell) compared to the level observed when the formation of extrachromosomal DNA molecules is not induced in the same cell (or cell of the same type), or an increase in the level (preferably in terms of titer, yield, or productivity, or in terms of titer per cell, yield per cell, or productivity per cell) compared to the level in control cells, for example, control cells as described elsewhere herein.

[0192] If necessary, the cellular product, product production, or level of the product (preferably potency, yield, or productivity) can be quantified per cell number (or per cell population size or cell population density). In other words, the cellular product, product production, or level of the product (preferably potency, yield, or productivity) can be quantified in a manner that takes into account the number of cells (or cell population size or cell population density) used to produce the product.

[0193] A suitable method for measuring cell population size or cell number is to measure the optical density of the cell population, for example, the optical density at 450 nm (OD). 450 ), optical density at 600 nm (OD 600 ), or optical density at 650 nm (OD 650 Methods involving the measurement of ) are well known in this field.

[0194] In the method of the present invention, if one or more origins of replication on a chromosome are inactivated (for example, because the origins of replication are excised to form part of the extrachromosomal DNA molecule of the present invention), the cells may exhibit a different (higher or lower) peak cell density than cells in which the origins of replication have not been removed.

[0195] In embodiments, the peak cell density (or maximum cell density, maximum cell density, or cell density plateau) of a cell culture used in the present invention or the method of the present invention is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, about 100% (i.e., nearly the same or the same), greater than 100%, at least 100%, at least 105%, at least 110%, about 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, and at least 150% of the peak cell density of the cell culture or control cell culture in which excision and formation have not been performed or could not occur. This includes values ​​up to these percentages "at most".

[0196] Preferably, the peak cell density (or maximum cell density, maximum cell density, or cell density plateau) of a cell culture used in the present invention or the method of the present invention is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, about 100% (i.e., nearly the same or the same), greater than 100%, at least 100%, at least 105%, at least 110%, about 110%, at least 115%, or at least 120% of the peak cell density of the cell culture or a control cell culture in which, for example, excision and formation have not been performed or could not occur. Values ​​up to these percentages "at most" are also included.

[0197] Preferably, the peak cell density (or maximum cell density, maximum cell density, or cell density plateau) of a cell culture used in the present invention or the method of the present invention is at least 90%, at least 95%, at least 100%, about 100% (i.e., nearly the same or the same), greater than 100%, at least 100%, at least 105%, at least 110%, or about 110% of the peak cell density of a control cell culture in which, for example, no excision and formation has been performed or could not occur. Values ​​up to these percentages "at most" are also included.

[0198] Suitable control cell cultures are as described elsewhere herein and may be, for example, cultures of the cells of the present invention in which excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules are not induced (e.g., do not occur or cannot occur).

[0199] In embodiments, the level of production of products produced by cells or modified cells used in the present invention or the methods of the present invention is higher as the peak cell density (or maximum cell density, maximum cell density, or cell density plateau) of the cell culture or modified cell culture approaches the peak cell density of the control cell culture, etc. For example, it is higher when it is at least 70% of the peak cell density of the control cell culture, etc., as described above.

[0200] In embodiments, the method of the present invention increases the yield or titer of products produced by cells, or improves the productivity of cells.

[0201] The term "potency" refers to the quantity or concentration of a product produced during or at the end of a bioprocess. Therefore, potency can be measured in "g / L" units or other appropriate units.

[0202] The term "yield" refers to the mass of the product relative to the mass of the substrate (e.g., carbon and energy source) supplied to the cells for a production method or bioprocess. Therefore, yield can be measured in "g / g" units or other appropriate units.

[0203] The term "productivity" refers to the rate at which cells produce products in a bioprocess. Therefore, productivity is the amount or concentration of product produced per unit time. Thus, productivity can be measured in units of "g / L / h" (i.e., grams per liter per hour) or other appropriate units.

[0204] Cells may already be producing products before the formation of extrachromosomal DNA molecules is induced. In this case, inducing the formation of extrachromosomal DNA molecules can be used to increase the level of the product or to overexpress the product. For example, as described elsewhere in this specification, it can be used to improve the titer, yield, and / or productivity of cells in the production of homologous or endogenous products, for example, compared to control cells.

[0205] Preferably, the product is not produced (or not produced in a significant amount, or not produced in a measurable amount) before the excision and formation steps are performed. Preferably, alternatively or additionally, the target gene is not expressed (or not expressed in a significant amount, or not expressed in a measurable amount) before the excision and formation steps are performed.

[0206] In a typical method of the present invention, during the growth stage, the target gene exists as a single copy in the genome, and when biomass accumulates to a desired level, popping is induced, thereby forming an extrachromosomal DNA molecule containing the origin of replication and the target gene. This extrachromosomal DNA molecule begins replication, and its copy number increases. Subsequently, once induced, the target gene (and / or desired product) is expressed at a level comparable to, or even exceeding, the expression level from a conventional expression plasmid. Thereafter (or by means of), the product can be produced and recovered (or isolated).

[0207] The copy number of a sequence or genetic element may be expressed as a ratio to the copy number of the genome. This copy number ratio can be calculated, for example, by the techniques described in the examples, or by other suitable methods known in the art. Therefore, the copy number of the extrachromosomal DNA molecule (or the copy number ratio of the extrachromosomal DNA molecule to the genome) can reach at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 during the process of the method of the present invention, preferably at least 5. Alternatively, the copy number (or copy number ratio) can reach at least the same level as that of control cells, for example, control cells containing a conventional expression plasmid using antibiotic selection as described herein. In embodiments, expression of the target gene may be induced when this copy number (or copy number ratio) is reached.

[0208] The activity of the active agents responsible for inducing the excision and formation steps (sometimes referred to herein as "popping") and, optionally, the inactivation of the origin of replication ("switching") may be controlled to avoid premature formation and subsequent loss of extrachromosomal DNA molecules during cell proliferation. For example, inactivation may be induced or controlled through the control of the active agent responsible for or involved in (e.g., catalyzing) the excision and / or formation steps. For example, the excision and / or formation steps may be induced or controlled via a promoter operably linked to a gene encoding an active agent responsible for or involved in the excision and formation steps ("popping" steps). In this case, the excision and / or formation steps can be induced by inducing the promoter. Therefore, a preferred promoter for this purpose is an inducible promoter.

[0209] In embodiments of the present invention, which include excising multiple nucleic acid sequences from a chromosome (or said chromosome), the excision of these nucleic acid sequences may occur at different times (or the excision events may be induced at different times). In embodiments of the present invention, which include a step of inactivating all replication origins within a cell, this inactivation event ("switching") and the excision of nucleic acid sequences that will form part of an extrachromosomal DNA molecule ("popping") may be induced at different times. However, it is preferable that switching and popping occur simultaneously. This can be achieved, for example, by placing the gene(s) responsible for switching and popping under the control of the same promoter. More preferably, switching and popping may occur as part of the same excision event.

[0210] In embodiments, the method of the present invention includes inducing the expression of a target gene and / or inducing the replication of an extrachromosomal DNA molecule. Inducing the replication of an extrachromosomal DNA molecule can also mean activating a replication origin (or said replication origin) of the extrachromosomal DNA molecule (if the replication origin is part of the extrachromosomal DNA molecule). Further embodiments relating to this point are described elsewhere in this specification. The induction of target gene expression and the induction of extrachromosomal DNA molecule replication may be performed simultaneously or sequentially. If performed sequentially, they may be carried out in any order. The excision and formation steps may be performed simultaneously or sequentially with one or both of these induction steps.

[0211] Therefore, in embodiments, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication, The above steps involve inducing the replication of extrachromosomal DNA molecules, The present invention provides a method comprising the step of inducing the expression of the target gene from the extrachromosomal DNA molecule described above.

[0212] If necessary, the method (and other methods of the present invention described herein) may further include a step of recovering the product, for example, as described elsewhere herein (or may end with such a step, or may have such a step as the final step).

[0213] Therefore, in embodiments, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication, A step of inducing the expression of the target gene from the above-mentioned extrachromosomal DNA molecule, The present invention provides a method comprising the step of inducing the replication of the above-mentioned extrachromosomal DNA molecule. If necessary, the method (and other methods of the present invention described herein) may further include a step of recovering the product, for example, as described elsewhere herein (or may end with such a step, or may have such a step as the final step).

[0214] In embodiments (for example, those relating to one or more nucleic acid sequences and / or extrachromosomal DNA molecules, and those relating to the methods of the present invention), a target gene and a gene for controlling (or responsible for controlling) the replication of a certain origin of replication (or the replication of such origin of replication) (e.g., the trfA gene) are operably linked to (or under the control of) the same promoter.

[0215] Preferably, the expression of the target gene and / or the replication of the extrachromosomal DNA molecule are induced (or activated) by the formation of the extrachromosomal DNA molecule. This can be achieved by designing the nucleic acid sequence that will be excised from the chromosome to form an extrachromosomal DNA molecule so that, when the extrachromosomal DNA molecule is formed (or during the process of its formation), the promoter is operably linked to a gene that controls (or is responsible for controlling) the activity of the target gene and / or a replication origin (or said replication origin).

[0216] This operable linkage between a promoter and a gene is typically achieved by ligating the promoter sequence to the gene sequence. For example, it can be achieved by ligating one or more excised nucleic acid sequences to form an extrachromosomal DNA molecule. Preferably, the operable linkage may be achieved by circularizing one excised nucleic acid sequence (or by ligating and circularizing multiple excised nucleic acid sequences) to form the extrachromosomal DNA molecule described herein. More preferably, the operable linkage may be achieved by recombining the promoter ahead of (or upstream of) the target gene when the extrachromosomal DNA molecule is formed. Potential strong promoters that can be used for this purpose include the tac promoter, the trc promoter, the hybrid P3 promoter, the Pveg promoter, the Placlq1 promoter, and the J23101 promoter. However, any strong non-inducible (or constitutive) promoter may be used.

[0217] In one embodiment, after performing the excision and formation steps of the method of the present invention, the expression of the target gene is increased (or is increased, increases, or increases at some point in time). In another embodiment, the target gene is not expressed (and / or no product is produced) before performing the excision and formation steps of the method of the present invention, but the target gene is expressed (and / or a product is produced) (or the expression of the target gene is increased after the process, increases after the process, or increases at some point after the process). In yet another embodiment, the target gene is not expressed (and / or no product is produced) before performing the excision and formation steps of the method of the present invention, and / or the target gene is expressed (and / or a product is produced) after performing the excision and formation steps of the method (or the expression of the target gene is increased after the process, increases after the process, or increases at some point after the process).

[0218] It is advantageous to suppress the expression of a target gene while it is present on the chromosome. This is because constitutive expression of recombinant proteins has several drawbacks. For example, constitutive expression of recombinant proteins depletes resources, potentially slowing cell proliferation. It also creates selective pressure against high expression, potentially leading to the dominance of non-expressing mutants.

[0219] When it is stated that the target gene is "not expressed," this term includes situations where the target gene is not significantly expressed, as well as situations where it is not measurably expressed.

[0220] Preferably, the threshold optical density level at which excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules should be initiated or induced after arrival (e.g., immediately after arrival) is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the peak cell density (or maximum cell density, maximum cell density, or cell density plateau) of the cell culture after the excision and formation steps have been performed, or at least 10%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the peak cell density of the control cell culture, e.g., the peak cell density of the cell culture in which the excision and formation steps have not been performed.

[0221] Preferably, the threshold optical density level at which excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules should be initiated or induced after arrival (e.g., immediately after arrival) is 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, or 85-90% of the peak cell density (or maximum cell density, maximum cell density, or cell density plateau) of the cell culture after the excision and formation steps have been performed, or of the peak cell density of the control cell culture, e.g., the peak cell density of the cell culture in which the excision and formation steps have not been performed. For example, the threshold optical density level at which excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules should be initiated or induced after arrival (e.g., immediately after arrival) is 2–5%.

[0222] The optical density levels corresponding to peak cell density or maximum cell density, as described above and elsewhere in this specification, are predetermined but vary from cell to cell under different conditions. Examples include different growth conditions such as culture medium and temperature, or the production of different products. Therefore, using the percentage of the maximum cell density achieved under selected or desired conditions is particularly advantageous in determining when the excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules should be initiated or induced.

[0223] The control cell culture may be, for example, a culture of the cells of the present invention in which excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules are not induced (e.g., do not occur or cannot occur).

[0224] In the embodiment, cells exist within a cell population. In fact, cells are typically part of a cell population, contain a cell population, or consist of a cell population.

[0225] In the preferred method of the present invention, the excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules are performed during the exponential growth phase of the cell population. During the exponential growth phase, the appropriate timing for performing (or inducing) the excision and formation steps can be easily determined, for example, to achieve the desired or maximum product production. For example, in some embodiments, it may be beneficial to perform (or induce) the excision and formation steps at the beginning (or first half) of the exponential growth phase when the cell density is low (or relatively low). Alternatively, in some embodiments, it may be beneficial to perform them later in the exponential growth phase (e.g., the second half, e.g., the end or final stage) when the cell density is high (e.g., higher or significantly higher than at the beginning of the exponential growth phase). The optimal timing for excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules may depend on the final (or maximum) cell density to be achieved. If the desired final cell density is high, it may be desirable to induce excision and formation later in the exponential growth phase; on the other hand, if the desired final cell density is low (or does not need to be very high), excision and formation can be induced earlier in the exponential growth phase.

[0226] It is well understood in the art that cell proliferation in culture can typically be modeled in four distinct phases: (1) the stagnation phase, (2) the exponential phase, (3) the stationary phase, and (4) the death phase. During the stagnation phase, cells adapt to the culture conditions, and cell division is minimal or absent. During the exponential phase, cell division occurs, doubling the cell number with each subsequent phase. The stationary phase is reached by factors that limit proliferation, such as depletion of essential nutrients. During the stationary phase, the rate of cell proliferation and the rate of cell death are equal (for example, neither may be substantially present); that is, the cell number remains nearly constant. During or in the death phase, the number of (live) cells decreases as more cells die.

[0227] In this technology, the timing of the start of the exponential growth phase can be determined in advance (for example, based on knowledge of the growth patterns of cells, cell lines, or cell systems under specific conditions / inoculation levels), or by measuring the optical density of the cell culture (for example, 450 nm (OD)). 450 ), 600nm (OD 600 ), or 650nm (OD 650 It is understood that proliferation can be predicted or understood by regularly monitoring it using methods such as [methods related to growth].

[0228] In the method of the present invention, the process by which cells transition from the proliferation stage to the production stage may be induced (or caused by, or occur simultaneously with, the steps of excising nucleic acid sequences from chromosomes and forming extrachromosomal DNA molecules) as described herein.

[0229] The expression of a target gene (and / or the production of its product, i.e., the desired product) or the induction of the expression of a target gene (and / or the induction of the production of the desired product) can occur at any appropriate time. For example, it can occur simultaneously with, after, or as a result of the formation of extrachromosomal DNA molecules.

[0230] In embodiments, the expression of the target gene (and / or the production of the desired product) may be initiated or induced simultaneously with (or approximately simultaneously with, immediately before, immediately after, or slightly before or slightly after) the excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules.

[0231] The production of a desired product may be initiated, for example, by inducing the production and / or activation of an enzyme or metabolic pathway responsible for the generation of the desired product, or by inducing the production of the product in other ways (for example, if the product is a protein). Preferably, the desired product and / or the enzyme are encoded by a target gene described herein. Therefore, production may be initiated, for example, by inducing the expression of the nucleotide sequence encoding the product (and / or the expression of the target gene).

[0232] The method of the present invention can be considered to include a step of producing a product. In embodiments in which the target gene codes for the product, the step of producing the product may be achieved by expressing the target gene from an extrachromosomal DNA molecule (or by inducing the expression of

[0233] In an embodiment, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication, A process of inducing the expression of the target gene from the above-mentioned extrachromosomal DNA molecule and thereby producing a product (for example, directly producing the product or indirectly producing the product), and, if necessary, The present invention provides a method including a step for recovering the above-mentioned product.

[0234] In an embodiment, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication, A step of inducing the expression of the target gene from the above-mentioned extrachromosomal DNA molecule, The process of producing a product (for example, directly producing a product or indirectly producing a product), and, if necessary, The present invention provides a method including a step for recovering the above-mentioned product.

[0235] Alternatively, the timing of the initiation or induction of the target gene (and / or the initiation or induction of the production of the desired product) may not necessarily be tied to the excision of nucleic acid sequences from chromosomes and the formation of extrachromosomal DNA molecules, and may be carried out at any appropriate time, as long as the cells are able to express the target gene measurably or significantly (or produce the product at a measurable or significant level). For example, in embodiments, product production is initiated or induced during the exponential growth phase of the cell population (or nearly at its initiation, just before, just after, slightly before, or slightly after). (For example, it may be initiated or induced by inducing the production and / or activation of an enzyme or metabolic pathway responsible for the production of the desired product, or by inducing product production in other ways (for example, if the product is a protein, by inducing the expression of the gene or nucleic acid sequence encoding the product). In other words, product production may be induced or carried out, for example, in the first half of the exponential growth phase, or when the cell density has reached or is before reaching 50% of the maximum cell density (or OD). In other embodiments, product production may be induced or carried out, for example, in a later part of the exponential growth phase of a cell population where the cell density is higher (e.g., the latter half of the exponential growth phase), or it may be induced or carried out after the cell density has reached 50% of the maximum cell density (or OD). Preferably, the desired product and / or the enzyme is encoded by a target gene described herein.

[0236] Alternatively, the induction of the expression of the target gene (and / or the production of the desired product) may not necessarily be tied to the timing of the exponential phase. For example, the target gene may be expressed constitutively or endogenously (and / or the product may be produced constitutively or endogenously), in which case the initiation or induction step is not necessarily required. Alternatively, initiation or induction may begin before or after the start of the exponential phase of cell culture growth.

[0237] In alternative embodiments, the expression of the target gene (and / or the production of the desired product) may be initiated or induced at any other point in time during the exponential growth phase, or during another phase, such as the lag phase (for example, it may be possible to induce expression (and / or production) when cells cultured overnight are diluted in fresh medium, or immediately after dilution), or during the stationary phase.

[0238] Therefore, the expression of the target gene (and / or product production) may be initiated or induced at a predicted, convenient, or suitable time after inoculation of cells into the culture medium, or it may be initiated or induced after the optical density of the cell culture reaches a certain or desired threshold level (e.g., immediately after or shortly thereafter).

[0239] Preferably, the threshold optical density level at which the expression of the target gene should be initiated or induced (and / or product production should be initiated or induced) at or after arrival (or immediately after or shortly after arrival) is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% of the peak cell density (or maximum cell density, maximal cell density, or cell density plateau) of the cell culture, the cell culture after excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules, or a control cell culture, e.g., a cell culture in which excision and formation have not been performed.

[0240] Preferably, the threshold optical density level at which the expression of the target gene should be initiated or induced (and / or product production should be initiated or induced) at or after arrival (or immediately after or shortly after arrival) is 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, or 85-90% of the peak cell density (or maximum cell density, maximal cell density, or cell density plateau) of the cell culture, the cell culture after excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules, or a control cell culture, e.g., a cell culture in which excision and formation have not been performed.

[0241] The optical density levels corresponding to peak cell density or maximum cell density, as described above and elsewhere in this specification, are predetermined but vary from cell to cell under different conditions. Examples include different growth conditions such as culture medium and temperature, or the production of different products. Therefore, using the percentage of the maximum cell density achieved under selected or desired conditions is particularly advantageous in determining when product production (and / or expression of the target gene) should be initiated or induced.

[0242] The control cell culture may be, for example, a culture of the cells of the present invention in which excision of nucleic acid sequences from chromosomes and formation of extrachromosomal DNA molecules are not induced (e.g., do not occur or cannot occur).

[0243] It will be understood that in some embodiments, the user does not need to induce the expression of the target gene (and / or the production of the product), and this step may be optional. This is because, for example, the expression of the target gene (and / or the induction or production of the product) occurs automatically at a specific point in time during the culture. For example, this is the case when the production of the product is related to a promoter that is sensitive to internal stimuli or internal conditions. For example, the promoters include the PCP_2836 promoter (Ma et al., 2018, Microbial Cell Factories, BioMed Central Ltd., 17(1), p.185. doi:10.1186 / s12934-018-1031-7.), the phoPR promoter (Paul et al., 2004, Journal of Bacteriology, American Society for Microbiology Journals, 186(13), pp.4262-4275. doi:10.1128 / JB.186.13.4262-4275.2004), and the P170 promoter (Madsen et al., 1999, Molecular Microbiology, Blackwell Publishing Ltd., 32(1), pp.75-87. The promoter may be induced when the cell culture enters a specific growth stage, as in the case of doi:10.1046 / j.1365-2958.1999.01326.x). Preferably, the promoter may be induced when the cell culture enters the exponential growth phase or during the exponential growth phase. Other preferred conditions include self-inducing media (e.g., those described in Studier et al., 2005 (Protein Expression and Purification, 2005, 41:207-234)) and self-inducing systems (e.g., SILEX described in Briand et al., 2016 (Scientific Reports, 6:33037)).

[0244] In embodiments, the expression of the target gene is induced by the formation of an extrachromosomal DNA molecule. More preferably, as in the POP8 design, for example, the target gene is operably ligated to a promoter, preferably a constitutive promoter, as a result of the formation of the extrachromosomal DNA molecule. This is advantageous because it means that the human operator performing this method does not need to actively induce the expression of the target gene (e.g., by inoculation of a substrate). It is also advantageous that the target gene is not expressed while it is present on the chromosome. This is beneficial because constitutive recombinant protein expression has several drawbacks. For example, constitutive recombinant protein expression can deplete resources and thus slow cell proliferation. It can also create selective pressure against high expression, potentially leading to the dominance of non-expressing mutants.

[0245] Alternatively, if the product is produced constitutively, continuously, or constitutively under the control of, for example, a constitutive or endogenous promoter, for example, if the product is an endogenous product (however, constitutive, continuous, or constitutive production is also applicable to the production of heterologous products), the user may not need to induce the production of the product (and / or the expression of the target gene) within the cell. In this case, excision of nucleic acid sequences from chromosomes within the cell and formation of extrachromosomal DNA molecules (and, if necessary, irreversible inactivation of chromosomal origins of replication or all chromosomal origins of replication, whether performed as part of the excision and formation steps or separately from those steps) can increase or enhance the expression level of the target gene (and / or increase the production or level of the desired product), for example, improving the cell titer, yield, and / or productivity in the production of the product.

[0246] These methods may further include steps for obtaining products from cells, for example, from the culture medium (or supernatant) or the intracellular environment of the cells, by means of recovery, isolation, purification, harvesting, or other methods. Methods for carrying out such steps are well known in the art.

[0247] In an embodiment, the present invention is a method for producing a product using cells, The process involves excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell described above. A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication, A step of inducing the expression of the target gene from the above-mentioned extrachromosomal DNA molecule, The present invention provides a method including a step for recovering the above-mentioned product.

[0248] Isolated or purified cells (or populations of cells) are also provided. In some embodiments, such cells are not from or do not correspond to a naturally occurring cell line. Some embodiments include further steps of culturing, growing, or preparing such cell lines or cell lines, and optionally, formulating such cultured, grown, or prepared cell lines or cell lines into a composition (e.g., a stable formulation for product production, e.g., industrial or commercial product production) or pharmaceutical composition containing such cell lines or cell lines. Alternatively, such cells can be preserved for future use, for example, by lyophilization or freezing.

[0249] In another embodiment, the present invention provides cells suitable for use in any aspect or embodiment of the method of the present invention.

[0250] In another embodiment, the present invention provides a cell comprising a chromosome containing a target gene and an origin of replication, wherein the cell (or chromosome) is modified (e.g., genetically modified) such that the target gene and the origin of replication can be excised from the chromosome to form an extrachromosomal DNA molecule containing the excised target gene and the origin of replication (or the target gene and the origin of replication can be excised from the chromosome to form an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication).

[0251] In another embodiment, the present invention provides a cell (or a chromosome within the cell) that is modified to include a chromosome containing a target gene and an origin of replication, wherein the target gene and the origin of replication are excisable from the chromosome, and an extrachromosomal DNA molecule containing the excised target gene and the origin of replication can be formed.

[0252] In another embodiment, the present invention provides a cell comprising a chromosome containing a target gene and an origin of replication.

[0253] In another embodiment, the present invention relates to a cell comprising a chromosome containing a nucleic acid sequence flanked by site-specific recombination sites for recognition by site-specific recombinase, The above nucleic acid sequence includes the target gene, origin of replication, and promoter. The promoter is positioned such that it can be operably linked to the target gene by site-directed recombination using the site-directed recombination site, by site-directed recombination, excision, and / or The present invention provides cells in which the promoter is positioned such that it can be operably linked to a gene for regulating the activity of the origin of replication by site-directed recombination, including excision and circularization of the nucleic acid sequence using the site-directed recombination site.

[0254] In embodiments, the chromosome comprises a nucleic acid sequence flanked by site-specific recombination sites for recognition by site-specific recombinase, the flanked nucleic acid sequence comprising a target gene and an origin of replication. The flanked nucleic acid sequence may include any of the components mentioned in any embodiment or aspect described herein, for example, any feature said to be contained within one or more nucleic acid sequences excised from the chromosome, as mentioned elsewhere in this specification. Preferably, the flanked nucleic acid sequence comprises a promoter, which is positioned such that when the flanked nucleic acid sequence is excised and circularized (for example, to form an extrachromosomal DNA molecule) by site-specific recombination using site-specific recombination sites (i.e., site-specific recombination sites flanking the nucleic acid sequence), the promoter is operably linked to a gene for regulating the activity of the origin of replication and / or to the target gene. Such promoters are also described elsewhere in this specification.

[0255] In one embodiment, the target gene and the origin of replication are flanked by site-specific recombination sites for recognition by site-specific recombinase.

[0256] In embodiments, the site-specific recombination site includes a site-specific recombination site for recognition by serine recombinase, preferably the serine recombinase being γδ, Bxb1, φC31, or TP901, and / or the site-specific recombination site includes a site-specific recombination site for recognition by tyrosine recombinase, preferably the tyrosine recombinase being Cre, Dre, Flp, KD, B2, or B3.

[0257] In the embodiment, the site-specific recombination site comprises one site-specific recombination site and / or comprises two or more pairs of site-specific recombination sites.

[0258] In one embodiment, the cell further comprises a gene encoding a site-specific recombinase.

[0259] In embodiments, a gene encoding a site-directed recombinase (and / or a target gene and / or a gene for controlling the activity of the origin of replication) is operably linked to a promoter. Preferably, the promoter is temperature-sensitive, pH-sensitive, photosensitive, or chemically sensitive, and more preferably, the promoter is regulated by a phage-lambda cl857 repressor.

[0260] In another embodiment, the present invention provides cells suitable for use in the method of the present invention.

[0261] In another embodiment, the present invention provides cells on which the excision and formation steps of the present invention have been performed. Accordingly, the present invention provides cells containing extrachromosomal DNA molecules as defined in any embodiment or aspect shown herein. The cells may also contain one or more chromosomes as defined in any embodiment or aspect shown herein, after the excision and formation steps of the present invention have been performed. For example, the cells may be cells that do not contain a functional origin of replication in any of the (one or more) chromosomes of the cells.

[0262] In another embodiment, the present invention provides a method of any aspect and embodiment described herein, wherein the cells are as defined in any one of the aspects and embodiments described herein.

[0263] The cells of the present invention described herein may also be referred to as modified cells.

[0264] In another embodiment, the present invention provides a polynucleotide vector comprising a target gene, an origin of replication, and site-specific recombination sites flanking the target gene and the origin of replication. This polynucleotide vector can be inserted into cells, for example, by homologous recombination, to produce cells of the present invention that can be used in the methods of the present invention.

[0265] In another embodiment, the present invention provides a use for producing products of any of the embodiments and models described herein of cells or polynucleotide vectors. Exemplary products are as described elsewhere in this specification.

[0266] In the examples herein, E. coli cells are used to demonstrate the present invention. The function of extrachromosomal DNA molecules such as plasmids is also characterized in other bacterial species and eukaryotic cells such as yeast, filamentous fungi, and mammalian cells. Therefore, in embodiments, the cells may be prokaryotic cells (e.g., bacterial cells, preferably E. coli) or eukaryotic cells (e.g., yeast cells, fungal cells (e.g., filamentous fungal cells), or mammalian cells). In such embodiments, the components used are adapted or selected to function in the chosen cell type.

[0267] Certain cells (e.g., eukaryotic cells and certain bacterial cells) may have more than one chromosome. Therefore, when the present invention is applied to cells having multiple chromosomes, the method of the present invention (including the excision and formation steps) is typically applied to one of the chromosomes in the cell. However, in such cells, if the objective of the method is to inactivate all chromosomal origins of replication within the cell, it may be necessary to inactivate (e.g., excise) origins of replication from two or more (usually all) chromosomes within the cell. Similarly, if a cell contains multiple chromosomal origins of replication (e.g., has multiple chromosomes or a single chromosome with multiple origins of replication), it may be necessary to inactivate (e.g., excise) two or more (usually all) chromosomal origins of replication.

[0268] Cells usable in the method of the present invention (i.e., cells capable of performing the excision and formation steps described herein) may be cells modified to obtain this capability. For example, this capability may be conferred by incorporating one polynucleotide sequence (or more polynucleotide sequences) into a chromosome (i.e., cells of the present invention may be produced). For example, the polynucleotide sequence (or more polynucleotide sequences) may include a target gene, an origin of replication, and, optionally, means for excising the polynucleotide sequence (e.g., adjacent site-specific recombination sites). The polynucleotide sequence(s) can be incorporated into cells by standard methods in the art, such as recombineering or homologous recombination, for example, using the phage-lambda Red recombinase system (see, e.g., Progress in Biophysics and Molecular Biology 147 (2019), 33e46) or the PCR and phage-lambda Red recombinase system of Datsenko and Wanner (PNAS, 2000, 97(12), 6640-6645). The cells of the present invention can also be generated using non-homologous site-specific recombination as described elsewhere herein.

[0269] In embodiments, the cells of the present invention are capable of producing a product and / or expressing a target gene as described herein (or having the ability to produce a product, or producing a product, and / or having the ability to express a target gene, or expressing a target gene). For example, a desired product or target gene obtained according to the method of the present invention. Examples of desired products and target genes are described elsewhere in this specification. Thus, cells that produce a product (preferably a product encoded by a target gene as described herein) are a further embodiment of the present invention.

[0270] In embodiments, the cell includes a gene encoding a product or a gene encoding an enzyme required to produce the product. Preferably, the gene of interest encodes the product or, for example, an enzyme for producing the product (e.g., an enzyme in the reaction pathway that produces (or brings about) the production of the product). Suitable genes encoding the product or enzyme are also described elsewhere in this specification. For example, the product (and the gene encoding it) may be an endogenous product, a homologous product, or a heterologous product.

[0271] In embodiments, a gene (e.g., a gene encoding a product, or a target gene) is operably linked to a promoter. Further embodiments of suitable promoters are described elsewhere in this specification (including embodiments described herein in relation to promoters for the control of site-specific recombinases; such embodiments may be applied as embodiments of promoters for the control of a target gene, with necessary modifications), and may be, for example, native or heterologous promoters, and constitutive or inducible promoters.

[0272] In another embodiment, the present invention provides a polynucleotide vector comprising a replication origin and a target gene flanked by site-directed recombination sites, preferably the target gene being a heterologous target gene and / or, preferably the target gene not being a selectable marker. Suitable and preferred site-directed recombination sites are described elsewhere in this specification.

[0273] In embodiments of the method of the present invention, the formation of an extrachromosomal DNA molecule induces the expression (or increased expression) of a target gene.

[0274] In the method for producing the product described in this specification (product production method) (or use in product production), any additional steps necessary for product production can be included. For example, the cells for producing the product can be cultured, grown, or propagated by other methods, or prepared by appropriate methods well-known in the art for the purpose of producing the product (e.g., an appropriate amount or sufficient amount of the product), or growing or expanding the cell population. Thus, these methods may further include the step of growing the cells by culturing, growing, or other methods.

[0275] The cells of the present invention can be manipulated to produce a product of interest, such as a therapeutically effective and useful product, in a useful or significant amount. Therefore, such cells have a clear therapeutic use and can be administered, for example, to a subject or patient, particularly a human patient, for whom administration of the product is therapeutically effective or useful. For example, the use of cells as live pharmaceuticals such as probiotics is recognized in the art, and the use of the cells of the present invention in any therapy for which the use of such live pharmaceuticals is suitable or appropriate is contemplated by the present invention.

[0276] Thus, in another aspect, the present invention provides the cells of the present invention for use in therapy.

[0277] In another aspect, the present invention provides the cells of the present invention for use in the treatment or prevention of a disease or disorder. In a preferred embodiment, the disease or disorder is cancer, a metabolic disease, or an immune disorder.

[0278] In another aspect, the present invention provides the use of the cells of the present invention in the manufacture of a medicament for the treatment or prevention of a disease or disorder. In a preferred embodiment, the disease or disorder is cancer, a metabolic disease, or an immune disorder.

[0279] In another embodiment, the present invention provides a method for treating or preventing a disease or condition, comprising the step of administering an effective amount of the cells of the present invention to a target, preferably the disease or condition being cancer, a metabolic disorder, or an immune disorder.

[0280] In embodiments, the above-mentioned disease or condition is a metabolic disease (or metabolic disorder). Preferably, the metabolic disease (or metabolic disorder) is an acid-base imbalance, metabolic brain disease, calcium metabolism disorder, DNA repair failure, glucose metabolism disorder, hyperlactatemia, iron metabolism disorder, lipid metabolism disorder, malabsorption syndrome, metabolic syndrome, congenital metabolic disorder, mitochondrial disease, phosphorus metabolism disorder, porphyria, proteostasis deficiency, metabolic skin disease, wasting syndrome (or cachexia), or water-electrolyte balance disorder.

[0281] In embodiments, the disease or condition described above is an immune disorder (or immune disease). Preferably, the immune disorder (or immune disease) is an allergy, asthma, autoimmune disease (e.g., lupus, scleroderma, hemolytic anemia, vasculitis, type 1 diabetes, Graves' disease, rheumatoid arthritis, multiple sclerosis, Goodpasture syndrome, pernicious anemia, myopathy, or Lyme disease), autoinflammatory syndrome, or immunodeficiency syndrome.

[0282] As used herein, the term “subject” (or “patient”) includes any mammal, e.g., human and any livestock, domesticated, or laboratory animal. Specifically, examples include mice, rats, pigs, cats, dogs, horses, sheep, rabbits, cattle, and monkeys (or other primates). However, the patient is preferably a human subject.

[0283] In the therapeutic methods and embodiments relating to use described herein, appropriate subjects are those who have, are suspected of having, or are at risk of having (or are susceptible to) the disease or condition to be treated.

[0284] The administration of cells or cell lines in the therapeutic methods and uses of the present invention is carried out in a pharmaceutically, therapeutically, or physiologically effective amount to the subject in need of treatment. Therefore, the methods and uses may include an additional step of identifying the subject in need of treatment.

[0285] Treatment of a disease or condition (e.g., treatment of an existing disease) carried out in accordance with the present invention includes the cure of the disease or condition, or the alleviation or mitigation of the disease (e.g., a reduction in the severity of the disease) or the alleviation or mitigation of the symptoms of the disease.

[0286] The methods and uses of the present invention are suitable for the prevention of disease and the active treatment of disease (e.g., treatment of pre-existing diseases). Therefore, prophylactic treatment is also included in the present invention. For this reason, in the methods and uses of the present invention, treatment includes, as appropriate, prophylaxis or prevention.

[0287] In the embodiment, the cells are bacterial cells, preferably Gram-negative bacterial cells or Gram-positive bacterial cells.

[0288] In the embodiment, the bacterial cells are of the genera Escherichia sp., Bacillus sp., Lactococcus sp., Streptococcus sp., Lactobacillus sp., Corynebacterium sp., Streptomyces sp., Pseudomonas sp., Clostridium sp., Xanthomonas sp., or Enterobacteriaceae.

[0289] In the embodiment, the bacterial cell is of the genus Escherichia coli, and more preferably Escherichia coli.

[0290] The term "plasmid" refers to an extrachromosomal DNA molecule, often in the form of a circular double-stranded DNA. Such elements may be self-replicating sequences, genomic integration sequences, phage sequences, or nucleotide sequences, and may be linear or circular, composed of single-stranded or double-stranded DNA or RNA, of any origin, and may have several nucleotide sequences joined or recombined to form a unique structure that allows for the introduction of a promoter fragment and a target or related DNA sequence (e.g., one relating to a selected gene product) into a cell, along with a suitable 3' untranslated sequence.

[0291] Throughout this application, the terms “a” and “an” are used to mean “at least one,” “at least one,” “one or more,” or “more than one” with respect to the component or process being referenced, unless an upper limit is specifically stated thereafter.

[0292] Furthermore, where the terms “comprise,” “comprises,” “has,” “having,” and other equivalent terms are used herein, in some more specific embodiments these terms include the terms “consists of” or “consists essentially of” and other equivalent terms. Methods including certain steps also include, as appropriate, methods consisting of these steps.

[0293] As used herein, the terms “increase” or “enhance” (or equivalent terms) include a measurable increase or rise compared to a suitable control. Suitable controls can be easily identified by those skilled in the art, and suitable examples are provided herein. Preferably, the increase is significant, for example, clinically or statistically significant, for example, having a probability value of 0.05 or less compared to a suitable control level or value.

[0294] As used herein, the terms “decrease” or “reduce” (or equivalent terms) include a measurable decrease or reduction compared to a suitable control. Suitable controls can be readily identified by those skilled in the art, and suitable examples are provided herein. Preferably, the decrease is significant, for example, clinically or statistically significant, for example, having a probability value of 0.05 or less compared to a suitable control level or value.

[0295] Methods for determining the statistical significance of differences between test groups or differences in the level or value of a particular parameter are well known in the art and described in the literature. For example, in this specification, a decrease or increase is generally considered statistically significant if the probability value is 0.05 or less when statistically compared using an appropriate significance test, such as the Student's t-test, the Mann-Whitney U-rank sum test, the chi-squared test, Fisher's exact test, one-way ANOVA, or two-way ANOVA.

[0296] The present invention will be further described below with reference to the following drawings in the following non-limiting embodiments. Description of the drawing [Brief explanation of the drawing]

[0297] [Figure 1]Figure 1. Design of POP2. The GOI (mRFP1) is inserted into the bacterial chromosome at a specific pre-modified position. In the chromosome, the GOI is located near the oriV, which is the plasmid replication origin, and the trfA gene required for plasmid replication. However, since trfA lacks a promoter and is not transcribed, oriV is inactive. When integrase expression is induced by changing the temperature from 30°C to 37°C, a predetermined fragment between the attP site and the attB site is excised from the genome and circularized to form the POP2 plasmid. During plasmid formation, a functional promoter is placed in front of the trfA gene, the TrfA protein is expressed, and plasmid replication from oriV is initiated. As a result, the plasmid copy number increases and the production of the POI increases. [Figure 2]Figure 2. Excision of the POP2 plasmid increases plasmid copy number and protein expression. A. Determination of copy number ratios of genome amplicons and POP2 plasmid-specific amplicons by qPCR analysis. Plasmid-to-genome copy number ratios were compared in a genome-mRFP1 strain in which plasmid-specific amplicons remain in the genome, a POP2 strain in which the POP2 plasmid is excised from the chromosome upon temperature change, and a wild-type E. coli strain holding a self-replicating mRFP1-KanR plasmid. The ratios of plasmid-specific amplicons to genome-specific amplicons before temperature change and 6 hours after temperature change are shown. The mean and standard deviation of the three biological replications are plotted. B. Evaluation of the protein production capacity of the POP2 strain compared with chromosome expression and conventional plasmid-based expression. Cultures were seeded after diluting the culture 1000-fold overnight and pre-grown in a microplate incubator at 30°C (white plot area). At 0 hours, the temperature was changed to 37°C (gray plot area) to induce excision of the POP2 plasmid in the POP2 strain. The production of fluorescent mRFP1 protein was induced by adding HSL 2 hours after temperature change and monitored by the increase in fluorescence. The mean and standard deviation of three biological replications are plotted. C. Growth curve of mRFP1 protein expression experiment. D. SDS-PAGE analysis of protein expression in wild-type E. coli, genomic mRFP1 strain, POP2, and wild-type E. coli residing mRFP1-KanR. Samples were collected 14 hours after induction of mRFP1 synthesis and visualized on a 12% SDS acrylamide gel. Purified 6His-mRFP1 was used as a control to confirm mRFP1 identity on SDS-PAGE. [Figure 3A] Figure 3. Designs for POP1, POP3A, POP3B, and POP4. In all setups, oriC is removed from the chromosome during plasmid pop-out. A. The POP1 plasmid contains both oriC and trfA. [Figure 3B] In B.POP3A, the plasmid contains the trfA gene, but oriC forms a separate minicircle. [Figure 3CD]In C.POP3B, the trfA gene remains within the chromosome and is transcribed after excision of the POP3B plasmid; oriC forms a separate minicircle. D.POP4 plasmid contains only oriC as the origin of replication. [Figure 4] Figure 4. POP1 yields the highest protein expression level. A. qPCR analysis of plasmid-versus-genome copy number changes in genomic mRFP1, POP1, POP3A, POP3B, and POP4 strains (before plasmid excision (0 hours) and after excision (6 hours)). The mean and standard deviation of three biological replicas are plotted. B. Evaluation of protein production capacity of wild-type E. coli, POP1, POP3A, POP3B, and POP4 strains carrying the mRFP1-KanR plasmid. Cultures were seeded overnight after diluting the culture 1000-fold and pre-grown in a microplate incubator at 30°C (white plot area). At 0 hours, the temperature was changed to 37°C (gray plot area) to induce plasmid excision in each POP strain. Two hours after the temperature change, HSL was added to induce the production of fluorescent mRFP1 protein, which was monitored by the increase in fluorescence. The mean and standard deviation of three biological replicas are plotted. C. Growth curves of mRFP1 protein expression experiments. D. SDS-PAGE analysis of protein expression in wild-type E. coli and POP1, POP3A, and POP3B strains. Samples were collected 14 hours after induction of mRFP1 synthesis and visualized on a 12% SDS acrylamide gel. The identity of mRFP1 was confirmed on SDS-PAGE using purified 6His-mRFP1 as a control. [Figure 5A]Figure 5. All POP designs are stable without antibiotic selection. The stability of each POP strain was estimated by comparing the protein synthesis capacity after 4 passages of 24-hour pre-culture with the protein synthesis capacity after 1 passage of 24-hour pre-culture. A. Growth curves of cultures and fluorescence due to mRFP1 expression after 1 or 4 passages of 24-hour pre-culture. Cultures were seeded after 1000-fold dilution of the 1st or 4th passage and pre-grown in a microplate incubator at 30°C (white plot area). At 0 hours, the temperature was changed to 37°C (gray plot area) to induce plasmid excision in each POP strain. Production of the fluorescent mRFP1 protein was induced by adding HSL 2 hours after the temperature change and monitored by the increase in fluorescence. Culture growth was monitored by measuring the optical density at 650 nm. The mean and standard deviation of two biological replicas are plotted. [Figure 5B] B. SDS-PAGE analysis of mRFP1 protein expression in wild-type Escherichia coli and POP2, POP1, POP3A, and POP3B strains. Samples were collected 14 hours after induction of mRFP1 synthesis. Two biological replicas were visualized on a 12% SDS-acrylamide gel for each strain and each passage condition. [Figure 6] Figure 6. scFv and hFGF-2 protein expression in the POP1 setup. SDS-PAGE analysis of scFv and hFGF-2 protein expression in wild-type E. coli, as well as POP1-scFv and POP1-hFGF-2 strains. Samples were collected 20 hours after induction of protein synthesis and visualized on a 12% SDS-acrylamide gel. [Figure 7] Figure 7. Designs of POP5, POP6, and POP7. A pMB1-type origin of replication lacking an RNAII promoter is positioned next to the attP site. When the fragment between the attP and attB sites is excised from the genome and circularized, a functional promoter is positioned in front of the pMB1-type origin of replication, RNAII is synthesized, and plasmid replication begins from the pMB1-type origin of replication. [Figure 8]Figure 8. POP6 has the highest plasmid-to-genome copy number ratio. A. qPCR analysis of plasmid-to-genome copy number changes in genomic mRFP1, POP5, POP6, and POP7 strains (before plasmid excision (0 hours) and after excision (6 hours)). The mean and standard deviation of two biological replicas are plotted. B. Evaluation of protein production capacity of POP5, POP6, and POP7 strains compared to chromosome expression. Cultures were seeded overnight after diluting the culture 500-fold and pre-grown in a microplate incubator at 30°C (white plot area). At 0 hours, the temperature was changed to 37°C (gray plot area) to induce plasmid excision in each POP strain. Two hours after the temperature change, HSL was added to induce the production of fluorescent mRFP1 protein, which was monitored by the increase in fluorescence. The mean and standard deviation of three biological replicas are plotted. C. Growth curves of mRFP1 protein expression experiments. D. SDS-PAGE analysis of protein expression in wild-type Escherichia coli and genomic mRFP1, POP5, POP6, and POP7 strains. Samples were collected 14 hours after induction of mRFP1 synthesis and visualized on a 12% SDS-acrylamide gel. The identity of mRFP1 was confirmed by SDS-PAGE using purified 6His-mRFP1 as a control. [Figure 9]Figure 9. The POP8 design enables automated induction of GOI from excised POP plasmids. A. POP8 design. The GOI (mRFP1) does not have a promoter when inserted into the genome. The promoter is located downstream and is positioned before the GOI after POP plasmid formation. TrfA is expressed from the same mRNA as the GOI. B. Evaluation of protein production capacity of genomic mRFP1 strain and POP8 strain. Cultures were seeded overnight after diluting the culture 1000-fold and pre-grown in a microplate incubator at 30°C (white plot area). At 0 hours, the temperature was changed to 37°C (gray plot area) to induce plasmid excision and mRFP1 expression in the POP8 strain. Production of fluorescent mRFP1 protein was monitored by the increase in fluorescence. The mean and standard deviation of three biological replications are plotted. C. Growth curves of mRFP1 protein expression experiments. D. SDS-PAGE analysis of mRFP1 protein expression in wild-type E. coli and POP8 strain. Samples were collected 14 hours after induction of mRFP1 synthesis and visualized on a 12% SDS acrylamide gel. [Examples]

[0298] Examples material and method Bacterial strains, plasmids, and growth media The bacterial strains and plasmids are shown in Table 1. E. coli DH5α and BW23473 were used for plasmid cloning and propagation. Genome modification and protein expression experiments were performed in E. coli MG1655. E. coli was grown in lysogenic broth (LB) medium. If plasmid maintenance was necessary during cloning or in the case of plasmid control strains, appropriate amounts of antibiotics (ampicillin 100 μg / ml, chloramphenicol 25 μg / ml, kanamycin 25 μg / ml) were added to the medium.

[0299] DNA manipulation Short oligonucleotide sequences for cloning, sequencing, and qPCR analysis were ordered from Metabion International AG. (Mutant λP) R The codon-optimized sequences for φC31 integrase with the (T41C) promoter, Bxb1 integrase with the λPL promoter, cI857 repressor with the PkatG promoter, attB and attP sites with synthetic promoters, and terminator were ordered from Twist Bioscience as synthetic DNA fragments (Table 2). The trfA and oriV sequences were amplified from plasmid pAJ144. The pMB1 family origins of replication were amplified as follows, excluding the RNAII promoter: the pBR322 ori sequence was amplified from pAJ35 with or without the bom and rop regions; and the pUC ori was amplified from pUC19. The codon-optimized sequences for scFv herceptin (with a His tag at the C-terminus) and hFGF-2 (amino acids 135-288, with 10 His tags and enterokinase cleavage sites at the N-terminus) were ordered from Twist Bioscience as synthetic DNA fragments. These fragments were assembled by overlap extension PCR, gel-purified, and incorporated into chromosomes using the reconciliation method (Datsenko and Wanner, PNAS, 2000, 97(12), 6640-6645). The final modified genomic regions are shown in Figures 1, 3, 7, and 9, and their sequences are shown in Table 3. Plasmid mRFP1-KanR was constructed using the CPEC method [1]. The final sequence is shown in Table 4.

[0300] Measurement of copy number The cultures were incubated overnight at 30°C in a shaking incubator, then diluted 500-fold (POP1-POP4 and their respective control strains) or 250-fold (POP5-POP7 and their respective control strains) in 15 mL of fresh medium, and incubated further at 30°C for 2 hours. Subsequently, the temperature was changed to 37°C to induce POP formation and, where applicable, switching (oriC excision). The point of temperature change was defined as time 0. Culture samples for copy number measurement were collected at time 0 and 6 and stored in a freezer at -20°C. OD650 was measured, and the same amount of biomass (0.8 OD units) was subjected to quantitative PCR analysis. A 0.8-unit cell pellet was resuspended in 150 μL of EDTA buffer (10 mM Tris, 0.1 mM EDTA, pH 8), lysed at 95°C for 10 minutes, and the cell lysate was serially diluted 10-fold, 100-fold, 1000-fold, and 10000-fold with PCR-grade water. The cells were then analyzed by qPCR assay using HOT FIREPol® Probe Universal qPCR Mix (Solis BioDyne). Hydrolysis probes labeled with FAM-BHQ1 and HEX-BHQ1 were designed to detect genome-specific and plasmid-specific DNA sequences, respectively (Table 2). The reaction was performed using a LighCycler 480 System (Roche Diagnostics), mixed and executed according to the manufacturer's recommendations. The plasmid-to-genome copy number ratio was calculated using the following formula. Plasmid-to-genome ratio = 2^(Ct ゲノム -Ct プラスミド ) [2] Plasmid-to-genome ratios were calculated for each dilution and averaged across dilutions.

[0301] Measurement of growth and protein production Cultures of POP strains and control strains were incubated overnight at 30°C in a shaking incubator. They were then diluted 1000-fold (POP1-POP4 and POP8 with their respective control strains) or 500-fold (POP5-POP7 with their respective control strains) in fresh LB medium and incubated for a further 2 hours at 30°C on 96-well plates. The temperature was then changed to 37°C (at hour 0). Two hours after the temperature change, mRFP1 synthesis was induced by adding 1 μM N-(3-oxooctanoyl)-L-homoserine lactone (HSL, Sigma-Aldrich, O1764, final concentration). mRFP1-related fluorescence, reflecting growth curves and protein levels, was measured using a BioTek Synergy H1 plate reader. Optical density at 650 nm and fluorescence intensity (excitation wavelength 580 / 13.5 nm, fluorescence wavelength 610 / 13.5 nm) were measured in 100 μL of culture. The amount of synthesized mRFP1 was estimated using SDS-PAGE. Fourteen hours after induction of protein synthesis (sixteen hours after temperature change), 40 μL samples were taken from the POP culture and control culture, and 10 μL of 5x SDS sample buffer was added. The samples were denatured at 98°C for 10 minutes. 10 μL of the samples were separated on a 12% SDS-PAGE gel and visualized using InstantBlue® Coomassie Protein Stain (Abcam). Cultures of POP1-scFv, POP1-hFGF-2, and a control strain were incubated overnight at 30°C in a shaking incubator. After dilution 500-fold in fresh LB medium, incubation was continued on a 96-well plate at 30°C for 2 hours, then the temperature was changed to 37°C. After incubation at 37°C for 2 hours, the temperature was reduced to 26°C, and 1 μM HSL was added to induce the synthesis of scFv and hFGF-2 proteins. Twenty hours after the induction of protein synthesis, 40 μL samples were taken from each culture and 10 μL of 5x SDS sample buffer was added. The samples were denatured at 98°C for 10 minutes. 10 μL of the samples were separated on a 12% SDS-PAGE gel and visualized using InstantBlue® Coomassie Protein Stain (Abcam). [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0302] result Plasmids must contain a functional origin of replication (ori) to replicate. Many different origins of replication with various regulatory mechanisms have been described to date. These include, but are not limited to, pBR322, pMB1, ColE1, R6K, oriV, p15A, pSC101, and pUC

[10] . Some origins of replication require the presence of specific proteins for replication (in addition to the cellular replication mechanism), and these proteins are usually encoded on the same plasmid. We have taken advantage of this and used oriV (originally derived from the RK2 plasmid) in our design. oriV requires the protein TrfA for replication, and if TrfA is not expressed, oriV is inactive and does not initiate DNA replication.

[0303] The inventors incorporate oriV, the trfA gene, the target gene, and other desired components into the E. coli chromosome (Figure 1). These are flanked by the serine recombinase recognition sites attB and attP. In this configuration, there is no active promoter before the trfA gene, and the trfA gene is not expressed. When the inventors induce the expression of the corresponding serine recombinase in the cell, this catalyzes irreversible recombination between the attB and attP sites, causing the intermediate DNA sequence to be excised by strand exchange (flipping out), and the DNA to be rejoined to form a hybrid attR / attL site

[11] . As a result, a circular plasmid is formed and separated from the bacterial chromosome. This recombination event places a functional promoter before the trfA gene, resulting in the expression of the TrfA protein. TrfA begins replication from oriV, and the plasmid begins replication independently of chromosomal DNA replication. The inventors refer to this setup as a pop-out plasmid (POP), and the design shown in Figure 1 is POP2.

[0304] In these examples, the inventors used serine recombinases derived from phage phiC31 and Bxb1 to construct POPs, but other types of genome editing could also be used for the same purpose. Possible candidates include, but are not limited to, other serine recombinases

[12] , tyrosine recombinases

[13] , CRISPR / Cas

[14] , or any other type of enzyme that permanently removes a desired sequence from a chromosome to form a circular plasmid.

[0305] The activity / expression of serine recombinase (or other types of editing) can be controlled to prevent the plasmid from forming prematurely during cell proliferation and subsequently being lost. For this purpose, we used a temperature-sensitive phage lambda cI857 repressor. At 30°C (or below), cI857 is controlled by the target promoter (P R or P LIt binds to and represses ), but at 37°C (or above), the repression is released and expression is initiated. For the same purpose, other types of gene expression control methods may be used, including, but are not limited to, induction by inducers (IPTG, arabinose, homoserine lactones, anhydrotetracycline, etc.)

[15]

[16]

[17] , targeted proteolysis

[18] , and various modifications of CRISPR / Cas9

[19] .

[0306] The inventors tested POP formation by measuring the copy number of the DNA sequence incorporated into the POP plasmid and comparing it to the copy number of the DNA sequence remaining in the chromosome (Figure 2A). When cultured at 30°C (up to 0 hours), both sequences are part of the E. coli chromosome, and their ratio remains close to 1. After changing the temperature to 37°C and 6 hours after POP2 formation, the plasmid-to-genome ratio increased fivefold, indicating a clear amplification of the POP2 plasmid. Simultaneously, there was no change in copy number in the control strain where both sequences remained on the chromosome. While a ratio of 5 plasmid copies per genome copy may seem low, it is the natural copy number for this particular plasmid configuration. As evidence, the inventors constructed an mRFP1-KanR plasmid similar to the excised POP2 plasmid, transformed wild-type parent E. coli with it, and confirmed that its copy number was similar to that of the excised POP2 plasmid (Figure 2A).

[0307] To evaluate how POP2 formation and copy number increase affect protein production capacity, the inventors used a fluorescence reporter system encoding monomeric red fluorescent protein 1 (mRFP1) under the control of a homoserine lactone (HSL)-inducible promoter. By design, upon a POP2 excision event, the mRFP1 gene becomes part of the POP2 plasmid along with the luxR transcription regulatory gene (Figure 1). mRFP1 production is induced by the addition of HSL to the culture medium and can be monitored by measuring red fluorescence (excitation wavelength: 580 nm, fluorescence wavelength: 610 nm).

[0308] When the mRFP1 gene remains as a single copy within the chromosome, its expression level is very low (Figure 2B). However, when the mRFP1 gene is located on POP2, the fluorescence signal reaches a 10-fold higher level, indicating much higher protein expression at the same culture density (Figures 2B, 2C). In fact, its expression level reaches that of a conventional plasmid (mRFP1-KanR plasmid) that requires antibiotic selection. This is further supported by SDS-PAGE analysis, which shows strong expression in lanes where mRFP1 is located on POP2 (Figure 2D).

[0309] Our expression system does not require selection for maintenance. In the POP design, the GOI remains integrated within the bacterial chromosome during biomass growth and replicates stably in sync with the rest of the chromosome. When the POP plasmid is excised at the end of the growth phase, it replicates until it reaches the copy number specified by TrfA. However, because there are not enough cell divisions remaining, the POP plasmid is not lost due to incomplete distribution or lack of selection.

[0310] If cells continue to divide for a sufficiently long time after POP formation, plasmids may be lost from the cells. To prevent this, the inventors combined POP technology with their patent-pending switcher technology (Patent Application WO2022117827A1)

[20] , which enables the maintenance of a metabolically active state while halting cell division and proliferation. The switcher technology is based on the controlled removal of oriC from chromosomes using serine recombinase. By combining these two technologies, two different designs, POP1 and POP3, were obtained.

[0311] In POP1, both POP and oriC are removed from the chromosome as a single unit (Figure 3A). The resulting plasmid contains both oriC and an active TrfA-regulated oriV origin of replication. In contrast, in POP3, the excision of oriC and POP is two separate events performed by the same serine recombinase (Figures 3B, 3C). The POP3 plasmid contains only oriV and no oriC. In POP3A, the trfA gene is transferred to the POP plasmid, while in POP3B it remains on the chromosome. Regardless of the position of the trfA gene at POP induction, an active promoter is incorporated ahead of it, inducing TrfA protein expression. In POP4, no plasmid-specific origin of replication is introduced, and only the chromosomal oriC becomes part of the pop-out plasmid, driving its replication (Figure 3D).

[0312] To evaluate the copy number changes in POP1, POP3, and POP4, the inventors performed qPCR-based measurements as described above. Six hours after temperature change, a significant increase in the copy number-to-genome ratio was observed in all POP versions (Figure 4A). This demonstrates chromosome-independent replication of the POP plasmid. The magnitude of this effect is at least partially due to a decrease in the relative abundance of the genome. That is, after removal of oriC, cells grow in length and biomass increases, but the genome remains at one copy per (elongated) cell.

[0313] An increase in copy number also leads to an increase in protein expression levels. At similar culture densities (Figure 4C), the highest mRFP1 expression levels were measured in the POP1 strain, followed by POP3A and POP3B (Figure 4B). Expression in POP4 remained at a similar level to that of the control plasmid. This expression was further supported by SDS-PAGE analysis (Figure 4D).

[0314] The increased protein expression levels in POP1 and POP3 (compared to POP2) may be achieved by two factors. The first factor is that the gene quantity increases due to the increase in copy number in POP1 and POP3 strains. The second factor is that protein expression increases in cells from which oriC has been removed, even under otherwise identical conditions

[20] . The high plasmid copy number-to-genome ratio in POP4 does not lead to a similar increase in mRFP1 expression. The exact reason for this remains unclear.

[0315] Long-term stability is a crucial characteristic for any protein expression system used in industrial setups. Many cell doublings can occur before sufficient biomass is accumulated in large-scale production. The inventors mimicked the number of doublings required for mass production by performing daily continuous passaging with 1000-fold dilutions for four consecutive days. These passagings were performed without selection. The inventors tested the stability of the POP system by comparing protein expression in the 4-day passaged culture with that of a single passaged culture (Figure 5). In both cultures, the expression levels of the reporter protein mRFP1 were very similar, demonstrating the good stability of the POP expression system (Figure 5B). Similar results were confirmed by SDS-PAGE analysis (Figure 5C).

[0316] In conclusion, by combining the genomic stability of POPs, the high copy number of GOIs on demand, and the physiological characteristics of switcher cells, a high-level protein expression system that does not require antibiotics can be obtained.

[0317] The inventors constructed the POP system using E. coli, but this system should also function with other bacteria, as well as eukaryotes such as yeast, filamentous fungi, and mammalian cells.

[0318] To confirm that the high protein production using the POP system is not purely mRFP1-specific, we tested the expression of two additional recombinant proteins in the POP1 design setup. The mRFP1 gene in the POP1 strain was replaced with either a single-chain fragment variable antibody (scFv Herceptin) or a gene encoding recombinant human fibroblast growth factor 2 (hFGF-2). Both proteins showed strong expression in the POP system (Figure 6).

[0319] The inventors verified that the concept of POP is not limited to plasmid types whose replication depends on the Rep protein, such as the RK2 plasmid containing TrfA. In the POP1 design, the trfA gene and RK2 oriV were replaced with a pMB1 type origin of replication (G. Cesareni, DW Banner, Regulation of plasmid copy number by complementary RNAs, Trends in Biochemical Sciences, Vol. 10, No. 8, 1985, pp. 303-306). The pMB1 origin of replication was incorporated into the genome without placing a promoter before the RNAII gene (Figure 7). If RNAII is not synthesized, the pMB1 origin of replication is not activated. Once the POP plasmid is formed, a strong constitutive promoter induces RNAII transcription, thereby enabling plasmid replication. The inventors constructed three POP strain variants to test the pMB1 type replication origin: the POP5 design included the pBR322 replication origin, the POP6 design included the pBR322 replication origin and the copy number control elements bom and rop, and the POP7 design included the pUC replication origin (Figure 7).

[0320] To determine the plasmid-to-genomic DNA copy number ratio in POP5, POP6, and POP7 strains, the inventors performed qPCR-based measurements as described above. Within 6 hours of temperature change, a significant increase was observed in all plasmid-to-genomic ratios (Figure 8A), with the highest ratio measured in POP6. As expected, the increase in plasmid copy number also led to an increase in protein expression levels compared to genome expression. However, despite a twofold difference in plasmid copy number among the POP strains, the expression level of the mRFP1 protein remained similarly high at similar culture densities (Figure 8C) (Figure 8B). This was also confirmed by SDS-PAGE analysis (Figure 8D). In this situation, it is likely that cellular physiological factors other than plasmid copy number limit protein production capacity. In conclusion, these findings demonstrate that the POP concept functions appropriately for the production of various proteins and can be designed using different plasmid replication mechanisms.

[0321] We were able to design a novel method for inducing protein expression through DNA rearrangement during plasmid formation. In the POP8 strain, there is no promoter in front of the mRFP1 gene on the genome (Figure 9A). When the plasmid is excised from the genome and circularized, a constitutively active promoter is positioned in front of the mRFP1 gene, and protein expression is initiated. After temperature changes, strong mRFP1 expression was detected by fluorescence measurement (Figure 9B) and also by SDS-PAGE (Figure 9D). This setup allows the use of any promoter as an inducible promoter, eliminating the need for small molecule inducers such as IPTG.

[0322] References 1. Gutterson NI, Koshland DE. Replacement and amplification of bacterial genes with sequences altered in vitro. Proceedings of the National Academy of Sciences of the United States of America. 1983;80: 4894. doi:10.1073 / PNAS.80.16.4894 2. Hersh MN, Ponder RG, Hastings PJ, Rosenberg SM. Adaptive mutation and amplification in Escherichia coli: two pathways of genome adaptation under stress. Research in Microbiology. 2004;155: 352-359. doi:10.1016 / J.RESMIC.2004.01.020 3. Andersson DI, Hughes D. Gene Amplification and Adaptive Evolution in Bacteria. https: / / doi.org / 101146 / annurev-genet-102108-134805. 2009;43: 167-195. doi:10.1146 / ANNUREV-GENET-102108-134805 4. Mignon C, Sodoyer R, Werle B. Antibiotic-Free Selection in Biotherapeutics: Now and Forever. Pathogens 2015, Vol 4, Pages 157-181. 2015;4: 157-181. doi:10.3390 / PATHOGENS4020157 5. Oliveira PH, Mairhofer J. Marker-free plasmids for biotechnological applications - implications and perspectives. Trends in Biotechnology. 2013;31: 539-547. doi:10.1016 / J.TIBTECH.2013.06.001 6. Wild J, Hradecna Z, Szybalski W, Blattner FR, Helinski D, Valla S, et al. Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones. Genome Research. 2002;12: 1434-1444. doi:10.1101 / GR.130502 7. Wild J, Szybalski W. Copy-Control Tightly Regulated Expression Vectors Based on pBAC / oriV. Methods in molecular biology (Clifton, NJ). 2004;267: 155-167. doi:10.1385 / 1-59259-774-2:155 8. Yoon YG, Cho JH, Kim SC. Cre / loxP-mediated excision and amplification of large segments of the Escherichia coli genome. Genetic Analysis: Biomolecular Engineering. 1998;14: 89-95. doi:10.1016 / S1050-3862(97)10005-5 9. Posfai G, Koob M, Hradecna Z, Hasan N, Filutowicz M, Szybalski W. In vivo excision and amplification of large segments of the Escherichia coli genome. Nucleic Acids Research. 1994;22: 2392-2398. doi:10.1093 / NAR / 22.12.2392 10. Solar G del, Giraldo R, Ruiz-Echevarria MJ, Espinosa M, Diaz-Orejas R. Replication and Control of Circular Bacterial Plasmids. Microbiology and Molecular Biology Reviews. 1998;62: 434. doi:10.1128 / MMBR.62.2.434-464.1998 11. Merrick C, Zhao J, Rosser S. Serine Integrases: Advancing Synthetic Biology. ACS Synthetic Biology. 2018. doi:10.1021 / acssynbio.7b00308 12. Yang L, Nielsen AAK, Fernandez-Rodriguez J, McClune CJ, Laub MT, Lu TK, et al. Permanent genetic memory with >1-byte capacity. Nature Methods. 2014;11: 1261-1266. doi:10.1038 / nmeth.3147 13. Fogg PCM, Colloms S, Rosser S, Stark M, Smith MCM. New Applications for Phage Integrases. Journal of Molecular Biology. 2014;426 VN-: 2703-2716. doi:10.1016 / j.jmb.2014.05.014 14. Yao R, Liu D, Jia X, Zheng Y, Liu W, Xiao Y. CRISPR-Cas9 / Cas12a biotechnology and application in bacteria. Synthetic and Systems Biotechnology. KeAi Communications Co.; 2018. pp. 135-149. doi:10.1016 / j.synbio.2018.09.004 15. Lutz R, Bujard H. Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR / O, the TetR / O and AraC / I1-I2 Regulatory Elements | Nucleic Acids Research | Oxford Academic. Nucleic Acids Research. 1997;25: 1203-1210. 16. Marschall L, Sagmeister P, Herwig C. Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints. Applied Microbiology and Biotechnology. 2017;101: 501-512. doi:10.1007 / s00253-016-8045-z 17. Cox RS, Surette MG, Elowitz MB. Programming gene expression with combinatorial promoters. Mol Syst Biol. 2007;3: 145. doi:10.1038 / msb4100187 18. Cameron DE, Collins JJ. Tunable protein degradation in bacteria. Nat Biotechnol. 2014;32: 1276-1281. doi:10.1038 / nbt.3053 19. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-γuided platform for sequence-specific control of gene expression. Cell. 2013;152: 1173-1183. doi:10.1016 / j.cell.2013.02.022 20. Kasari M, Kasari V, Karmas M, Joers A. Decoupling Growth and Production by Removing the Origin of Replication from a Bacterial Chromosome. ACS Synthetic Biology. 2022;11: 2610-2622. doi:10.1021 / ACSSYNBIO.1C00618 / SUPPL_FILE / SB1C00618_SI_010.TXT

Claims

1. A method of producing products using cells, The process of excising one or more nucleic acid sequences containing the target gene and the origin of replication from the chromosome within the cell, A step of forming an extrachromosomal DNA molecule containing the excised target gene and the excised origin of replication, A method comprising the step of inducing the expression of the target gene from the extrachromosomal DNA molecule.

2. The method according to claim 1, wherein the expression of the target gene from the extrachromosomal DNA molecule is induced by the formation of the extrachromosomal DNA molecule.

3. The method according to claim 1, wherein the expression of the target gene from the extrachromosomal DNA molecule is induced simultaneously with or after the formation of the extrachromosomal DNA molecule.

4. The method according to any one of the preceding claims, wherein the target gene is a heterologous target gene.

5. The method according to any one of the preceding claims, wherein the target gene encodes the product or an enzyme required to produce the product.

6. The method according to any one of the prior claims, wherein the one or more nucleic acid sequences do not include a selectable marker.

7. The method according to any one of the preceding claims, wherein the one or more nucleic acid sequences include a second origin of replication.

8. The method according to claim 7, wherein the extrachromosomal DNA molecule includes the excised second origin of replication.

9. The method according to any one of the preceding claims, wherein the replication origin or the second replication origin is an endogenous or non-inducible replication origin.

10. The method according to any one of the preceding claims, wherein, as a result of the method, the chromosome does not contain a functional origin of replication.

11. The method according to any one of the preceding claims, wherein the excision step and the forming step are performed simultaneously or sequentially.

12. The method according to any one of the preceding claims, wherein the extrachromosomal DNA molecule is a plasmid.

13. The method according to any one of the preceding claims, wherein the one or more nucleic acid sequences is a single nucleic acid sequence.

14. The method according to any one of the preceding claims, comprising the step of inducing replication of the extrachromosomal DNA molecule.

15. The method according to claim 14, wherein the replication of the extrachromosomal DNA molecule is induced by the formation of the extrachromosomal DNA molecule.

16. The method according to claim 14 or claim 15, wherein the excision step and the forming step are performed simultaneously or sequentially with one or both of the induction steps.

17. The method according to any one of the preceding claims, wherein the cells are present within a cell population, the excision step and / or the formation step are performed during the exponential growth phase of the cell population, preferably, the cells are bacterial cells present within a bacterial cell population, and the excision step and / or the formation step are performed during the exponential growth phase of the bacterial cell population.

18. The method according to any one of the preceding claims, wherein the origin of replication and / or the second origin of replication is non-inducible, preferably an origin of replication on a chromosome, i.e., an oriC, and more preferably an oriC of Escherichia coli.

19. The method according to any one of the preceding claims, wherein the origin of replication and / or the second origin of replication is inducible, preferably an origin of replication or oriV on a plasmid, and more preferably an origin of replication of plasmid RK2, pBR322, pMB1, ColE1, R6K, p15A, pSC101 or pUC or their oriV.

20. The method according to any one of the preceding claims, comprising the step of recovering the aforementioned product.

21. A cell containing a chromosome that includes a nucleic acid sequence flanked by site-specific recombination sites for recognition by site-specific recombinase, The nucleic acid sequence includes the target gene, the origin of replication, and the promoter. The promoter is positioned so as to be operablely linked to the target gene by site-directed recombination using the site-directed recombination site, by site-directed recombination, excision and circularization of the nucleic acid sequence, and / or A cell in which the promoter is positioned so as to be operably linked to a gene for regulating the activity of the origin of replication by site-directed recombination, including excision and circularization of the nucleic acid sequence using the site-directed recombination site.

22. The cell according to claim 21, wherein the site-specific recombination site includes a site-specific recombination site for recognition by serine recombinase, preferably the serine recombinase being γδ, Bxb1, φC31, or TP901, and / or the site-specific recombination site includes a site-specific recombination site for recognition by tyrosine recombinase, preferably the tyrosine recombinase being Cre, Dre, Flp, KD, B2, or B3.

23. The cell according to claim 21 or claim 22, wherein the site-specific recombination site comprises a pair of site-specific recombination sites, and / or the site-specific recombination site comprises two or more pairs of site-specific recombination sites.

24. The cell according to any one of claims 21 to 23, further comprising a gene encoding the site-specific recombinase.

25. The cell according to claim 24, wherein the gene encoding the site-specific recombinase is operably linked to a promoter, preferably the promoter is temperature-sensitive, pH-sensitive, photosensitive, or chemosensitive, and more preferably the promoter is regulated by the phage-lambda cl857 repressor.

26. The method according to any one of claims 1 to 20, wherein the cells are as defined in any one of claims 21 to 25.

27. A polynucleotide vector comprising a target gene, an origin of replication, and site-specific recombination sites flanking the target gene and the origin of replication, wherein the polynucleotide vector comprises a nucleic acid sequence flanked by site-specific recombination sites for recognition by site-specific recombinase as defined in any one of claims 21 to 23.

28. Use of a cell according to any one of claims 21 to 25 or a polynucleotide vector according to claim 27 for producing a product.

29. Cells for use in the treatment or prevention of a disease or condition, preferably the disease or condition being cancer, metabolic disease, or immune disorder, according to any one of claims 21 to 25.

30. The cell is a bacterial cell, preferably a member of the genera Escherichia coli, Bacillus, Lactococcus, Streptococcus, Lactobacillus, Corynebacterium, Streptomyces, Pseudomonas, Clostridium, Xanthomonas, or Enterobacteriaceae, according to any one of claims 1 to 29, cell, polynucleotide vector, use, or cell for use.

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