Plasmids with an increased replication rate

Abstract

The invention relates to a plasmid vector comprising a) an origin of replication; and b) a PAS region, comprising i) a primosomal assembly sequence PAS-forward (SEQ ID NO: 2), which is located 3´ of the origin of replication and in the direction of replication; ii) a primosomal assembly sequence PAS-reverse (SEQ ID NO: 3), which is located 3´ of the primosomal assembly sequence PAS-forward and in the direction opposite the replication; and iii) a sequence X between the primosomal assembly sequence PAS-forward and the primosomal assembly sequence PAS-reverse, said sequence sharing a sequence identity of at most approximately 75% with SEQ ID NO: 1.

Description

[0001] Plasmids with an increased replication rate

[0002] The present invention relates to a plasmid vector comprising a) a replication origin; and b) a PAS region comprising i) a primosome assembly sequence PAS-forward (SEQ ID NO:2) located 3' to the replication origin and in the direction of replication; ii) a primosome assembly sequence PAS-reverse (SEQ ID NO:3) located 3' to the primosome assembly sequence PAS-forward and in the direction opposite to replication; and iii) a sequence X between the primosome assembly sequence PAS-forward and the primosome assembly sequence PAS-reverse, which has at most about 75% sequence identity with SEQ ID NO:1.

[0003] The principle behind many new therapies is the use of genetic information to synthesize a drug in a target cell. For this purpose, the genetic information is provided in the form of a target gene. To achieve efficient transfer of the target gene into a target cell, the gene to be transferred must first be synthesized in sufficient quantity. Plasmid DNA (pDNA) or plasmid vectors represent flexible and easily modifiable sources of recombinant DNA for a wide variety of research, industrial, and pharmaceutical applications. Particularly in the fields of modern medicine and gene therapy, plasmids serve, for example, as important raw materials in the production of mRNA or viral vectors, and can also be used as active ingredients themselves, for instance, in the form of DNA vaccines.With the development and application of new technologies, the demand for high-quality plasmid DNA has increased significantly in recent years. Since the supply of large quantities of highly purified plasmid DNA is often a limiting factor, improving existing processes for plasmid DNA production is of central importance.

[0004] Plasmid DNA is classically obtained via fermentation using recombinant bacteria, such as Escherichia coli. For this purpose, a selected bacterial strain is transformed with a plasmid carrying the target gene and then cultured under suitable conditions. During bacterial division and multiplication, the bacteria replicate and amplify the contained plasmid and thus the target gene it carries. This process is called cloning and allows for the efficient and sequence-accurate duplication of any target gene. The frequency and efficiency of plasmid replication determine the amount of plasmid that can be obtained from each individual bacterial cell. These parameters depend, among other things, on the properties of the bacterial cell, but especially on the properties of the plasmid used.

[0005] Plasmids contain regulatory elements that are recognized by proteins of the respective host cell and lead to the expression of cis-acting effectors. A DNA sequence containing the elements necessary for replication is called the origin of replication. The regulatory elements encoded within the origin of replication can be either protein or RNA effectors. Regardless of their molecular structure, however, the mechanism of action is similar: One element assumes a positive regulatory function and initiates replication. A second element influences the expression or function of the first element and represses replication. The relative abundance of the two elements in the cell influences the number of plasmid copies in that cell.The copy number of plasmids containing, for example, the pMBl or the closely related ColEl origin of replication, is influenced by the interaction of the complementary RNAs RNAI1I and RNAI2I. RNAI1I is a 553 base pair RNA whose transcription is coordinated by the relatively weak RNAI1I promoter. After transcription, RNAI1I binds to the homologous sequence on the plasmid and initiates replication in a primer-like manner. RNAI2I is a 108 base pair RNA completely homologous to RNAI1I, whose expression is coordinated by the relatively strong RNAI2I promoter. Through interaction of the homologous regions of RNAI1I and RNAI2I, the former inhibits the binding of the latter to the plasmid and thus prevents replication initiation. This interaction is stabilized by the gene product of the rop (repressor of primer) gene, which is encoded on many plasmids.Since the promoter of RNAI is stronger than the promoter of RNAI I, an increasing number of plasmid copies leads to an overabundance of RNAI, resulting in a complete blockade of replication initiation. The number of plasmid copies is limited by this regulatory mechanism. To achieve a higher number of plasmid copies and thus more efficient replication of the target gene on a plasmid, the regulatory mechanism of plasmid replication must be adapted to maximize plasmid replication without creating an excessive metabolic burden for the bacterial cells used for cloning. To avoid plasmid loss due to increased metabolic burden, lower feeding rates are used in the initial bulking phase of fermentation in some pDNA production processes.These adjustments, however, result in a longer fermentation time and thus a longer overall production process. To further increase production efficiency and thus reduce production costs in plasmid production, in addition to increasing the plasmid titer, shortening the fermentation and process time is also suitable.

[0006] Currently, plasmid DNA is typically produced in bacteria through a fermentative process. To achieve the highest possible titers of plasmid DNA, and thus of the target gene, so-called "high-copy" plasmids such as pUC19 or pVAXl are used. These plasmids represent variants of naturally occurring plasmids that have arisen either through random mutagenesis and selection or through directed design. Fundamental principles for creating high-copy plasmids are based on disrupting the interaction between regulatory elements or adjusting their relative concentrations within the cell. Furthermore, the integration of replication-enhancing sequences has also proven to be an effective means of increasing the number of plasmids produced.

[0007] The number of copies can be identified.

[0008] Many plasmids used for cloning therapeutically relevant target genes, such as pUC19, pVAXl, or pBluescript, possess a variant of the pMBl origin of replication, the so-called pUC origin. These pUC plasmids differ from pMBl plasmids, i.e., plasmids with a pMBl origin such as pBR322, by the absence of the coding sequence for the regulatory protein Rop. Consequently, the Rop protein, which stabilizes the interaction between RNAI and RNAI1I and thus increases the effectiveness of RNAI, is missing (Tomi zawa et al. Control of ColEl plasmid replication: enhancement of binding of RNAI to the primer transcript by the Rom protein. Cell. 1984 Oct; 38(3): 871-8). The weakening of the RNAI / RNAI I interaction due to the absence of Rop thus has a positive effect on the number of plasmid copies in the cell.Furthermore, the RNAI I encoded on pUC plasmids possesses a single base exchange that causes a structural change in RNAII at temperatures above 37 °C. This structural change reduces the affinity of RNAI for RNAII, thus requiring a greater predominance of RNAI to halt replication. This reduced binding affinity of the regulatory RNAs can therefore lead to an increased plasmid copy number (Muesing et al.; A single base-pair alteration is responsible for the DNA overproduction phenotype of a plasmid copy-number mutant. Cell. 1981 Apr;24 (l):235-42). As a result, a greater number of plasmids, and thus a higher overall plasmid titer, can be obtained from each individual cell in a single production process. Due to the temperature-dependent increase in plasmid copy number described above, a cultivation temperature of 37 °C is used in many state-of-the-art fermentation processes.

[0009] Deregulation of the RNAI / RNAII interaction is a highly effective means of increasing plasmid copy number. While plasmids with the pMBl origin of replication have a copy number of 10–20 per cell, the copy number of pUC plasmids is several hundred per cell (Shao B et al. Single-cell measurement of plasmid copy number and promoter activity. Nat Commun. 2021 Mar 5; 12 (1): 1475). Because of this beneficial effect, many known high-copy plasmids contain adaptations that further weaken the interaction between RNAI and RNAII, either additionally or in other ways.

[0010] In addition to the positively regulatory RNAII transcript, the RNAI transcript can also be affected by structural changes that lead to an increase in the plasmid copy number per cell. A spontaneous single-base exchange in the RNAI terminator region results in a significant increase in the plasmid copy number in plasmid pBR322 and the closely related plasmid ColEl. This exchange results in impaired termination of the RNAI transcript, leading to the formation of a considerably longer RNA molecule instead of the 104-base-pair RNAI. Due to the altered tertiary structure of RNAI, binding to RNAII is reduced, resulting in an increase in the plasmid copy number (Boros et al. High-copy-number derivatives of the plasmid cloning vector pBR322. Gene. 1984 Oct;30 (l-3):257-60).

[0011] Substitutions in the overlap region of RNAI and RNAII can also influence plasmid copy number. The interaction between RNAI and RNAII is largely coordinated by bases located at the tips of three stem loops, situated in the 3' region of the RNAI transcript and the 5' region of the RNAI1 transcript, respectively. EP1326989 describes the isolation of plasmid variants exhibiting substitutions in stem loop 2 (inc2) through undirected mutagenesis and subsequent selection. These plasmid variants showed a significant increase in plasmid copy number compared to the parental variant, which is attributed to a reduced affinity between the regulatory transcripts.

[0012] In addition to the base substitutions described above, point mutations in stem loop 1 (incü) and stem loop 3 (incl) are also known. All these substitutions are located in the overlap region of RNAI and RNAII, thus maintaining the complementarity of the corresponding transcripts. Nevertheless, due to the lower free energy of AU interactions compared to GC interactions, they lead to a reduction in the interaction strength and thus the affinity (Trivedi et al. High-Level Production of Plasmid DNA by Escherichia coli DH5a ShsacB by Introducing inc Mutations. Appl Environ Microbiol. 2014 Dec;80 (23):7154-60). Besides the interaction strength between RNAI and RNAI1, another important factor for the plasmid copy number is their relative availability. This is determined, among other things, by the relative strength of the corresponding promoters.Replacing the relatively weak RNAI1 promoter with a promoter of higher activity leads to an increase in the plasmid copy number (Castelloni et al. Analysis of dominant copy number mutants of the plasmid pMBl. Nucleic Acids Res. 1985 Jul 25; 13(14): 5353-67). An extreme form of adjustment of the relative transcript amount involves the complete removal of RNAI-based regulation. This is possible because the RNAI / RNAI1 overlap region is involved in plasmid replication in a regulatory, but not functional, way. Both the 108-base-pair-long RNAI gene and the 36-base-pair-long promoter region can be deleted. In this case, the plasmid copy number present in the cell is determined only by the strength of the RNAI1 gene. The activity of the RNAI I promoter is regulated, which, if an excessively strong RNAI I promoter is used, leads to the occurrence of cell-damaging, so-called "runaway plasmid replication".The use of inducible promoters allows for inducible control of the plasmid copy number by producing only small amounts of RNAI1 transcript in an early, non-induced phase, thereby preventing runaway plasmid replication (Panayotos, DNA replication regulated by the priming promoter. Nucleic Acids Res. 1984 Mar 26; 12(6): 2641-8).

[0013] Another possibility for the controllable reduction of the amount of active RNAI transcript lies in the use of a competitive target sequence in the form of an antisense RNA. This antisense RNA corresponds to the sequence complementary to RNAI and thus ultimately to the 108 base pair long 5' region of RNAI I. This principle was applied very early on to increase the plasmid copy number of pBR322 (Bachvarov et al. Construction of a ColEl plasmid bearing inducible high-copy-number phenotype. Folia Microbiol (Praha). 1990; 35(3): 177-82). By integrating an additional, truncated RNAI I gene, which was placed under the control of an inducible promoter, the copy number of pBR322 could be increased fourfold after induction. The inducible, truncated RNAI I gene can alternatively be expressed not from the same plasmid, but from a second, non-target plasmid or the bacterial chromosome.

[0014] An increase in the plasmid replication rate can also be achieved by overexpression of genes whose gene products catalyze the synthesis of nucleoid biosynthesis precursors. The chromosomal integration of the genes rpiA and zwf in DH5a is associated with increased plasmid replication rates. Such adaptations to the production process require the use of genetically modified microorganisms. These genomic adaptations can sometimes lead to changes in the relative molar concentrations of the nucleotides present in the cell. The establishment of imbalances within the naturally occurring nucleotide pool is associated with greatly increased mutation rates in bacterial cells.

[0015] The integration of reproduction-enhancing elements on the plasmid backbone has also been successfully used to increase the plasmid copy number. Deletion analysis has shown that the SV40 enhancer sequence is critical for the increased plasmid titers that can be achieved with the plasmid pNTCUltra (Williams et al. Generic plasmid DNA production platform incorporating low metabolic burden seedstock and fed-batch fermentation processes. Biotechnol Bioeng. 2009 Aug 15; 103(6): 1129-43). The integration of this SV40 enhancer sequence enables plasmid titers that are two times higher than those of other high-copy plasmids such as pVAXl (Thermo Fisher Scientific) or gWi z (Genlantis).These modified plasmid backbones, due to their improved maximum plasmid titers, contribute to the more economical production of individual plasmids, but require a long fermentation process to achieve maximum production capacity. To enable more efficient production, for example of pharmaceutically relevant plasmids, shortening the fermentation process time is of great importance.

[0016] Specially adapted plasmid backbones or plasmid vectors, which allow for high plasmid titers and high replication rates without the need to specifically adapt bacterial strains, are therefore of great advantage for the production of plasmid DNA.

[0017] One object of the invention is to provide plasmid vectors that enable high replication rates with substantially unchanged maximum plasmid titers. An alternative object of the present invention is to provide plasmid vectors that enable the maximum production rate to be reached earlier with substantially unchanged maximum plasmid titers. Another alternative object of the present invention is to provide plasmid vectors that enable the maximum relative plasmid titers to be reached earlier with substantially unchanged maximum plasmid titers.

[0018] The problems of the invention are solved by a plasmid vector comprising a) a replication origin; and b) a PAS region comprising i) a primosome assembly sequence PAS-forward (SEQ ID NO: 2) which is localized 3' to the replication origin and in the direction of replication; ii) a primosome assembly sequence PAS-reverse (SEQ ID NO: 3) which is localized 3' to the primosome assembly sequence PAS-forward and in the direction opposite to replication; and iii) a sequence X between the primosome assembly sequence PAS-forward and the primosome assembly sequence PAS-reverse which has at most about 75% sequence identity with SEQ ID NO: 1.

[0019] The plasmid vector according to the invention comprises a plasmid backbone into which a specific DNA sequence, namely a specific PAS region, has been inserted 3' of the origin of replication. The integration of this specific PAS region enables the maximum production rate to be reached earlier, with essentially unchanged high plasmid titers. Thus, with plasmids possessing the PAS region according to the invention, up to approximately 90% of the maximum plasmid titer can be achieved after approximately 24 hours of fermentation, whereas with analogous plasmids lacking the PAS region according to the invention, usually no more than approximately 60% of the maximum plasmid titer is reached after 24 hours of fermentation.

[0020] Thus, the present invention enables a reduction in fermentation time at very high, economically satisfactory plasmid titers by increasing the replication rate. This allows the fermentation process to be shortened by approximately half the time typically required in the prior art without significant loss of product yield. This enables both the more economical production of individual plasmids and increased flexibility within a production plant.

[0021] Definitions

[0022] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as they would normally be understood by a person skilled in the field of the present invention. Any technical feature mentioned in the following definitions can be applied to any embodiment of the invention.

[0023] The terms “nucleic acid sequence”, “nucleotide sequence”, “DNA sequence” and “RNA sequence” are known to a person skilled in the art and refer to an individual and specific sequence of nucleotides.

[0024] The term "nucleic acid" refers to a sequence of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-stranded and double-stranded DNA or RNA, genomic DNA, cDNA, mRNA, saRNA, gRNA, siRNA, miRNA, or circRNA, which may include purine and pyrimidine bases, nucleotide analogs, or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0025] The terms "plasmid vector," "plasmid," and "plasmid DNA" are used interchangeably herein and describe a construct of extrachromosomal genetic material, preferably a circular DNA duplex molecule, that can replicate in a cell independently of chromosomal DNA. The term "plasmid vector" is used in its broadest sense and encompasses any vehicle for a nucleic acid or target gene that, for example, allows the nucleic acid or target gene to be introduced into prokaryotic and / or eukaryotic host cells and, if necessary, integrated into a genome. Plasmid vectors can be, for example, expression vectors, cloning vectors, transfer vectors, or storage vectors, or combinations thereof. An expression vector can be used for the production of expression products, such as mRNA, peptides, polypeptides, or proteins.A cloning vector is typically a vector containing a cloning site that can be used to insert nucleic acid sequences into the vector, for example, a nucleic acid sequence with an open reading frame. A transfer vector is a vector suitable for transferring nucleic acid molecules into cells or organisms. A storage vector is a vector that allows for the convenient storage of a nucleic acid molecule, such as an mRNA molecule. Thus, a storage vector might contain a sequence that corresponds to, for example, a desired mRNA sequence or a portion thereof.

[0026] The term "sequence identity" refers to the similarity of two nucleotide sequences, or amino acid sequences, expressed as a percentage. Sequence identity depends on the number of identical positions between the two sequences, taking into account the number and length of gaps that must be introduced to achieve optimal sequence alignment. As used here, sequence identity is determined using the Clustal Omega program with the default settings (Sievers et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7:539, 2011). It is known to those skilled in the art that sequence identity using the Clustal Omega program for nucleotide or amino acid sequences can be easily determined on the EMBL-EBI website.

[0027] (https: / / www.ebi.ac.uk / j-dispatcher / msa / clustalo) can be determined.

[0028] The term “homologous genes” or “homologous sequences” here means that the DNA sequences of these genes or DNA segments are at least 70%, preferably at least 80%, preferably at least 90% and most preferably at least 95% identical, i.e. they exhibit a corresponding sequence identity.

[0029] For the purposes of the present invention, the term "randomized sequence" means a nucleotide sequence in which each position has an independent and equal probability of being any nucleotide. The random nucleotides can be any nucleotides, e.g., G, A, C, T, U, or chemical analogues thereof, in any order, where G represents guanyl nucleotides, A represents adenyl nucleotides, T represents thymidyl nucleotides, C represents cytidyl nucleotides, and U represents uracyl nucleotides.

[0030] The term "origin of replication" refers to a specific sequence at which DNA replication is initiated. The term origin of replication encompasses both prokaryotic origins, such as those found in bacteria, and eukaryotic origins, such as those found in mammals.

[0031] The terms “promoter,” “promoter region,” or “promoter sequence” are used interchangeably herein and describe a DNA regulatory region located upstream (5’) of a coding sequence of a gene or a non-coding sequence, capable of binding an RNA polymerase and triggering the transcription of a downstream (3’) coding or non-coding sequence. Suitable promoters can be derived from any organism, including prokaryotic and eukaryotic organisms. A promoter can direct the transcription of a prokaryotic or eukaryotic gene. A promoter may include additional recognition or binding sites for other factors involved in the regulation of gene transcription. A promoter may be a constitutively active promoter, i.e., a promoter that is constitutively in an active state, or it can be an inducible promoter, i.e.A promoter whose state is controlled by an external stimulus, for example, being switched from an inactive to an active state. Such an external stimulus could be, for example, a specific temperature, a compound, or a protein. Constitutively active and inducible promoters are known to those skilled in the art. Examples of inducible promoters include the lac promoter, which is inducible by isopropyl β-D-thiogalactopyranoside (IPTG), the tetracycline-regulated promoter, the rhamnose-inducible promoter, and the arabinose promoter, which is inducible by arabinose.

[0032] As used herein, “operatively associated with a promoter” means that the promoter causes or regulates the transcription of DNA that codes for a gene.

[0033] A "PAS region" here means a nucleotide sequence bounded in the 5' direction by a primosome assembly sequence on the light strand and in the 3' direction by a primosome assembly sequence on the heavy strand, encompassing both primosome assembly sequences and, if applicable, the nucleotide sequence located between them. The light strand is defined as the strand of a plasmid that has a lower number of purine bases relative to the complementary strand, and the heavy strand is defined as the strand of a plasmid that has a higher number of purine bases relative to the complementary strand.

[0034] In this context, a "primosome assembly sequence" (PAS) means a sequence to which at least one, preferably several, proteins involved in replication bind in order to assemble a

[0035] to teach replication initiation.

[0036] A “titer” or “plasmid titer” within the meaning of the present invention is defined as the amount of plasmid product obtained during the fermentation of a bacterial strain. The titer is expressed as the concentration of the plasmid product per fermentation volume in nmol / L or mg / ml.

[0037] The terms "production rate," "replication rate," and "replication efficiency" are used interchangeably here and describe a time derivative of the plasmid titer obtained during fermentation. The production rate is given as plasmid titer per fermentation time (mg / L / h).

[0038] Detailed description of the invention

[0039] The present invention relates to a plasmid vector comprising a) a replication origin; and b) a PAS region comprising i) a primosome assembly sequence PAS-forward (SEQ ID NO:2) located 3' of the replication origin and in the direction of replication; ii) a primosome assembly sequence PAS-reverse (SEQ ID NO:3) located 3' of the primosome assembly sequence PAS-forward and in the direction opposite to replication; and iii) a sequence X between the primosome assembly sequence PAS-forward and the primosome assembly sequence PAS-reverse, which has at most about 75% sequence identity with SEQ ID NO:1.

[0040] The plasmid vector according to the invention comprises at least one

[0041] Origin of replication. Preferably, the plasmid vector comprises exactly one origin of replication. The origin of replication can be any origin that mediates autonomous replication in the host cell in question. Examples of suitable origins of replication are pUC, pMBl, ColEl, or pl5A, or a functionally active variant thereof. Preferably, the origin of replication is a pUC origin of replication. In a particularly preferred embodiment, the pUC origin of replication comprises the SEQ ID NO: 4.

[0042] The plasmid vector of the present invention comprises a specific PAS region. The PAS region includes a primosome assembly sequence PAS-forward (SEQ ID NO: 2). The PAS-forward is located 3' from the origin of replication and in the direction of replication. The distance of the primosome assembly sequence PAS-forward from the origin of replication can be chosen arbitrarily. In a preferred embodiment, the distance of the PAS-forward from the origin of replication is between about 110 and about 200 base pairs, more preferably between about 115 and about 150 base pairs, further preferably between about 115 and about 140 base pairs, and even more preferably between about 118 and 130 base pairs.

[0043] The PAS region further includes a primosome assembly sequence PAS-revers ( SEQ ID NO : 3 ) which is located 3 ' of the primosome assembly sequence PAS-forward and in the direction opposite to replication .

[0044] Furthermore, the PAS region comprises a sequence X between the primosome assembly sequence PAS-forward and the primosome assembly sequence PAS-reverse. The sequence X according to the invention is characterized in that it has at most approximately 75% sequence identity with SEQ ID NO: 1. In a further preferred embodiment, the sequence X has at most 75% sequence identity with SEQ ID NO: 1. In a further preferred embodiment, the sequence X has at most approximately 70%, preferably at most 70%, sequence identity with SEQ ID NO: 1. In a further preferred embodiment, the sequence X has at most approximately 65%, preferably at most 65%, sequence identity with SEQ ID NO: 1. In a further preferred embodiment, the sequence X has at most approximately 60%, preferably at most 60%, sequence identity with SEQ ID NO: 1.In a further preferred embodiment, sequence X exhibits at most approximately 55%, preferably at most 55%, sequence identity with SEQ ID NO:1. In a further preferred embodiment, sequence X exhibits at most approximately 50%, preferably at most 50%, sequence identity with SEQ ID NO:1.

[0045] In a preferred embodiment, the sequence X has a length of about 100 to about 200 base pairs, more preferably a length of about 120 to about 180 base pairs, more preferably a length of about 130 to about 170 base pairs, more preferably a length of about 140 to about 160 base pairs, even more preferably a length of about 148 to about 150 base pairs, and most preferably a length of about 149 base pairs. In a particularly preferred embodiment, the sequence X has a length of 149 base pairs.

[0046] According to the invention, the PAS region 3' of an origin of replication is integrated into a plasmid vector. The PAS region comprises a primosome assembly sequence PAS-forward (PAS-for) with the sequence SEQ ID NO: 2, a primosome assembly sequence PAS-revers (PAS-rev) with the sequence SEQ ID NO: 3, and a sequence located between PAS-forward and PAS-revers (sequence X). Preferably, the plasmid vector has no other PAS region besides the PAS region according to the invention. In a further preferred embodiment, the plasmid vector has no other primosome assembly sequence besides the primosome assembly sequences of the PAS region.

[0047] The PAS region according to the invention is based on the PAS region of plasmid pBR322 (Marians et al. Maximal Limits of the Escherichia coli Replication Factor Y Effector Site Sequences in pBR322 DNA, J Biol Chem. 1982 May 25;257 (10):5656-62). Surprisingly, it was found that a modification of this sequence results in an increased production rate and improved replication efficiency. Starting from the PAS region of plasmid pBR322 (SEQ ID NO: 5), a sequence segment (SEQ ID NO: 1) was modified such that the sequence identity of the resulting modified sequence (sequence X) to the corresponding sequence segment within the PAS region of plasmid pBR322 (SEQ ID NO: 1) is at most 75%. The remaining sequence segments are identical to the PAS region of plasmid pBR322.

[0048] For the PAS region of plasmid pBR322, which is modified according to the invention, no functionality related to the replication rate is known in the prior art. Surprisingly, the modification of the sequence SEQ ID NO:1, compared to analogous plasmid vectors with the unmodified PAS region of plasmid pBR322, results in a new functionality, namely an increase in the production rate. According to the invention, up to approximately 90% of the maximum production rate can be obtained after only about 24 hours of fermentation, whereas with the unmodified sequence, approximately 30 to 42 hours are typically required to reach the maximum production rate. The modification of the PAS region of plasmid pBR322, i.e., the reduction of the sequence identity of sequence X, can be carried out starting from SEQ ID NO:1 using any method known in the field.In one embodiment, the reduction of sequence identity is achieved, for example, by substitution of base pairs. Preferably, the reduction of sequence identity is achieved by randomized substitution or randomization of the sequence SEQ ID NO : 1 or one.

[0049] Sequence segment of sequence SEQ ID NO:1. In one implementation, for example, to generate sequence X, the sequence order of variable-sized segments within SEQ ID NO:1 is randomized, and the resulting randomized sequences are used to substitute the corresponding sequence order of SEQ ID NO:1. In another implementation, to generate sequence X, the sequence order of the entire SEQ ID NO:1 is randomized, and the resulting randomized sequence is used to substitute SEQ ID NO:1. The randomization of the base sequence can be performed, for example, using the online tool "Shuffle DNA" (https: / / www.bioinformatics.org / sms2 / shuffle_dna.html). This randomization does not add or remove bases not present in the sequence.The relative proportion of G / C bases in relation to A / T bases is not changed; only the sequence of bases contained in the sequence is changed.

[0050] In a preferred embodiment, the sequence SEQ ID NO:1 is substituted by a modified sequence X. The modified sequence X can have more or fewer base pairs, or the same number of base pairs as SEQ ID NO:1. In a preferred embodiment, the modified sequence X has the same number of base pairs as SEQ ID NO:1. According to the invention, the integration of the modified sequence X into the plasmid vector takes place between the primosome assembly sequences PAS-forward (SEQ ID NO:2) and PAS-reverse (SEQ ID NO:3) located 3' of the origin of replication.

[0051] In a preferred embodiment, the plasmid vector according to the invention comprises the sequence SEQ ID NO: 6 as sequence X. In this embodiment, the PAS region preferably has the SEQ ID NO: 7.

[0052] In a further preferred embodiment, the plasmid vector according to the invention comprises the sequence SEQ ID NO: 8 as sequence X. In this embodiment, the PAS region preferably has the SEQ ID NO: 9.

[0053] In another preferred embodiment, the plasmid vector according to the invention comprises the sequence SEQ ID NO: 10 as sequence X. In this embodiment, the PAS region preferably has the SEQ ID NO: 11.

[0054] In a further preferred embodiment, the plasmid vector according to the invention comprises the sequence SEQ ID NO: 12 as sequence X. In this embodiment, the PAS region preferably has the SEQ ID NO: 13.

[0055] In yet another preferred embodiment, the plasmid vector according to the invention comprises the sequence SEQ ID NO: 14 as sequence X. In this embodiment, the PAS region preferably has the SEQ ID NO: 15.

[0056] In a further preferred embodiment, the plasmid vector according to the invention comprises sequence X with SEQ ID NO: 16. In this embodiment, the PAS region preferably has SEQ ID NO: 17. In a further preferred embodiment, sequence X is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16. Sequence X is particularly preferred from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. Sequence X is even more preferred from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12.

[0057] In a preferred embodiment, the plasmid vector according to the invention does not contain a rop gene. Within the scope of this invention, this means that the plasmid vector contains at most approximately 50%, preferably at most approximately 40%, more preferably at most approximately 30% of the open reading frame of the rop gene. In embodiments in which partial sequences of the open reading frame of the rop gene are contained in the plasmid vector, transcription and translation of these partial sequences does not yield a functional ROP gene.

[0058] Protein.

[0059] In a preferred embodiment, the plasmid vector of the invention comprises an insertion sequence with at least one recognition site for at least one restriction endonuclease. The insertion sequence comprises a nucleic acid region that has one or more recognition sequences or recognition sites for one or more restriction endonucleases. In a preferred embodiment, the plasmid vector comprises an insertion sequence with several consecutive recognition sequences for several different restriction endonucleases. Each of the recognition sequences can be used for cleavage of the plasmid vector and integration of a nucleic acid. Preferably, a nucleic acid with an open reading frame sequence is integrated.The nucleic acid can encode any gene of interest, for example a reporter gene such as GFP, a gene for the expression of any target protein, a gene that serves as a template for the production of mRNA, or a gene for the production of a viral particle.

[0060] In a further preferred embodiment, the plasmid vector according to the invention comprises a selection marker, wherein the selection marker is preferably selected from the group consisting of an antibiotic resistance gene, a toxin, an antitoxin, and a reporter element. The selection marker is suitable for distinguishing those host cells containing the plasmid vector from those host cells that do not contain the plasmid vector. Suitable selection markers are known to those skilled in the art. For example, genes encoding antibiotic resistance, a toxin, an antitoxin, or a reporter element are suitable as selection markers. Furthermore, auxotrophy markers encoding an essential gene that is deleted in the respective bacterial strain containing the plasmid are suitable as selection markers.

[0061] Suitable genes that confer antibiotic resistance are known to a person skilled in the art. The preferred antibiotic against which resistance is conferred by the selection marker is chosen from the group consisting of ampicillin, tetracycline, kanamycin, geneticin, chloramphenicol, spectinomycin, hygromycin, sulfonamide, trimethoprim, bleomycin, zeocin, gentamicin, and blistidin.

[0062] Suitable selection markers also include toxins such as sacB and hok, antitoxins such as sacB antisense RNA and sok, and reporter elements such as GFP.

[0063] In a preferred embodiment, the plasmid vector according to the invention exhibits a higher replication rate in fermentation culture after 24 h compared to an analogous plasmid vector with the unmodified sequence SEQ ID NO : 1, i.e., with a plasmid vector that is identical to the plasmid vector according to the invention except for the modified sequence X. A higher replication rate means that at a certain time point in the fermentation culture, for example after about 24 h, preferably at least 120%, more preferably at least 130%, even more preferably at least 140%, particularly preferably at least 150%, and especially preferably at least 160% of the plasmid titer is obtained, compared to an analogous plasmid vector with the unmodified sequence SEQ ID NO : 1. with a plasmid vector that is identical to the plasmid vector according to the invention, except for the modified sequence X.

[0064] In another preferred embodiment, the plasmid vector of the present invention reaches at least about 70% of the maximum plasmid titer after 24 h in fermentation culture, which is reached after about 30 h to 42 h, preferably after about 42 h. More preferably, the plasmid vector reaches at least about 75% of the maximum plasmid titer after 24 h in fermentation culture, which is reached after about 30 h to 42 h, preferably after about 42 h. More preferably, the plasmid vector reaches at least about 80% of the maximum plasmid titer after 24 h in fermentation culture, which is reached after about 30 h to 42 h, preferably after about 42 h. Even more preferably, the plasmid vector according to the invention reaches at least about 85% of the maximum plasmid titer in fermentation culture after 24 h, which is reached after about 30 h to 42 h, preferably after about 42 h.

[0065] In another aspect, the present invention relates to a method for cloning DNA sequences using the plasmid vector of the invention. This involves the fermentative production of the plasmid vector according to the invention, which comprises a DNA sequence of interest. Here, a DNA sequence of interest is cloned into a plasmid vector according to the invention, preferably into an insertion sequence present on the plasmid vector. The plasmid vector thus obtained with the inserted DNA sequence of interest is introduced into a suitable bacterial strain, and the resulting bacteria with the introduced plasmid vector are cultured in a suitable fermentation medium. After a certain period, the bacteria are harvested, the cell pellet is isolated, the cells are lysis, and the plasmid vector is isolated.Methods for cloning DNA sequences, fermentation processes, and methods for isolating plasmids are well known to those skilled in the art. Furthermore, analytical methods for identifying, quantifying, and determining the purity of plasmid DNA are well known to those skilled in the art, for example, UV / VIS spectroscopy, HPLC, fluorescence spectroscopy, agarose gel electrophoresis, capillary electrophoresis, or a combination of these methods.

[0066] Any bacterial strain suitable for plasmid production can be used for a fermentation culture. Suitable bacterial strains for plasmid production are well known to those skilled in the art. The bacterial strain is preferably characterized by being a Gram-negative bacterium, more preferably a strain of the genus Enterobacteriaceae, particularly preferably a strain of the species Escherichia coli (E. coli), especially preferably E. coli K12 or E. coli B.

[0067] The process for the fermentative production of plasmid DNA is preferably carried out such that the fermentation volume or production scale is at least about 0.5 liters, preferably at least about 10 liters, more preferably at least about 300 liters, particularly preferably at least about 1000 liters, and even more preferably at least about 10,000 liters. Media for cultivating the production strain in shake flasks and fermenters are known to those skilled in the art from the practice of microbial cultivation. They typically consist of a carbon source, a nitrogen source, and additives such as vitamins, salts, and trace elements, which optimize cell growth and plasmid production. In principle, all common media known to those skilled in the art for the cultivation of microorganisms are suitable as fermentation media.Complex media, mineral salt media, or minimal salt media, to which a specific proportion of complex components such as yeast extract is added, can be used. Furthermore, additional components can be added to the medium to improve cell growth, for example, vitamins, salts, amino acids, and / or trace elements.

[0068] In principle, all sugars, sugar alcohols, or organic acids, or their salts, that are usable by host cells can be used as a carbon source for fermentation. This includes all forms of monosaccharides, encompassing hexoses such as glucose, mannose, fructose, or galactose, as well as pentoses such as xylose, arabinose, or ribose, and all di- and polysaccharides derived from them, such as sucrose, lactose, maltose, maltodextrin, starch, or the monomers or oligomers released from them by enzymatic or chemical hydrolysis. Other usable carbon sources are acetic acid and its derived acetate salts, ethanol, glycerol, citric acid and their salts, or pyruvate and their salts. Preferred carbon sources for fermentation are selected from the group consisting of glucose, fructose, sucrose, mannose, xylose, and arabinose, as well as mixtures thereof.Particularly preferred carbon sources for fermentation are glucose and sucrose, with glucose being especially preferred. The carbon source can be completely introduced into the fermentation medium at the beginning of the fermentation, or none or only a portion of the carbon source can be introduced initially and added during the course of the fermentation. A preferred embodiment involves introducing a portion of the carbon source initially and adding a portion during the course of the fermentation.

[0069] Any suitable substance can be used as a nitrogen source for fermentation. Suitable nitrogen sources include ammonia, either gaseous or in aqueous solution as NH4OH, or salts of ammonia, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, or ammonium nitrate. Other suitable nitrogen sources include nitrate salts, such as KNO3, NaNOa, ammonium nitrate, Ca(NOa)2, and Mg(NOa)2. Further suitable nitrogen sources include urea, complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soy peptone, or casamino acids.

[0070] Furthermore, salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, and / or iron can be added to the fermentation medium, as well as trace amounts of salts of the elements molybdenum, boron, cobalt, manganese, zinc, copper, and / or nickel. Organic acids such as citrate, amino acids such as isoleucine, and vitamins such as vitamin B1 or vitamin B6 can also be added. Other additives include all types of fatty acids, monocarboxylic acids, or dicarboxylic acids, in the form of free fatty acids or salts thereof.

[0071] During fermentation, various parameters can be influenced, such as the

[0072] Nutrient supply, oxygen partial pressure, pH value and temperature of the culture are continuously monitored and controlled.

[0073] Fermentation takes place under pH and temperature conditions that promote the growth and plasmid vector production of the respective host cells. The pH of the fermentation culture is usually preferably between approximately pH 5 and pH 9. More preferred is a pH range of approximately pH 5.5 to pH 8. Particularly preferred is a pH range of approximately pH 6.0 to pH 7.5.

[0074] The temperature of the culture is typically preferably between about 20°C and about 40°C. A temperature range between about 25°C and about 37°C is preferred, and a temperature range between about 30°C and about 37°C is particularly preferred.

[0075] The cultivation period in the process according to the invention is preferably between about 10 h and about 96 h. A cultivation period of about 20 h to about 72 h is preferred. A cultivation period of about 20 h to about 48 h is further preferred. More preferably, the cultivation period is about 20 h to about 28 h, even more preferably about 22 h to about 26 h, still more preferably about 23 h to about 25 h, and most preferably about 24 h.

[0076] The growth of the production strain can take place in an anaerobic cultivation without oxygen supply, or in an aerobic cultivation with oxygen supply. The inventive method is preferably carried out in an aerobic cultivation with oxygen.

[0077] In the aerobic cultivation of host bacteria for the production of the plasmid vector according to the invention, an oxygen saturation of at least about 10% (v / v), particularly preferably at least about 25% (v / v), and even more preferably at least about 50% (v / v) is preferably maintained. Methods and devices for regulating oxygen saturation in a fermentation culture are known to those skilled in the art. In one embodiment, the regulation of oxygen saturation is carried out automatically, according to the prior art, by a combination of gas supply and stirring speed.

[0078] Oxygen supply can be achieved by any suitable method, for example, by introducing compressed air or pure oxygen. Preferably, in aerobic cultivation according to the invention, oxygen supply is achieved by introducing compressed air. The compressed air supply range for aerobic cultivation is, for example, from approximately 0.05 volumes of compressed air per volume of fermentation medium per minute (vvm) to approximately 10 vvm. Preferably, the compressed air supply ranges from approximately 0.2 vvm to approximately 8 vvm, more preferably from approximately 0.4 to approximately 6 vvm, and particularly preferably from 0.8 to 5 vvm.

[0079] The stirring speed during fermentation in the process according to the invention is, for example, about 700 revolutions per minute (rpm), preferably about 1800 rpm, more preferably about 2500 rpm, and particularly preferably about 3000 rpm.

[0080] In one embodiment of the process according to the invention, the cultivation of the host cells takes place in so-called batch mode. To obtain biomass, a culture medium is inoculated with a starter culture of host cells carrying a plasmid vector according to the invention. Cell growth then occurs without further feeding of nutrient sources. In another embodiment of the process according to the invention, cultivation takes place in so-called fed-batch mode, also known as fed-batch mode. In this embodiment, biomass is obtained by feeding additional nutrient sources after an initial growth phase in batch mode. These are referred to as feed or inflow.The infusion can consist of the carbon source, the nitrogen source, one or more vitamins or trace elements essential for production, or a combination of these components. These components can be added to the infusion as a mixture or separately. Additionally, other media components and specific additives that enhance plasmid production, such as amino acids, can be added. The infusion can be administered continuously or discontinuously in portions. A combination of continuous and discontinuous infusion is also possible.

[0081] Preferably, the method for cloning DNA sequences using the plasmid vector according to the invention is a fermentative method in fed-batch mode.

[0082] In the fed-batch method preferred according to the invention for cloning DNA sequences using the plasmid vector according to the invention, preferred carbon sources in the feed are selected from glucose, sucrose, glucose- or sucrose-containing vegetable hydrolysates, as well as mixtures thereof in any desired mixing ratio. A particularly preferred carbon source in the feed is glucose.

[0083] Preferably, in the process according to the invention, the carbon source is added to the culture in such a way that the concentration of the carbon source in the fermenter does not exceed approximately 10 g / L during the production phase. Preferably, the maximum concentration of the carbon source in the fermenter during the production phase is approximately 2 g / L, more preferably approximately 0.5 g / L, and particularly preferably approximately 0.1 g / L.

[0084] The invention will be further described below with reference to exemplary embodiments and the accompanying illustrations, without being limited by this.

[0085] Figure 1 shows a schematic representation of a section of the plasmid vector according to the invention with the origin of replication and the PAS region with the primosome assembly sequence PAS-for, the primosome assembly sequence PAS-rev and the sequence X between PAS-for and PAS-rev.

[0086] Figure 2 shows a schematic plasmid map of the pVAXl plasmid. pCMV refers to the cytomegalovirus promoter. Kanamycin refers to the kanamycin resistance gene. BGH pA refers to the polyA region of the bovine growth hormone gene.

[0087] Figure 3 shows a schematic representation of different PAS regions. PASBR322 denotes the PAS region of the control plasmid pVAX1-PASBR322. PAS 09, PAS 10, PAS 18, PAS31, PAS38, and PAS39 denote PAS regions with different modified sequence segments. The light bar indicates the sequence segment that is modified relative to SEQ ID NO: 1.

[0088] Figure 4 shows the plasmid titer [nmol / L] of different plasmid vectors according to the invention in fermentation culture at 24 hours and at 42 hours. Figure 5 shows the relative plasmid titer of different plasmid vectors according to the invention after fermentation over 24 hours. The percentage of this titer is given in relation to the titer obtained after 42 hours.

[0089] Examples

[0090] All enzymes and kits were used according to the manufacturers' instructions. Unless otherwise stated, all standard methods, molecular biological and microbiological procedures used, such as bacterial cell transformation, polymerase chain reaction (PCR), gene synthesis, DNA isolation and purification, DNA modification using restriction enzymes, Klenow fragment and / or ligase, were carried out in the manner described in the literature, recommended by the respective manufacturers, or in a manner well known to those skilled in the art.

[0091] Determining the sequence identity

[0092] To determine sequence identity, SEQ ID NO:1 was used as the reference sequence. All sequences X to be compared with SEQ ID NO:1 were compared to the reference sequence in a pairwise sequence alignment (mBed algorithm; cluster size for mBed Guide Trees: 100) created using the Clustal Omega 1.2.2 alignment tool integrated into Geneious Prime® 2023.0.2 Build 2023-01-09 11:52. Within the sequence comparison, all positions with identical bases in both the reference sequence and the sequence being compared were counted. The number of these identical positions was then divided by the total number of bases in the reference sequence to determine the percentage of sequence identity.

[0093] Example 1: Production of the plasmid production strain E. coli WCM105 AfliC AyddS AendA

[0094] The bacterial strain E. coli WCM105 AfliC AyddS AendA, described in EP2418276 Bl, was used as the starting strain for the production of the bacterial strain E. coli WCM105 AfliC AyddS AendA. This strain was in turn produced from the strain E. coli WCM105. E. coli WCM105 is producible according to EP0338410B1.

[0095] Production of the strain E. coli WCM105 AfliC AyddS AendA: : cat pKD46

[0096] First, 443 base pairs of the coding region of the chromosomal wild-type endA gene (SEQ ID NO: 18) in the strain E. coli WCM105 AfliC AyddS were replaced by an expression cassette containing the gene for a chloramphenicol acetyltransferase (cat; UniProt No. P62577). For this purpose, a derivative of the plasmid pKD3 (Datsenko et al. Proc Natl Acad Sei USA. 2000 Jun 6; 97(12): 6640-5, https: / / www.addgene.org / 45604 / ) was used as a template for amplification of this cat cassette by PCR. The pKD3 derivative was generated as follows: Regions PCR01 (SEQ ID NO: 21) and PCR02 (SEQ ID NO: 24), each 250 base pairs long and homologous to the wild-type endA gene, or to upstream and downstream regions, were first amplified using the oligonucleotides Pp489 (SEQ ID NO: 19) and Pp490 (SEQ ID NO: 20) and Pp493 (SEQ ID NO: 22) and Pp494 (SEQ ID NO: 23), respectively, and chromosomal DNA of strain WCM105 AfliC AyddS as a template.In parallel, the cat expression cassette (SEQ ID NO: 27) was amplified using the oligonucleotides Pp491 (SEQ ID NO: 25) and Pp492 (SEQ ID NO: 26) and pKD3 as a template. The resulting amplicons were then combined by overlap extension PCR (Horton et al. 2013, Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction, BioTechniques 54, 129-33) to form a combined amplicon PCR04 (SEQ ID NO: 28), and this was inserted into the plasmid pKD3, which had been cut with the restriction enzymes Paei and Ndel (Thermo Fisher Scientific). The resulting plasmid was designated pKD3-AendA. PCR for amplification of the cat cassette was performed using pKD3-AendA as a template and the oligonucleotides Pp495 (SEQ ID NO:29) and Pp496 (SEQ ID NO:30).The first 250 nucleotides of amplicon PCR05 are homologous to the 5'-side open reading frame (ORF) sequence of the endA gene, and the last 250 nucleotides of amplicon PCR05 are homologous to the 3'-side-side sequence of the endA ORF. This resulted in the linear DNA fragment PCR05 (SEQ ID NO:31), which contained the cat cassette.

[0097] The strain E. coli WCM105 AfliC AyddS was transformed with the plasmid pKD46 (Coli Genetic Stock Center CGSC#: 7739), resulting in the strain E. coli WCM105 AfliC AyddS pKD46. Competent cells of the strain WCM105 AfliC AyddS pKD46, prepared according to the procedure described by Datsenko and Wanner (so), were transformed with the linear DNA fragment PCR05 (SEQ ID NO:31) containing the cat cassette. Selection for integration of the cat cassette into the chromosome of WCM105 AfliC AyddS pKD46 at the position of the wild-type endA ORF was performed on LB agar plates containing 20 mg / L chloramphenicol. In this way, cells were obtained in which the wild-type endA ORF had been partially replaced by the cat cassette (E. coli WCM105 AfliC AyddS endA: : cat pKD46) .That the integration occurred at the correct position in the chromosome was confirmed by PCR using the oligonucleotides Pp505 (SEQ ID NO: 32) and Pp506 (SEQ ID NO: 33) and chromosomal DNA from the chloramphenicol-resistant cells E. coli AfliC AyddS endA: : cat pKD46 as a template. Cells of the strain E. coli WCM105 AfliC AyddS endA: : cat pKD46 now expressed the cat gene, encoding a chloramphenicol acetyltransferase instead of the endA wild-type gene. Subsequently, the plasmid pKD46 was cured by cultivation at a non-permissive temperature of 37 °C, and the strain E. coli WCM105 AfliC AyddS endA: : cat was isolated by selection on LB agar plates and LB agar-chloramphenicol (20 mg / L) plates. In a next step, the cat cassette was removed from the chromosome of E. coli WCM105 EfliC EyddS endA : cat. For this purpose, the strain E. coli WCM105 EfliC EyddS endA: : cat was modified with the plasmid pCP20 (Cherepanov et al.).Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant, Gene. 1995;158:9-14.) Bacterial cells that had eliminated the cat cassette from the chromosome were isolated by selecting chloramphenicol-sensitive cells after parallel cultivation on LB agar and LB agar-chloramphenicol (20 mg / L) plates. Confirmation that the deletion occurred at the correct position on the chromosome was obtained by PCR using the oligonucleotides Pp505 (SEQ ID NO: 32) and Pp506 (SEQ ID NO: 33) and chromosomal DNA from the chloramphenicol-sensitive cells E. coli WCM105 EfliC EyddS EendA as a template. The sequence of the original endA locus was verified by sequencing the PCR product. The resulting strain was designated E. coli WCM105 EfliC EyddS EendA.

[0098] Example 2: Production of plasmid vectors with different PAS regions

[0099] Production of the plasmid vector pVAXl-PASBR322 using the PAS-

[0100] Region of the plasmid pBR322

[0101] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of a control plasmid containing the PAS region of plasmid pBR322. pVAXl was linearized by cleavage with the restriction enzymes Bsp68I and Hindi (Thermo Fisher Scientific). Using pBR322 as a template and the oligonucleotides Pp237 (SEQ ID NO: 34) and Pp073 (SEQ ID NO: 35), the 444 base-pair amplicon PCR06 (SEQ ID NO: 36), containing the PAS region of pBR322, was generated by PCR. The first 21 nucleotides of Pp237 are homologous to the 5'-side-of-the-soapy sequence of the Bsp68I cleavage site, and the first 21 nucleotides of Pp073 are homologous to the 3'-side-of-the-soapy sequence of the Hindi cleavage site. The amplicon PCR06 was integrated into the linearized plasmid pVAXl via in-fusion cloning (Takara Bio).Plasmids that had successfully integrated the amplicon were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAXl-seq-rv (SEQ ID NO: 38). Verification of the integrated sequence was performed by Sanger sequencing using the oligonucleotides pVAXl-seq2-rv (SEQ ID NO: 39) and pVAXl-seq23-fw (SEQ ID NO: 40). The resulting control plasmid was designated pVAXl-PASBR322.

[0102] Production of plasmid vectors with modified PAS regions

[0103] Production of the plasmid vector pVAXl-PAS09

[0104] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS09. To create a PAS region with a modified sequence, the sequence of 64 consecutive nucleobases located 22 base pairs apart (3') of the PAS-for sequence was randomized. The base sequence randomization was performed using the online tool "Shuffle DNA".

[0105] (https: / / www.bioinformatics.org / sms2 / shuffle_dna.html). During randomization, no bases not present in the sequence were added, and no bases present in the sequence were removed. The relative proportion of G / C bases to A / T bases remained unchanged; only the order of the bases in the sequence was altered. This process generated the randomized sequence segment PAS09 (SEQ ID NO: 41). Using the bioinformatics software Geneious Prime (Geneious Prime® 2023.0.2 Build 2023-01-09 11:52), the 64 nucleobases located at 22 base pairs 3' intervals within the PAS-for sequence in the PAS region BR322 were substituted by the randomized sequence segment PAS09. Through this process, the PAS region 09 (SEQ ID NO: 7) was initially generated in silico. A DNA fragment containing the PAS region 09 (SEQ ID NO: 60) was then created using gene synthesis (GeneArt).This DNA fragment was integrated into the pVAXl plasmid, linearized by restriction enzyme cutting with Bsp68I and Hindi (Thermo Fisher Scientific), via infusion cloning (Takara Bio). Plasmids that successfully integrated the fragment were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO:37) and pVAXl-seq-rv (SEQ ID NO:38). The integrated sequence was verified by Sanger sequencing using the oligonucleotides pVAXl-seq2-rv (SEQ ID NO:39) and pVAXl-seq23-fw (SEQ ID NO:40). The resulting plasmid was designated pVAXl-PAS09.

[0106] Production of the plasmid vector pVAXl-PASlO

[0107] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PASlO. The sequence of 63 consecutive nucleobases, located 86 base pairs 3' apart in the PAS-for sequence, was randomized using the online tool "Shuffle DNA" as previously described. This process generated the randomized sequence segment PAS10 (SEQ ID NO: 58). Within the PAS region BR322, the 63 nucleobases located 86 base pairs 3' apart in the PAS-for sequence were substituted by the randomized sequence segment PAS10 (SEQ ID NO: 58) as described. This process initially generated PAS region 10 in silico (SEQ ID NO: 9).Using infusion cloning, a fragment (SEQ ID NO: 61) generated by gene synthesis (GeneArt) and containing the PASIO region was integrated, as previously described, into the plasmid pVAXl linearized by restriction enzyme cut with Bsp68I and Hindi. Plasmids that successfully integrated the fragment were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAXl-seq-rv (SEQ ID NO: 38). The integrated sequence was verified by Sanger sequencing using the oligonucleotides pVAX-seq2-rv (SEQ ID NO: 39) and pVAX-seq23-fw (SEQ ID NO: 40). The resulting plasmid was designated pVAXl-PASlO.

[0108] Production of the plasmid vector pVAXl-PAS18

[0109] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of plasmid pVAXl-PAS18. Within the PAS region BR322, the sequence of 64 consecutive nucleobases located 22 base pairs 3' apart from the PAS-for sequence was replaced, as previously described, by the randomized sequence segment PAS09 (SEQ ID NO: 41), and the sequence of 63 consecutive nucleobases located 86 base pairs 3' apart from the PAS-for sequence was replaced, as previously described, by the randomized sequence segment PAS10 (SEQ ID NO: 58). These processes initially generated PAS region 18 (SEQ ID NO: 11) in silico. Using in fusion cloning, a fragment (SEQ ID NO: 62) created by gene synthesis (GeneArt), which includes the PAS18 region, was integrated into the plasmid pVAXl linearized by cutting with the restriction enzymes Bsp68I and Hindi, as previously described.Plasmids that had successfully integrated the fragment were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAXl-seq-rv (SEQ ID NO: 38). Verification of the integrated sequence was achieved by Sanger sequencing using the oligonucleotides pVAX-seq2-rv (SEQ ID NO: 39) and pVAX-seq23-fw (SEQ ID NO: 40). The resulting plasmid was designated pVAXl-PAS18.

[0110] Production of the plasmid vector pVAXl-PAS31

[0111] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS31. The sequence of 22 consecutive nucleobases located immediately 3' of the PAS-for sequence was randomized using the online tool "Shuffle DNA" as previously described. This process generated the randomized sequence segment PAS31 (SEQ ID NO: 59). Within the PAS region BR322, the 22 nucleobases located immediately 3' of the PAS-for sequence were substituted by the randomized sequence segment PAS31 (SEQ ID NO: 59) as previously described.Within PAS region BR322, the sequence of 64 consecutive nucleobases located at intervals of 22 base pairs 3' of the PAS-for sequence, as previously described, was replaced by the randomized sequence segment PAS09 (SEQ ID N0:41), and the sequence of 63 consecutive nucleobases located at intervals of 86 base pairs 3' of the PAS-for sequence, as previously described, was replaced by the randomized sequence segment PAS10 (SEQ ID NO: 58). These processes initially generated PAS region 31 (SEQ ID NO: 13) in silico. Using the oligonucleotides Pp335 (SEQ ID NO: 42) and Pp338 (SEQ ID NO: 43) or Pp339 (SEQ ID NO: 44) and pVAX-seq2-rv (SEQ ID NO: 39), as well as pVAX1-PAS18 as a template, the amplicons PCR07 (SEQ ID NO: 45) and PCR08 (SEQ ID NO: 46) were generated. The resulting amplicons were then analyzed by overlap-extensity PCR (Horton et al.).2013) to form a combined amplicon (SEQ ID NO: 47) and this was integrated into the pVAXl plasmid linearized by restriction enzyme cut with Alw44I and Hindi (Thermo Fisher Scientific) using in fusion cloning as previously described. Plasmids that had successfully integrated the combined amplicon were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAXl-seq-rv (SEQ ID NO: 38). The integrated sequence was verified by Sanger sequencing using the oligonucleotides pVAX-seq2-rv (SEQ ID NO: 39) and pVAX-seq23-fw (SEQ ID NO: 40). The resulting plasmid was designated pVAXl-PAS31.

[0112] Production of the plasmid vector pVAXl-PAS38

[0113] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS38. Within the PAS region BR322, the sequence of 64 consecutive nucleobases located 22 base pairs apart (3' of the PAS-for sequence) was substituted, as previously described, by the randomized sequence segment PAS10 (SEQ ID NO: 58). This process initially generated the PAS region 38 (SEQ ID NO: 15) in silico. Using the oligonucleotides Pp335 (SEQ ID NO: 42) and Pp384 (SEQ ID NO: 48) or Pp385 (SEQ ID NO: 49) and pVAX-seq2-rv (SEQ ID NO: 39), as well as pVAX1-PASBR322 as a template, the amplicons PCR09 (SEQ ID NO: 50) and PCR10 (SEQ ID NO: 51) were generated. The resulting amplicons were then analyzed by overlap-extensity PCR (Horton et al.).2013) to form a combined amplicon (SEQ ID NO: 52) and this was integrated into the pVAXl plasmid linearized by restriction enzyme cut with Alw44I and Hindi (Thermo Fisher Scientific) using in fusion cloning as previously described. Plasmids that had successfully integrated the combined amplicon were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAXl-seq-rv (SEQ ID NO: 38). Verification of the integrated sequence was performed by Sanger sequencing using the oligonucleotides pVAX-seq2-rv (SEQ ID NO: 39) and pVAX-seq23-fw (SEQ ID NO: 40). The resulting plasmid was designated pVAXl-PAS38.

[0114] Production of the plasmid vector pVAXl-PAS39

[0115] The plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS39. Within the PAS region BR322, the sequence of 63 consecutive nucleobases located 86 base pairs apart (3' of the PAS-for sequence) was substituted, as previously described, by the randomized sequence segment PAS09 (SEQ ID NO: 41). This process initially generated the PAS region 39 (SEQ ID NO: 17) in silico. Using the oligonucleotides Pp 335 (SEQ ID NO: 42) and Pp386 (SEQ ID NO: 53) or Pp387 (SEQ ID NO: 54) and PppVAX-seq2-rv (SEQ ID NO: 39), as well as pVAX1-PASBR322 as a template, the amplicons PCR11 (SEQ ID NO: 55) and PCR12 (SEQ ID NO: 56) were generated. The resulting amplicons were then analyzed by overlap extension PCR (Horton et al.).2013) to form a combined amplicon (SEQ ID NO: 57) and this was integrated into the pVAXl plasmid linearized by restriction enzyme cut with Alw44I and Hindi (Thermo Fisher Scientific) using in fusion cloning as previously described. Plasmids that had successfully integrated the combined amplicon were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAXl-seq-rv (SEQ ID NO: 38). Verification of the integrated sequence was performed by Sanger sequencing using the oligonucleotides pVAX-seq2-rv (SEQ ID NO: 39) and pVAX-seq23-fw (SEQ ID NO: 40). The resulting plasmid was designated pVAXl-PAS39.

[0116] Example 3: Fermentative production of plasmid DNA on a 1 L scale. For the production of the plasmid vectors pVAXl-PASBR322, pVAXl-PAS09, pVAXl-PASlO, pVAXl-PAS18, pVAXl-PAS31, pVAXl-PAS38 and pVAXl-PAS39, the strain E. coli WCM105 EfliC EyddS EendA was individually transformed with the plasmids pVAXl, pVAXl-PASBR322, pVAXl-PAS09, pVAXl-PASlO, pVAXl-PAS18, pVAXl-PAS31, pVAXl-PAS38 and pVAXl-PAS39 using the CaC12 method. Selection for plasmid-containing cells was performed using kanamycin (20 mg / 1 ).

[0117] The production of the individual, transformed strains was carried out in 1 stirred tank fermenters (Eppendorf). 0.6 L of a mineral salt medium (5 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.5 g / L NaCl, 1.5 g / L MgSO4 7 H2O, 0.25 g / L CaCl2 2 H2O, 0.075 g / L FeSO4 7 H2O, 1 g / L NasCitrate 2 H2O, and 50 mg / L kanamycin) including 15 g / L glucose, enriched with complex components and 10 g / L yeast extract (ProCel 251 MG), were inoculated with a pre-culture cultivated in fermentation medium to an OD600 of approximately 0.05. Inoculation represents point 0 of the fermentation process, or the start of fermentation. During fermentation, a temperature of 30 °C was maintained, and the pH was kept constant at approximately 7.0 by adding NH4OH or HaPCh. The culture was stirred at 700 rpm at the beginning and aerated with 2 µg of compressed air purified through a sterile filter.Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation. The target value for O₂ saturation during fermentation was set to 30%. After the O₂ saturation dropped below the target value, a regulatory cascade was initiated to bring the O₂ saturation back up to the target value. During this process, the stirring speed was increased to a maximum of 1,800 rpm. Glucose feeding was started after the initial glucose had been metabolized. Before feeding began, the temperature was increased from 30 °C to 37 °C. After the addition of 100 g of pure glucose, the...

[0118] Glucose feeding rate reduced by 60%.

[0119] Samples were taken after 24 and 42 hours of cultivation. The cells were separated from the fermentation medium by centrifugation, and the plasmid DNA content in the cell pellet was determined. For this purpose, the plasmid DNA was first isolated using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific), and the concentration in the elution fraction was then determined using UV / VIS spectrometry. To calculate the plasmid titer in the initial sample, the measured plasmid concentration was first multiplied by the elution volume and then divided by the volume of the fermentation broth used for sampling.

[0120] Figure 4 shows the plasmid titer (nmol / L) of the plasmid vector pVAXl, the plasmid vector pVAXl-PASBR322, and the plasmid vectors according to the invention, pVAXl-PAS 09, pVAXl-PAS l O, pVAXl-PAS 18, pVAXl-PAS31, pVAXl-PAS38, and pVAXl-PAS39, in fermentation culture at 24 hours and at 42 hours. It can be seen that the plasmid titers of the plasmid vectors according to the invention are significantly higher at 24 hours than the plasmid titers of the control plasmid vector pVAXl-PASBR322.

[0121] Figure 5 shows the relative plasmid titer of the plasmid vector pVAXl, the plasmid vector pVAXl-PASBR322, and the plasmid vectors according to the invention, pVAXl-PAS 09, pVAXl-PAS l O, pVAXl-PAS 18, pVAXl-PAS31, pVAXl-PAS38, and pVAXl-PAS39, in fermentation culture after fermentation for 24 hours. The percentage of this titer relative to the titer obtained after 42 hours is indicated. It can be seen that relative plasmid titers significantly higher than those of the control plasmid vector pVAXl-PASBR322 can be achieved with the plasmid vectors according to the invention.

Claims

Patent claims 1. Plasmid vector comprising a) a replication origin; and b) a PAS region comprising i) a primosome assembly sequence PAS-forward (SEQ ID NO:2) located 3' of the replication origin and in the direction of replication; ii) a primosome assembly sequence PAS-reverse (SEQ ID NO:3) located 3' of the primosome assembly sequence PAS-forward and in the direction opposite to replication; iii) a sequence X between the primosome assembly sequence PAS-forward and the primosome assembly sequence PAS-reverse, which has at most about 75% sequence identity with SEQ ID NO:

1.

2. Plasmid vector according to claim 1, wherein the sequence X has at most about 70% sequence identity to SEQ ID NO:

1.

3. Plasmid vector according to claim 1, wherein the sequence X has at most about 65% sequence identity with SEQ ID NO:

1.

4. Plasmid vector according to claim 1, wherein the sequence X has at most about 60% sequence identity with SEQ ID NO:

1.

5. Plasmid vector according to any of the preceding claims, wherein the plasmid vector does not contain a rop gene.

6. Plasmid vector according to any of the preceding claims, wherein the origin of replication is a pUC origin of replication.

7. Plasmid vector according to one of the preceding claims, wherein the plasmid vector comprises an insertion sequence with at least one recognition site for at least one restriction endonuclease.

8. Plasmid vector according to one of the preceding claims, further comprising a selection marker, wherein the selection marker The preferred choice is from the group consisting of an antibiotic resistance gene, a toxin, an antitoxin, and a reporter element.

9. Plasmid vector according to any of the preceding claims wherein the sequence X is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO:

16.

10. Plasmid vector according to one of claims 1-8 wherein the sequence X is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO:

14.

11. Plasmid vector according to one of claims 1-8 wherein the sequence X is selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO:

12.

12. Plasmid vector according to one of the preceding claims, wherein the plasmid vector exhibits a higher replication rate in fermentation culture after 24 h compared to an analogous plasmid vector with the unmodified sequence SEQ ID NO:

1.

13. Plasmid vector according to one of the preceding claims, wherein the plasmid vector in fermentation culture reaches at least about 70% of the maximum plasmid titer reached after about 42h after 24 h.

14. Method for cloning DNA sequences using the plasmid vector according to any one of claims 1-13.

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