WO2025252302A1 - Plasmides ayant un taux de réplication accru - Google Patents

Plasmides ayant un taux de réplication accru

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Publication number
WO2025252302A1
WO2025252302A1 PCT/EP2024/065398 EP2024065398W WO2025252302A1 WO 2025252302 A1 WO2025252302 A1 WO 2025252302A1 EP 2024065398 W EP2024065398 W EP 2024065398W WO 2025252302 A1 WO2025252302 A1 WO 2025252302A1
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WIPO (PCT)
Prior art keywords
seq
sequence
plasmid
pas
plasmid vector
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PCT/EP2024/065398
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German (de)
English (en)
Inventor
Ralph KRAFCZYK
Christian BOSCH
Hagen RICHTER
Johanna Koch
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Wacker Chemie AG
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Wacker Chemie AG
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Application filed by Wacker Chemie AG filed Critical Wacker Chemie AG
Priority to CN202480056951.2A priority Critical patent/CN121794386A/zh
Priority to KR1020267006667A priority patent/KR20260051065A/ko
Priority to PCT/EP2024/065398 priority patent/WO2025252302A1/fr
Publication of WO2025252302A1 publication Critical patent/WO2025252302A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • 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.
  • Plasmid DNA 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 example, in the form of DNA vaccines.
  • Plasmid DNA is classically obtained via fermentation using recombinant bacteria, such as Escherichia coli.
  • a selected bacterial strain is transformed with a plasmid carrying the target gene and then cultured under suitable conditions.
  • 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.
  • 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.
  • RNAI ⁇ sub>1 ⁇ /sub>I is a 553 base pair RNA whose transcription is coordinated by the relatively weak RNAI ⁇ sub>1 ⁇ /sub>I promoter. After transcription, RNAI ⁇ sub>1 ⁇ /sub>I binds to the homologous sequence on the plasmid and initiates replication in a primer-like manner.
  • RNAI ⁇ sub>2 ⁇ /sub>I is a 108 base pair RNA completely homologous to RNAI ⁇ sub>1 ⁇ /sub>I, whose expression is coordinated by the relatively strong RNAI ⁇ sub>2 ⁇ /sub>I promoter.
  • 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.
  • RNAI 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.
  • 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.
  • lower feeding rates are used in the initial bulking phase of fermentation in some pDNA production processes. However, these adjustments result in a longer fermentation time and thus a longer overall production process.
  • Plasmid titers are also suitable for shortening the fermentation and process duration.
  • plasmid DNA is typically produced in bacteria through a fermentative process.
  • 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.
  • the integration of replication-enhancing sequences has also proven to be an effective means of increasing the number of plasmids produced.
  • the number of copies can be identified.
  • plasmids used for cloning therapeutically relevant target genes possess a variant of the pMBl origin of replication, the so-called pUC origin.
  • pUC origin a variant of the pMBl origin of replication
  • 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 RNAI ⁇ sub>1 ⁇ /sub>I and thus increases the effectiveness of RNAI, is missing (Tomi zawa et al.
  • RNAI I encoded on pUC plasmids possesses A single base exchange occurs, which, at temperatures above 37 °C, causes a structural change in RNAII. This structural change reduces the affinity of RNAI for RNAII, thus requiring a greater predominance of RNAI to halt replication.
  • 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.
  • 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 strong increase in the plasmid copy number in plasmid pBR322 and the closely related plasmid ColEl. This exchange This results in impaired termination of the RNAI transcript, leading to the formation of a significantly longer RNA molecule instead of the 104 base pairs long 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).
  • 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.
  • RNAI Ribonucleic 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 RNAI1/RNAI1 overlap region, while regulatory, is not functionally involved in plasmid replication.
  • Both the 108 base pair RNAI gene and the 36 base pair promoter region can be deleted.
  • the plasmid copy number in the cell is regulated solely by the strength or activity of the RNAI promoter.
  • Using an excessively strong RNAI promoter leads to cell-damaging runaway plasmid replication.
  • the use of inducible promoters allows for inducible control of the plasmid copy number by producing only a small amount of RNAI 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).
  • RNAI transcript 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.
  • 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 (Prague). 1990; 35(3): 177-82).
  • RNAI1 By integrating an additional, truncated RNAI1 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 RNAI1 gene can alternatively be expressed not from the same plasmid, but from a second, non-target plasmid or the bacterial chromosome.
  • 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.
  • This SV40 enhancer sequence enables plasmid titers that are higher than those of other high-copy plasmids such as pVAXl (Thermo Fisher Scientific) or gWi z (Genlantis) by a factor of Two are increased.
  • modified plasmid backbones contribute to the more economical production of individual plasmids due to their improved maximum plasmid titers, but require a long fermentation process to reach maximum production capacity. To enable more efficient production, for example of pharmaceutically relevant plasmids, shortening the fermentation process time is of great importance.
  • 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.
  • 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' of the replication origin and in the direction of replication; ii) 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; and iii) a sequence X between the primosome assembly sequence PAS-forward and the primosome assembly sequence PAS-revers which has at most about 75% sequence identity with SEQ ID NO : 1 .
  • 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.
  • a specific DNA sequence namely a specific PAS region
  • the integration of this specific PAS region enables the maximum production rate to be reached earlier, with essentially unchanged high plasmid titers.
  • 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.
  • 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.
  • nucleic acid sequence refers to a person skilled in the art and refer to an individual and specific sequence of nucleotides.
  • 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 analogues, or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • Plasmid vector 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, are used.
  • a cloning vector is typically a vector that contains a cloning site, which 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 can contain a sequence that corresponds, for example, to a desired mRNA sequence or a portion thereof.
  • 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.
  • 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%, identical to the original DNA sequence. preferably to at least 90% and particularly preferably to at least 95% identical, i.e., exhibiting a corresponding sequence identity.
  • 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.
  • origin of replication refers to a specific sequence at which DNA replication is initiated.
  • origin of replication encompasses both prokaryotic origins, such as bacterial origins, and eukaryotic origins, such as those found in mammals.
  • promoter refers to 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, That is, a promoter that is constitutively in an active state, or it can be an inducible promoter, that is, a promoter whose state is controlled by an external stimulus, for example, being switched from an inactive to an active state.
  • 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.
  • inducible promoters examples include the lac promoter, which is inducible by isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), the tetracycline-regulated promoter, the rhamnose-inducible promoter, and the arabinose promoter, which is inducible by arabinose.
  • lac promoter which is inducible by isopropyl- ⁇ -D-thiogalactopyranoside (IPTG)
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • tetracycline-regulated promoter the tetracycline-regulated promoter
  • rhamnose-inducible promoter the rhamnose-inducible promoter
  • arabinose promoter which is inducible by arabinose.
  • operatively associated with a promoter means that the promoter causes or regulates the transcription of DNA that codes for a gene.
  • 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
  • the heavy strand is defined as the strand of a plasmid that has a higher number of purine bases relative to the complementary strand.
  • a “primosome assembly sequence” here means a sequence to which at least one, preferably several, primosomes are attached. Proteins involved in replication bind to form a
  • 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.
  • production rate means a time derivative of the plasmid titer obtained during fermentation.
  • the production rate is given as plasmid titer per fermentation time (mg/L/h).
  • 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.
  • the plasmid vector according to the invention comprises at least one
  • the plasmid vector comprises exactly a replication origin.
  • the replication origin can be any replication origin that mediates autonomous replication in the host cell in question. Examples of suitable replication origins are pUC, pMBl, ColEl, or pl5A, or a functionally active variant thereof.
  • the replication origin is a pUC replication origin.
  • the pUC replication origin comprises the SEQ ID NO: 4.
  • 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.
  • 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 .
  • a primosome assembly sequence PAS-revers SEQ ID NO : 3
  • 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.
  • sequence X has at most 75% sequence identity with SEQ ID NO:1.
  • sequence X has at most about 70%, preferably at most 70%, sequence identity with SEQ ID NO:1.
  • sequence X has at most about 65%, preferably at most 65%, sequence identity with SEQ ID NO:1.
  • sequence X has at most about 60%, preferably at most 60%, sequence identity with SEQ ID NO:1. In a further preferred embodiment, sequence X has at most about 55%, preferably at most 55%, sequence identity with SEQ ID NO:1. In a further preferred embodiment, sequence X has at most about 50%, preferably at most 50%, sequence identity with SEQ ID NO:1.
  • 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.
  • the PAS region 3' of a replication origin 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).
  • the plasmid vector has no other sequences besides the PAS region according to the invention.
  • the plasmid vector has no further primosome assembly sequences besides those of the PAS region.
  • 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.
  • 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%.
  • 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%.
  • sequence segments are identical to the PAS region of plasmid pBR322.
  • the modification of the PAS region of the plasmid pBR322, i.e., the reduction of the sequence identity of sequence X, can be performed starting from SEQ ID NO:1 by any method known in the field.
  • the reduction of sequence identity is achieved, for example, by substitution of base pairs.
  • the reduction of sequence identity is achieved by randomized substitution or randomization of the sequence SEQ ID NO:1 or one
  • 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.
  • 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.
  • 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.
  • the plasmid vector is inserted between the 3' of the origin of replication PAS-forward (SEQ ID NO: 2) and PAS-reverse (SEQ ID NO:3) primosome assembly sequences located at the origin of replication.
  • the plasmid vector according to the invention comprises the sequence SEQ ID NO: 6 as sequence X.
  • the PAS region preferably has the SEQ ID NO: 7.
  • the plasmid vector according to the invention comprises the sequence SEQ ID NO: 8 as sequence X.
  • the PAS region preferably has the SEQ ID NO: 9.
  • the plasmid vector according to the invention comprises the sequence SEQ ID NO: 10 as sequence X.
  • the PAS region preferably has the SEQ ID NO: 11.
  • the plasmid vector according to the invention comprises the sequence SEQ ID NO: 12 as sequence X.
  • the PAS region preferably has the SEQ ID NO: 13.
  • the plasmid vector according to the invention comprises the sequence SEQ ID NO: 14 as sequence X.
  • the PAS region preferably has the SEQ ID NO: 15.
  • the plasmid vector according to the invention comprises the sequence SEQ ID NO: 16 as sequence X.
  • the PAS region preferably has the SEQ ID NO: 17.
  • 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 selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 is particularly preferred.
  • Sequence X selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 12 is even more preferred.
  • the plasmid vector according to the invention does not contain a rop gene.
  • transcription and translation of these partial sequences does not yield a functional ROP gene.
  • 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.
  • 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.
  • 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 a 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.
  • a reporter gene such as GFP
  • a gene for the expression of a any target protein e.g., GFP
  • a gene that serves as a template for the production of mRNA e.g., a gene for the expression of a viral particle.
  • 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.
  • auxotrophy markers encoding an essential gene that is deleted in the respective bacterial strain containing the plasmid are suitable as selection markers.
  • 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.
  • 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.
  • 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 except for the modified sequence.
  • Sequence X is identical to the plasmid vector according to the invention.
  • 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, i.e., with a plasmid vector that is identical to the plasmid vector according to the invention except for the modified sequence X.
  • 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 after 24 h in fermentation culture, which is reached after about 30 h to 42 h, preferably after about 42 h.
  • the present invention relates to a method for cloning DNA sequences using the plasmid vector of the invention.
  • a DNA sequence of interest is cloned into a plasmid vector according to the invention, preferably into an insertion sequence. which is 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 specific period, the bacteria are harvested, the cell pellet is isolated, the cells are lysed, and the plasmid vector is isolated.
  • 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.
  • the process for the fermentative production of plasmid DNA is carried out such that the fermentation volume or production scale is at least about 0.5 liters, preferably the fermentation volume is 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 familiar to those skilled in the art 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.
  • all common media known to those skilled in the art for cultivating microorganisms are suitable as fermentation media. These include complex media, mineral salt media, or minimal salt media to which a specific proportion of complex components, such as yeast extract, is added.
  • additional components can be added to the medium to improve cell growth, for example, vitamins, salts, amino acids, and/or trace elements.
  • 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.
  • 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 at the beginning and added during the course of the fermentation.
  • a preferred embodiment involves introducing part of the carbon source at the beginning and adding part during the course of the 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 and complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soy peptone, or casamino acids.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a The oxygen content is set to a 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).
  • Methods and devices for regulating the oxygen saturation in a fermentation culture are known to those skilled in the art.
  • the regulation of the oxygen saturation is carried out automatically, according to the prior art, by a combination of gas supply and stirring speed.
  • Oxygen supply can be achieved by any suitable method, for example, by introducing compressed air or pure oxygen.
  • 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.
  • 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.
  • 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.
  • the cultivation of the host cells is carried out in so-called batch mode.
  • 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 from nutrient sources.
  • cultivation takes place in the so-called fed-batch mode, also known as infeed mode.
  • biomass is obtained by adding additional nutrient sources after an initial growth phase in batch mode. These are referred to as feed or infeed.
  • the infeed can consist of the carbon source, the nitrogen source, one or more vitamins or trace elements important for production, or a combination of these components.
  • the components can be added to the infeed as a mixture or separately. Additionally, other media components and specific additives that increase plasmid production, such as amino acids, can also be added to the infeed.
  • the infeed can be supplied continuously or discontinuously in portions. A combination of continuous and discontinuous infeed is also possible.
  • the method for cloning DNA sequences using the plasmid vector according to the invention is a fermentative method in fed-batch mode.
  • 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.
  • the carbon source is added to the culture in such a way that the content of The carbon source in the fermenter during the production phase does not exceed approximately 10 g/L.
  • 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.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • Figure 4 shows the plasmid titer [nmol/L ] of different plasmid vectors according to the invention in fermentation culture at time 24 hours, as well as at time 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.
  • 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.
  • mBed algorithm cluster size for mBed Guide Trees: 100
  • Example 1 Production of the plasmid production strain E. coli WCM105 AfliC AyddS AendA
  • the starting strain for the generation of the bacterial strain E. coli WCM105 AfliC AyddS AendA was the one described in EP2418276 Bl.
  • the bacterial strain E. coli WCM105 AfliC AyddS is used, which in turn was generated from the strain E. coli WCM105.
  • E. coli WCM105 is producible according to EP0338410B1.
  • 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.
  • 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.
  • the 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) were synthesized.
  • the first 250 nucleotides of amplicon PCR05 are homologous to the 5'-side sequence of the open reading frame (ORF) of the endA gene, and the last 250 nucleotides of amplicon PCR05 are homologous to the 3'-side sequence of the endA ORF. This resulted in the linear DNA fragment PCR05 (SEQ ID NO:31), which contained the cat cassette.
  • 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.
  • the plasmid pKD46 was cured by cultivation at the non-permissive temperature 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/1 ) plates.
  • the cat cassette was removed from the chromosome of E. coli WCM105 EfliC EyddS endA : cat.
  • the strain E. coli WCM105 EfliC EyddS endA: : cat was transformed with the plasmid pCP20 (Cherepanov et al.
  • 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 named E. coli WCM105 EfliC EyddS EendA.
  • 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 cutting with the restriction enzymes Bsp68I and Hindi (Thermo Fisher Scientific).
  • the 444 base-pair amplicon PCR06 (SEQ ID NO: 36), containing the PAS region, was generated by PCR.
  • the amplicon PCR06 was generated from pBR322.
  • the first 21 nucleotides of Pp237 are homologous to the 5'-soapy sequence of the Bsp68I site, and the first 21 nucleotides of Pp073 are homologous to the 3'-located sequence of the Hindi site.
  • the amplicon PCR06 was integrated into the linearized plasmid pVAXl by in-fusion cloning (Takara Bio). Plasmids that 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).
  • 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 control plasmid was named pVAXl-PASBR322.
  • the plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS09.
  • 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”.
  • This process initially generated PAS region 09 (SEQ ID NO: 7) in silico.
  • a DNA fragment containing PAS region 09 (SEQ ID NO: 60) was then created using gene synthesis (GeneArt). This DNA fragment was integrated into the plasmid pVAXl, which had been linearized by restriction enzyme cutting with Bsp68I and Hindi (Thermo Fisher Scientific), using in-fusion cloning (Takara Bio). 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 performed 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.
  • the plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PASlO.
  • 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.
  • the plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of plasmid pVAXl-PAS18.
  • 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).
  • a fragment (SEQ ID NO: 62) generated by gene synthesis (GeneArt) and containing the PAS18 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). Verification of the integrated sequence was performed by Sanger sequencing using the oligonucleotides pVAX-seq2-rv (SEQ ID NO: 39) and pVAXl-seq23-fw. (SEQ ID NO: 40) . The resulting plasmid was named pVAXl-PAS18 .
  • 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.
  • PAS9 SEQ ID N0:41
  • PAS10 SEQ ID NO: 58
  • Plasmids containing the combined The amplicons that had successfully integrated 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 plasmid was designated pVAXl-PAS31.
  • the plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS38.
  • 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.
  • 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 pVAXl-PASBR322 as a template, the amplicons PCR09 (SEQ ID NO: 50) and PCR10 (SEQ ID NO: 51) were generated. The resulting amplicons were then combined into a single amplicon (SEQ ID NO: 52) by overlap-extension PCR (Horton et al.
  • Plasmids that had successfully integrated the combined amplicon were identified by colony PCR using the oligonucleotides Pp081 (SEQ ID NO: 37) and pVAX1-seq-rv (SEQ ID NO: 38). Verification of the integrated sequence was performed by Sanger sequencing using the oligonucleotides pVAX2-rv (SEQ ID NO: 39) and pVAX23-fw. (SEQ ID NO: 40) . The resulting plasmid was named pVAXl-PAS38 .
  • the plasmid pVAXl (Thermo Fisher Scientific, see Figure 2) was used as the starting vector for the production of the plasmid pVAXl-PAS39.
  • 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.
  • 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 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-PAS39.
  • Example 3 Fermentative production of plasmid DNA on a 1 L scale
  • the strain E 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 each of 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/L).
  • 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.
  • a mineral salt medium 5 g/L ( NH4 ) 2SO4 , 5 g/L KH2PO4, 0.5 g/L NaCl, 1.5 g/L MgSO4
  • Inoculation represents point 0 of the fermentation process, or the start of 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 initially stirred at 700 rpm and aerated with 2 ⁇ L of compressed air purified through a sterile filter. Under these initial conditions, the oxygen probe was calibrated to 100% saturation prior to 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 restore it to the target level. During this process, the stirring speed was increased to a maximum of 1,800 rpm.
  • Glucose feeding began after the initial glucose had been metabolized. Before feeding began, the temperature was increased from 30°C to 37°C. increased. After the administration of 100 g of pure glucose, 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.
  • 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.
  • 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 is given in relation to the titer obtained after 42 hours. It can be seen that with the plasmid vectors according to the invention, significantly higher relative plasmid titers can be achieved. are the plasmid titers of the control plasmid vector pVAXl-PASBR322 .

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Abstract

L'invention concerne un vecteur plasmidique comprenant a) une origine de réplication ; et b) une région PAS, comprenant i) une séquence d'assemblage primosomique PAS-vers l'avant (SEQ ID No 2), qui est située en 3' de l'origine de réplication et dans la direction de réplication ; ii) une séquence d'assemblage primosomique PAS-inversée (SEQ ID No 3), qui est située en 3' de la séquence d'assemblage primosomique PAS-vers l'avant et dans la direction opposée à la réplication ; et iii) une séquence X entre la séquence d'assemblage primosomique PAS-vers l'avant et la séquence d'assemblage primosomique PAS-inversée, ladite séquence partageant une identité de séquence d'au plus approximativement 75 % avec SEQ ID No 1.
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EP0338410B1 (fr) 1988-04-19 1994-09-28 Consortium für elektrochemische Industrie GmbH Mutants secrétants d'escherichia coli
EP1326989A1 (fr) 2000-10-04 2003-07-16 Boehringer Ingelheim International GmbH Vecteurs d'expression a origine de replication cole1 modifiee utilises dans le controle du nombre de copies de plasmides
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EP0338410B1 (fr) 1988-04-19 1994-09-28 Consortium für elektrochemische Industrie GmbH Mutants secrétants d'escherichia coli
EP1326989A1 (fr) 2000-10-04 2003-07-16 Boehringer Ingelheim International GmbH Vecteurs d'expression a origine de replication cole1 modifiee utilises dans le controle du nombre de copies de plasmides
EP2418276B1 (fr) 2006-02-02 2013-11-20 Wacker Chemie AG Procédé destiné à la fabrication d'une broche
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