Bacterial strains for DNA production

Abstract

Compositions for the production of plasmid nucleic acids, as well as methods for making and using the same, are provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit under Section 119(e) of the United States Patent Act as of the filing date of U.S. Patent Application No. 63 / 035,630, filed on June 5, 2020, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Escherichia coli (E. coli) has a long history in biotechnology and drug development, and has been used for many years as a host for plasmid DNA production. This is due to various reasons, including E. coli's genetic simplicity (e.g., its small gene count of less than 4,400), growth rate, safety, successful acceptance of foreign DNA, and ease of management. Furthermore, due to its long history and use, E. coli has become a well-defined organism that has been manipulated in various ways. For example, several different strains have been created for various purposes, including cloning, plasmid DNA production, and protein expression. Most commonly, E. coli K12 derivatives such as DH5α, JM108, and DH10β are used for plasmid DNA cloning and production because they possess specific genomic mutations desirable for cloning purposes. These primarily result in the inactivation of genes encoding nucleases, recombinases, and other enzymes that reduce the stability, purity, and cloning efficiency of the strain's DNA. [Overview of the project]

[0003] Engineered bacterial strains and vectors for enhanced plasmid DNA production are provided herein.

[0004] In one embodiment, the present invention is an engineered nucleic acid vector comprising a stationary-phase-inducible promoter and a primosome assembly site (PAS). In some embodiments, the vector further includes a point mutation on RNAII that induces the formation of a key stem-loop, SL4. In some embodiments, the native promoter of RNAII is disrupted. In some embodiments, the native promoter of RNAII is deleted.

[0005] In some embodiments, the stationary-phase induction promoter is P(osmY). In some embodiments, P(osmY) has the sequence of SEQ ID NO: 27. In some embodiments, PAS has the sequence of SEQ ID NO: 28.

[0006] In some embodiments, SL4 has the sequence of SEQ ID NO: 29. In some embodiments, the vector is plasmid 1(+PAS+P(osmY)).

[0007] In some embodiments, the vector is plasmid 2 (+PAS+P(osmY)+SL4). In some embodiments, the vector has a sequence that is at least 70% identical to sequence number 19 (the sequence of plasmid 1). In some embodiments, the vector has a sequence that is at least 70% identical to sequence number 20 (the sequence of plasmid 2).

[0008] In some embodiments, the vector is further arranged from 5' to 3' as follows: (a) origin of replication; (b) Promoter and (c) Contains antibiotic resistance genes

[0009] In some embodiments, the vector further includes an open reading frame (ORF) encoding the mRNA of interest.

[0010] In other aspects, genotype:| <repA|ori_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)> Recombinant plasmids containing |gamma>|beta>|exo>|a>| are provided.

[0011] In another embodiment, a recombinant plasmid is provided that contains a nucleic acid sequence having at least 70% identity with SEQ ID NO: 19.

[0012] In another embodiment, a recombinant plasmid is provided that contains a nucleic acid sequence having at least 70% identity with SEQ ID NO: 20.

[0013] A method for carrying out an in vitro transcription reaction is provided in another aspect of the present invention, the method using the engineered nucleic acid vector described herein.

[0014] In some embodiments, the present invention is a nucleic acid comprising a prsA variant. In some embodiments, the nucleic acid has 70% to 99% sequence identity with respect to prsA. In some embodiments, the nucleic acid has at least 70% sequence identity with respect to prsA* (SEQ ID NO: 23). In some embodiments, the nucleic acid has at least 80% sequence identity with respect to

[0015] In another embodiment of the present invention, a genetically modified microorganism containing a prsA variant is provided, wherein the genetically modified microorganism has a genome in which the repressor gene purR is disrupted. In some embodiments, the prsA variant has 70% to 99% sequence identity with prsA. In some embodiments, the prsA variant has at least 90% sequence identity with prsA* (SEQ ID NO: 23). In some embodiments, the prsA variant has SEQ ID NO: 23. In some embodiments, purR is deleted. In some embodiments, purR has SEQ ID NO: 25. In some embodiments, the EcoKI restriction system is deleted from the genome. In some embodiments, endA is deleted from the genome. In some embodiments, recA is deleted from the genome. In some embodiments, the genetically modified microorganism is a recombinant strain of Escherichia coli (E. coli).

[0016] In some embodiments of the present invention, recombinant strains of Escherichia coli (E. coli) are provided, comprising an E. coli genome having at least the following gene deletions: endA (ΔendA) and recA (ΔrecA). In some embodiments, E. coli is derived from MG1655. In some embodiments, the E. coli genome comprises a nucleic acid sequence of the MG1655 genome having at least the following gene deletions with respect to MG1655: endA (ΔendA) and recA (ΔrecA). In some embodiments, the E. coli genome comprises a nucleic acid sequence with at least 95% sequence identity with the MG1655 genome. In some embodiments, the EcoKI restriction system is deleted from the E. coli genome.

[0017] In some embodiments, the E. coli genome contains nucleic acid sequences that are at least 80% identical to those of the MG1655 genome. In some embodiments, the E. coli genome contains nucleic acid sequences that include the nucleic acid sequences of the MG1655 genome with respect to the EcoKI restriction deletion. In some embodiments, E. coli contains the prsA variant. In some embodiments, the E. coli genome contains nucleic acid sequences that are at least 80% identical to those of the MG1655 genome. In some embodiments, the E. coli genome contains the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the purR sequence is deleted from the E. coli genome. In some embodiments, the E. coli genome contains nucleic acid sequences that are at least 80% identical to those of the MG1655 genome. In some embodiments, the E. coli genome has the nucleic acid sequence of SEQ ID NO: 25, which is deleted with respect to the MG1655 genome.

[0018] In one embodiment, the present disclosure relates to a recombinant strain of E. coli comprising an E. coli genome having at least the following gene deletions: endA and recA.

[0019] In some embodiments, the E. coli genome further includes at least one gene deletion selected from the group consisting of mrr, hsdR, hsdM, hsdS, symE, and mcrBC.

[0020] In some embodiments, the E. coli genome further comprises at least two gene deletions selected from the group consisting of mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further comprises at least three gene deletions selected from the group consisting of mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further comprises at least four gene deletions selected from the group consisting of mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further comprises at least five gene deletions selected from the group consisting of mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further comprises the gene deletions: mrr, hsdR, hsdM, hsdS, symE, and mcrBC.

[0021] In some embodiments, the E. coli genome is derived from E. coli strain MG1655 or strain 1. In some embodiments, the E. coli genome is derived from E. coli strain 4.

[0022] In one aspect, the present disclosure relates to a recombinant strain of E. coli, wherein the E. coli genome further comprises a plasmid. In some embodiments, the plasmid can express prsA* or knockout purR. In some embodiments, the plasmid can express prsA* and knockout purR.

[0023] In one aspect, the present disclosure relates to the genotype: | <repA101|ori101_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)>|gamma>|beta>|exo>|60a>| recombinant plasmid.

[0024] In some embodiments, the E. coli genome disclosed herein can further express a gene of a positive selection marker based on a first environmental factor or a negative selection marker based on a second environmental factor, and the first environmental gene and the second environmental factor are not the same. In some embodiments, the E. coli genome disclosed herein can further express genes of a positive selection marker based on a first environmental factor and a negative selection marker based on a second environmental factor, and the first environmental gene and the second environmental factor are not the same. In some embodiments, the positive selection marker is a gene capable of conferring kanamycin resistance. In some embodiments, the negative selection marker can express levansucrase.

[0025] In some aspects, a genetically modified microorganism comprising strain 3 is provided.

[0026] In some aspects, a genetically modified microorganism comprising strain 4 is provided.

[0027] In some aspects, an engineered nucleic acid vector comprising a nucleic acid having at least 70% sequence identity to SEQ ID NO: 21 is provided.

[0028] In some aspects, an engineered nucleic acid vector comprising a nucleic acid having at least 80% sequence identity to SEQ ID NO: 21 is provided.

[0029] In some aspects, an engineered nucleic acid vector comprising a nucleic acid having at least 90% sequence identity to SEQ ID NO: 21 is provided.

[0030] In some aspects, an engineered nucleic acid vector comprising a nucleic acid having at least 95% sequence identity to SEQ ID NO: 21 is provided.

[0031] In some aspects, an engineered nucleic acid vector comprising a nucleic acid having SEQ ID NO: 21 is provided.

[0032] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid having at least 70% sequence identity with respect to SEQ ID NO: 22.

[0033] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid having at least 80% sequence identity with respect to sequence number 22.

[0034] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid having at least 90% sequence identity with respect to SEQ ID NO: 22.

[0035] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid having at least 95% sequence identity with respect to sequence number 22.

[0036] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid having sequence number 22.

[0037] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid sequence having at least 70% sequence identity with respect to any one of sequence numbers 1 to 15.

[0038] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid sequence having at least 80% sequence identity with respect to any one of sequence numbers 1 to 15.

[0039] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid sequence having at least 90% sequence identity with respect to any one of sequence numbers 1 to 15.

[0040] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid sequence having at least 95% sequence identity with respect to any one of sequence numbers 1 to 15.

[0041] In some embodiments, an engineered nucleic acid vector is provided that contains a nucleic acid sequence having at least 99% sequence identity with respect to any one of sequence numbers 1 to 15.

[0042] In some embodiments, an engineered nucleic acid vector is provided that contains one of the nucleic acid sequences of sequence numbers 1 to 15.

[0043] In some embodiments, an engineered nucleic acid vector is provided that includes a nucleic acid sequence having at least 70% sequence identity with respect to sequence number 10.

[0044] In some embodiments, an engineered nucleic acid vector is provided that includes a nucleic acid sequence having at least 70% sequence identity with respect to sequence number 11.

[0045] In some embodiments, an engineered nucleic acid vector is provided that includes a nucleic acid sequence having at least 95% sequence identity with respect to sequence number 10.

[0046] In some embodiments, an engineered nucleic acid vector is provided that includes a nucleic acid sequence having at least 95% sequence identity with respect to sequence number 11.

[0047] In some embodiments, an engineered nucleic acid vector is provided that includes a nucleic acid sequence having at least 99% sequence identity with respect to sequence number 10.

[0048] In some embodiments, an engineered nucleic acid vector is provided that includes a nucleic acid sequence having at least 99% sequence identity with respect to sequence number 11.

[0049] In some embodiments, an engineered nucleic acid vector containing the nucleic acid sequence of sequence number 10 is provided.

[0050] In some embodiments, an engineered nucleic acid vector comprising the nucleic acid sequence of sequence number 11 is provided.

[0051] Each limitation of the present invention may encompass various embodiments of the present invention. Therefore, each limitation of the present invention involving any one element or combination of elements is expected to be included in each aspect of the present invention. In its application, the present invention is not limited to the configuration details or arrangement of components shown in the following description or illustrated in the drawings. The present invention may have other embodiments and can be carried out or implemented in various ways. Furthermore, the expressions and terms used herein are for illustrative purposes only and should not be considered limiting. The use herein of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof means encompassing the items listed therein and their equivalents, as well as additional items.

[0052] The attached drawings are not drawn to scale. In the drawings, identical or nearly identical components shown in various diagrams are represented by similar numbers. For clarity, not all components are labeled in every drawing. The following are the drawings. [Brief explanation of the drawing]

[0053] [Figure 1] The biosynthesis of purines and pyrimidines in the wild-type E. coli K12 strain (A) is shown, and the carbon flux to purine synthesis from strain 4 by genome-derived overexpression of PrsA* and purR knockout is shown (B). [Figure 2] This paper illustrates exemplary positive and negative selection processes used to introduce gene knockouts into E. coli. [Figure 3] This shows the lineage from stocks 2, 3, and 4 to their parent stock, stock 1. [Figure 4] This graph shows the proportion of supercoiled monomers in various plasmids prepared from strain 1. [Figure 5A]The plasmid yield (A) obtained from a shaking flask culture containing strain 1 / plasmid 1 (sequence number 19) and from a single copy plasmid carrying the gene shown on the y-axis is shown. [Figure 5B] The plasma culture density (B) obtained from a shaking flask culture containing strain 1 / plasmid 1 (sequence number 19) and from a single copy plasmid carrying the gene shown on the y-axis is shown. [Figure 5C] The Ct difference (C) values ​​obtained from a shaking flask culture containing strain 1 / plasmid 1 (sequence number 19) and a single copy plasmid carrying the gene shown on the y-axis are shown. [Figure 6] This shows the plasmid copy number of PL-007948 in strain 1, which possesses a single copy plasmid for prsA* expression. [Figure 7A] Plasmid yields (A) for strains 3 and 1 containing plasmid 1 (SEQ ID NO: 19) after 16 hours in a shaking flask are shown. [Figure 7B] The culture densities (B) of strain 3 and strain 1 containing plasmid 1 (sequence number 19) after 16 hours in a shaking flask are shown. [Figure 8A] The optical density (A) obtained from stocks 1, 3, and 4, which possess PL-007948, is shown. [Figure 8B] Plasmid DNA yield (B) obtained from strains 1, 3, and 4 possessing PL-007948 is shown. [Figure 9A] This shows plasmid DNA (pDNA) produced by strains 3 and 1 of the Ambr250 lineage. A shows the dynamic profile of pDNA accumulation. [Figure 9B] This shows plasmid DNA (pDNA) produced by strains 3 and 1 of the Ambr250 lineage. B shows the statistical analysis of pDNA produced by strains 3 and 1 after 22 hours of EFT. [Figure 10] This shows the relative productivity of stock 3 and stock 1 over time. [Figure 11]This shows pDNA production by strains 4 and 1 of the Ambr250 lineage. A shows the dynamic profile of pDNA accumulation. B shows the statistical analysis of pDNA produced by strains 1 and 4 at 22-hour EFT. [Figure 12] This shows the relative productivity of stocks 1 and 4 over time. [Figure 13A] The diagram shows the process for a long-term pDNA stability experiment. The diagram shows that strains containing two different plasmids were grown, subcultured in fresh medium for several days (A), and then poly(A) tail Sanger sequencing was performed. [Figure 13B] The diagram showing the process of the long-term pDNA stability experiment shows the total number of generations (B) of the NEB strain (a strain similar to the commercially available strain), strain 1, and strain 4 that possess the plasmid shown. [Figure 13C] This diagram shows the process for long-term pDNA stability experiments. The diagram shows a process flow diagram modeling the expected number of generations (#) a cell strain will undergo, from an MCB vial to a 30-liter or 300-liter fermentation scale. "MCB" - Master Cell Bank, "WCB" - Working Cell Bank (C). [Figure 14] The growth profiles of strains 1 and 4, which possess the indicated plasmid, are shown. [Figure 15] The graph shows the time course of plasmid DNA production in strains 1 and 4, which possess plasmid 1. [Figure 16] This shows plasmid maps with modifications made to construct plasmid 1 and plasmid 2. [Figure 17A] Plasmid yield (A) obtained from strain 1 using various plasmids is shown. [Figure 17B] The final culture optical density (B) obtained at 16 hours using strain 1 with various plasmids is shown. [Figure 18]Plasmid production data for Modification 9 (SEQ ID NO: 10, Ori10) and Modification 10 (SEQ ID NO: 11, Ori11) are shown. A shows the increase in plasmid DNA (pDNA) in milligrams / liter (mg / l) compared to the parent plasmid (SEQ ID NO: 16), based on the control plasmid (PL_007984). B shows the improved overall productivity, measured in mg of pDNA per gram of wet cell weight (gWCW). [Modes for carrying out the invention]

[0054] E. coli has long been used as a host for plasmid DNA production. Several different strains have been created for a variety of purposes, including cloning, plasmid DNA production, and protein expression. Most commonly, E. coli K12 derivatives such as DH5α, JM108, and DH10β are used for plasmid DNA cloning and production. These primarily result in the inactivation of genes encoding nucleases, recombinases, and other enzymes that reduce the stability, purity, and cloning efficiency of the strain's DNA.

[0055] Furthermore, E. coli possesses regulatory pathways that restrict or regulate the expression of other products, which are sometimes desirable to have in large quantities (e.g., nucleotides). Therefore, it is difficult to increase the efficiency of E. coli in producing the desired product while the genes controlling these pathways are activated.

[0056] Numerous developments of strains and vector engineering that significantly improve plasmid DNA production are provided. Improvements include the identification and manipulation of an enzyme (PrsA) in E. coli that yields higher plasmid DNA yields when overexpressed. Variants of this enzyme that significantly disrupt feedback repression by downstream metabolites have been developed and incorporated into host cells. In the host, the variant enzyme can kineticate carbon through the cellular DNA biosynthesis pathway. Other engineering developments include knockout of PurR, a repressor of the DNA synthesis pathway from the genome. E. coli strains incorporating the engineered improvements described herein have shown significantly improved yields, with increases of more than twofold, for example, in plasmid yield, representing a fairly significant improvement.

[0057] While E. coli has been used in various ways, its use in specific applications remains limited, and in some cases, many desired features are left unmet. The improved strains described herein offer significant advantages over prior art strains. The strains disclosed herein include various combinations of manipulated components, for example, deletions or mutations of the EcoKI restriction system, endA deletion (ΔendA, an endonuclease that can degrade plasmid DNA during purification), recA deletion (ΔrecA, a recombinase that causes instability of DNA and poly(A) tails), addition of the PrsA enzyme, deletion of purR (encoding a transcriptional repressor in the nucleotide biosynthesis pathway), and / or deletions of one or more of mrr, hsdR, hsdM, hsdS, symE, and mcrBC.

[0058] Enhanced methods for plasmid DNA production, as well as tools and compositions involved in those methods, are also provided. A first-generation custom E. coli strain, referred to as strain 1, contains two gene deletions: ΔendA (an endonuclease that can degrade plasmid DNA during purification) and ΔrecA (a recombinase that is the main cause of DNA and poly(A) tail instability). This strain was further manipulated to remove the EcoKI restriction system in order to produce a new strain referred to herein as strain 2.

[0059] Naturally occurring E. coli possesses the EcoKI restriction system. EcoKI is a restriction-modification enzyme complex responsible for the identification and restriction of unmethylated foreign DNA, as well as the modification of naturally occurring hemimethylated DNA through methylation for self-recognition. If left untreated, the EcoKI system recognizes unmethylated DNA as foreign and degrades any DNA that also possesses its own EcoKI recognition site. While inactivating the EcoKI system from E. coli is not essential for cloning plasmid DNA, deletion of the EcoKI recognition site in the desired plasmid DNA significantly improves the efficiency of cloning and transformation.

[0060] Accordingly, in one embodiment, the disclosure relates to a recombinant strain of E. coli comprising an E. coli genome having at least the following gene deletions: endA and recA. The endA gene encodes an endonuclease 1 protein that, when expressed, can induce double-strand break activity. This activity can degrade, or otherwise impair, the plasmid DNA products of the E. coli possessing the gene. The recA gene encodes a recA protein associated with DNA repair and maintenance. However, recA plays a role in mediating homologous recombination of DNA, as well as homologous pairing, homologous recombination, DNA break repair, and the SOS response, through its property of promoting DNA repair, which triggers the cell cycle to inhibit the initiation of DNA repair and mutagenesis. The properties of both endA and recA are not beneficial for consistent and identical DNA plasmid products. In some embodiments, the recombinant strain of E. coli comprises an E. coli genome lacking endA and recA.

[0061] In some embodiments, this disclosure relates to recombinant strains of E. coli, the E. coli genome further includes exogenous DNA encoding a purine biosynthesis enzyme. The exogenous DNA is integrated into the E. coli genome. Integrating a prsA* expression cassette encoding a mutant purine biosynthesis enzyme into the genome of strain 2 or strain 1 provides a significant increase in plasmid DNA yield. A strain designed from strain 2 and to which prsA* is added is referred to as strain 3. Strain 3 can be further modified by knocking out purR, which encodes a transcriptional repressor of the nucleotide biosynthesis pathway. This strain, referred to herein as strain 4, may have further enhancements. When strain 4 was tested for plasmid DNA productivity together with strains 1 and 3, each of strains 1, 3, and 4 showed a higher and improved plasmid DNA yield than the original E. coli strain (see Figure 8A). Of the three strains tested, strain 1 showed a lower yield than strain 3, which showed a lower yield than strain 4. The stability of the poly-A tail was also found to improve in strain 4 after transformation and over multiple generations of propagation (see, for example, Table 4, which shows that strain 4 showed improved stability of the poly-A tail after transformation compared to the commercially available strain (control) and strain 1).

[0062] In some embodiments, the present invention encompasses E. coli strains containing a gene encoding the phosphoribosyl pyrophosphate synthetase protein (prsA). In other embodiments, the present invention encompasses E. coli strains containing a gene encoding the phosphoribosyl pyrophosphate synthetase protein (prsA*) variant. E. coli strains may contain the prsA variant. In some embodiments, E. coli strains may contain prsA and the prsA variant. PRPP (phosphoribosyl pyrophosphate) is a pentose phosphate formed from ribose 5-phosphate and one ATP by the enzyme phosphoribosyl pyrophosphate synthetase encoded by the gene prsA. The production of phosphoribosyl pyrophosphate synthetase is an initial step in the biosynthesis of purines, pyrimidines, and nicotinamide nucleotides, as well as in the biosynthesis of histidine and tryptophan.

[0063] A prsA variant refers to a nucleic acid encoding a variant of the enzyme phosphoribosyl pyrophosphate synthetase that differs from the naturally occurring enzyme phosphoribosyl pyrophosphate synthetase by at least one amino acid. Preferably, the prsA variant is resistant to negative feedback regulation by downstream metabolites of the DNA biosynthesis pathway. This resistance to negative feedback regulation prevents the pathway from shutting down to conserve energy, thereby accelerating the process of nucleic acid synthesis.

[0064] In some embodiments, the prsA variant has at least 70% sequence identity with prsA. In some embodiments, the prsA variant contains a sequence that has at least 70% sequence identity with prsA. In some embodiments, the prsA variant contains a sequence that has at least 70% sequence identity with prsA, but includes at least one nucleotide difference, i.e., deletion, insertion, or substitution. In some embodiments, the prsA variant contains prsA* (SEQ ID NO: 23). In some embodiments, the prsA variant is prsA* (SEQ ID NO: 23). prsA* is also referred to as prsA_D128A. In another embodiment, the prsA variant includes a nucleic acid sequence having at least 70% identity with respect to sequence number 23 (for example, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, and at least 99.9% identity).

[0065] "Percent identity," "sequence identity," "% identity," or "% sequence identity" (which may be used interchangeably herein) of two sequences (e.g., nucleic acids or amino acids) refers to a quantitative measurement of the similarity between two sequences (e.g., nucleic acids or amino acids). Percent identity can be determined using the algorithm of Karlin and Altschul, Proc.Natl.Acad.Sci.USA90:5873-77,1993, as modified by Karlin and Altschul, Proc.Natl.Acad.Sci.USA87:2264-68,1990. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J.Mol.Biol.215:403-10,1990. BLAST protein search can be performed using the XBLAST program, score=50, and word length=3 to obtain amino acid sequences homologous to the target protein molecule. If a gap exists between two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using the BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) may be used. Where percentage identity or a range (e.g., at least, more) is specified, unless otherwise specified, the endpoint is inclusive, and the range (e.g., at least 70% identity) includes all ranges within the cited range.

[0066] Some embodiments include E. coli strains containing genomes lacking the functional repressor gene purR. Genetic modification of E. coli strains to mitigate the effects of the feedback inhibitor / repressor purR may be useful to further promote plasmid DNA synthesis in the systems disclosed herein. In some embodiments, the purR gene is disrupted in E. coli by causing a frameshift mutation or by knocking out the gene. Disruption of gene function may be achieved by altering the normal encoding of the functional enzyme purR by the purR gene, thereby reducing or eliminating the production of the functional enzyme in the microorganism. Disruption broadly includes gene deletion, as well as gene modifications that affect mRNA transcription levels and / or stability and alter the promoter or repressor upstream of the polypeptide-encoding gene (e.g., introduction of a stop codon, frameshift mutation, introduction or removal of a portion of a gene, introduction of a degradation signal). In some embodiments, gene disruption is interpreted as any genetic modification to an amino acid sequence that results in at least a 50% reduction in the enzymatic function of DNA, mRNA encoded from DNA, and / or a gene encoded by a microorganism. In some embodiments, purR includes wild-type purR. In some embodiments, purR includes a sequence having at least 70% identity to wild-type purR. In some embodiments, purR includes a sequence having at least 70% identity to SEQ ID NO: 25. In some embodiments, purR includes the sequence of SEQ ID NO: 25. In some embodiments, purR has the sequence of SEQ ID NO: 25.

[0067] Therefore, in some embodiments, the E. coli strain expresses a prsA variant such as prsA* and / or purR expression is suppressed. In some embodiments, both plasmids can express prsA* or knock out purR.

[0068] In some embodiments, this disclosure relates to genotype: | <repA101|ori101_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)> Regarding recombinant plasmids containing |gamma>|beta>|exo>|60a>|.

[0069] In some embodiments, the recombinant plasmid contains a nucleic acid sequence having at least 70% identity with SEQ ID NO: 26. In some embodiments, the recombinant plasmid contains the nucleic acid sequence of SEQ ID NO: 26.

[0070] In some embodiments, this disclosure relates to recombinant strains of E. coli comprising plasmids, wherein the plasmid is genotype | <repA101|ori101_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)> |gamma>|beta>|exo>|60a>|, having nucleic acids that are at least 70% identical to SEQ ID NO: 26. In one embodiment, the disclosure relates to a recombinant strain of E. coli, wherein the plasmid has the genotype | <repA101|ori101_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)> This plasmid contains |gamma>|beta>|exo>|60a>|.

[0071] Both strains 3 and 4 were found to exhibit higher plasmid DNA yields compared to strain 1. Strain 3 produced higher pDNA than strain 4 after 16 hours of EFT (elapsed fermentation time). The yield of strain 3 was statistically higher than that of strain 1 within a 95% confidence interval. The specific productivity (mg / L) of strain 4, calculated as pDNA produced per gram of biomass, was found to be significantly higher than that of strain 1.

[0072] In some embodiments, the E. coli genome further includes at least one gene deletion selected from the group consisting of mrr, hsdR, hsdM, hsdS, symE, and mcrBC.

[0073] The mrr gene encodes the protein mrr, which is involved in the recognition and regulation of foreign DNA, particularly restricting (i.e., degrading) adenine and cytosine methylated DNA. The hsdR gene encodes the type I restriction enzyme EcoKI R protein, which produces endonuclease cleavage of nucleic acids (e.g., DNA), conferring random double-stranded fragments with terminal 5'-phosphates, and ATP is simultaneously hydrolyzed. The hsdM gene encodes the type I restriction enzyme EcoKI M protein, and the hsdS gene encodes the type I restriction enzyme EcoKI-specific (S) protein. The M and S subunits together form a methyltransferase (MTase) that methylates two adenine residues on the complementary strand of the binary DNA recognition sequence. In the presence of the R subunit, the complex also functions as an endonuclease, binding to the same target sequence but cleaving the DNA at a site somewhat distant from it. Whether the DNA is cleaved or modified depends on the methylation status of the target sequence. If the target site is not modified, the DNA is cleaved. When the target site is hemimethylated, the complex functions as a maintenance MTase that modifies the DNA so that both strands are methylated (UniProt, www.uniprot.org / uniprot / P05719). The symE gene encodes the toxic protein SymE, a protein involved in the degradation and recycling of damaged RNA. Overexpression of the SymE protein can be toxic to cells and affects colony formation ability and protein synthesis. The mcrBC gene encodes the 5-methylcytosine-specific restriction enzyme McrBC, subunit McrB. McrB is an endonuclease that cleaves DNA containing 5-methylcytosine or 5-hydroxymethylcytosine in one or both strands. In some embodiments, the E. coli genome further includes at least two gene deletions selected from the group including mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further includes at least three gene deletions selected from the group including mrr, hsdR, hsdM, hsdS, symE, and mcrBC.In some embodiments, the E. coli genome further includes at least four gene deletions selected from the group including mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further includes at least five gene deletions selected from the group including mrr, hsdR, hsdM, hsdS, symE, and mcrBC. In some embodiments, the E. coli genome further includes gene deletions: mrr, hsdR, hsdM, hsdS, symE, and mcrBC.

[0074] In some embodiments, the E. coli genome is derived from E. coli strain MG1655 or strain 1. In some embodiments, the E. coli genome is derived from E. coli strain 4 (strain 4>ΔendA ΔrecA Δmrr-mcr::P(J23119)>prsA* ΔpurR).

[0075] In several embodiments, engineered nucleic acid vectors having unique structural and functional attributes for enhanced plasmid production are provided. The nucleic acid vectors described herein have been engineered and synthesized using novel combinations of elements. The resulting nucleic acid vectors with one or more design modifications have been found to have significantly increased supercoil product yields.

[0076] Efforts in vector engineering for plasmid DNA production have primarily focused on increasing plasmid DNA copy number and plasmid higher-order coil formation. Herein, we have discovered that combinations of several modifications to plasmid structure result in significant and unexpected improvements in plasmid DNA yield and quality. These modifications include combinations of replacing the RNAII native promoter (replication primer) with a stationary-phase induction promoter, introducing point mutations that induce the formation of the critical stem-loop SL4 on RNAII necessary for initiating plasmid DNA replication, and / or incorporating a primosome assembly site into the plasmid backbone.

[0077] In several embodiments, these modifications to the plasmid origin (such as the plasmid shown in Figure 16) were used to construct new enhanced plasmids. Exemplary modified plasmids include plasmid 1 (+PAS+P(osmY)) and plasmid 2 (+PAS+P(osmY)+SL4). Plasmid 1 contains a stationary-phase induction promoter, a native RNAII promoter (replication primer) substituted with P(osmY), and a primosome assembly site (PAS) inserted into the backbone. Plasmid 2 is a modification of plasmid 1, further adding the introduction of four point mutations that promote the formation of SL4, a critical stem-loop on RNAII necessary for initiating pDNA replication. These plasmids were tested in various assays, and the plasmid DNA yields obtained with plasmids 1 and 2 were found to be significantly higher compared to the control plasmid plasmid, plasmid 1 (SEQ ID NO: 19) (Figures 17A and B). Furthermore, the introduction of PAS was shown to significantly increase the proportion of plasmid DNA, which is a higher-order coil-forming monomer (Figure 4).

[0078] The RNAII promoter initiates plasmid DNA replication. The copy number can be controlled by the relative ratio of RNAII (primer) to RNAI (inhibitor). It was determined that fine-tuning the intensity and timing of RNAII expression could reduce E. coli overload and increase plasmid yield. The RNAII promoter was subjected to various modifications to increase RNAII expression, including point mutations and the addition of a dedicated RNAII promoter. Attempts to completely remove the RNAII promoter and replace it with the stationary-phase-increased E. coli promoter proved largely toxic, resulting in the failure of the strains to survive. In stark contrast, replacing the native E. coli RNAII promoter with the stationary-phase promoter P(osmY) promoter showed significant improvement. The ratio of osmY transcripts was approximately 50 times higher in the stationary phase compared to the logarithmic phase.

[0079] In some embodiments, the present invention is a plasmid comprising a functional P(osmY) promoter. In some embodiments, the plasmid does not have a functional RNAII promoter. The functional P(osmY) promoter may contain a sequence having at least 70% sequence identity to SEQ ID NO: 27. In some embodiments, the P(osmY) promoter is SEQ ID NO: 27. In another embodiment, the P(osmY) promoter includes a nucleic acid sequence having at least 70% identity with respect to sequence number 27 (for example, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, and at least 99.9% identity).

[0080] Furthermore, stem-loop 4 (SL4) mutations are performed to block RNAI repression. SL4 mutations can increase the rate of SL4 formation, and therefore increase the replication rate.

[0081] The presence of poly(A) tails significantly affects plasmid higher-order coil formation and isomer distribution. The loss of higher-order coil formation was found to be offset by incorporating PAS into the plasmid. Adding PAS significantly increased the proportion of higher-order coil-forming monomers and slightly improved yield.

[0082] In addition to evaluating the novel strains disclosed herein with existing vector backbones, two strains, strain 1 and strain 4, were analyzed with plasmid 1, an optimally engineered vector. With plasmid 1 vector, both strains 1 and 4 produced similar amounts of plasmid DNA, which was twice as high as the plasmid DNA produced with the basic vector (Figure 15).

[0083] A “nucleic acid” is at least two nucleotides covalently linked to each other, and may optionally include a phosphodiester bond (e.g., a phosphodiester “backbone”). As used herein, the terms “nucleic acid sequence” and “polynucleotide” are used interchangeably and do not imply any length limitation. As used herein, the terms “nucleic acid” and “nucleotide” are used interchangeably. The terms “nucleic acid sequence” and “polynucleotide” encompass DNA (including cDNA) and RNA sequences. The nucleic acid sequences of the present invention include nucleic acid sequences removed from their naturally occurring environments, recombinant or cloned DNA isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0084] "Engineered nucleic acids" are nucleic acids that do not exist in nature. However, it should be understood that while engineered nucleic acids as a whole do not exist in nature, they may contain naturally occurring nucleotide sequences. In some embodiments, engineered nucleic acids contain nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, engineered nucleic acids contain bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids. "Recombinant nucleic acids" are molecules constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) and, in some embodiments, can replicate in living cells. "Synthetic nucleic acids" are molecules that have been amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that are chemically modified or otherwise modified, but can form base pairs with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules resulting from any of the aforementioned replications. Nucleic acids may contain naturally occurring nucleotides and / or nucleotides that do not exist in nature, such as modified nucleotides.

[0085] The engineered nucleic acids described herein can be produced using molecular biological methods. In some embodiments, the engineered nucleic acids are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, DG et al. Nature Methods, 343-345, 2009 and Gibson, DG et al. Nature Methods, 901-903, 2010). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, 3' extension activity of DNA polymerase, and DNA ligase activity. The 5' exonuclease activity chews back the 5' terminal sequence, exposing the complementary sequence for annealing. The polymerase activity fills the gap in the annealed region. The DNA ligase then seals the nick, covalently joining the DNA fragments. The overlapping sequences of adjacent fragments are much longer than those used in golden gate assembly, resulting in a higher rate of correct assembly.

[0086] The nucleic acid vectors of the present invention may also have one or more termination sequences, which may or may not be present. A termination sequence is a nucleic acid sequence that signals the end of an expression cassette or transcription region. A valid transcription vector typically contains one or more termination sequences. These termination sequences may include, for example, T7 and T4 termination sequences.

[0087] Preferred vectors of the present invention may also have one or more resistance markers, or markers specific to a particular vector. For example, a vector may originally have an ampicillin resistance marker. In some preferred embodiments of the present invention, the ampicillin marker is replaced with a different marker, such as a kanamycin resistance marker. In some embodiments, the E. coli genome disclosed herein may further express a gene for a positive selection marker based on a first environmental factor or a negative selection marker based on a second environmental factor, where the first and second environmental factors are not the same. In some embodiments, the E. coli genome disclosed herein may further express a gene for a positive selection marker based on a first environmental factor and a gene for a negative selection marker based on a second environmental factor, where the first environmental gene and the second environmental factor are not the same. In some embodiments, the positive selection marker is a gene that can confer kanamycin resistance. In some embodiments, the negative selection marker may express levansculas.

[0088] The vectors disclosed herein may also have any pathogen-derived sequences removed. Removing pathogen-derived sequences can have a positive effect on product yield.

[0089] An origin of replication (ori) can be included in a nucleic acid and can be modified as disclosed herein. In some embodiments, the nucleic acid may include several ori, for example, two ori. This could be, for example, a combination of a low-copy-number ori and a temperature-dependent ori, or an ori that enables growth in various host organisms.

[0090] In some embodiments, the plasmid contains an engineered nucleic acid vector. In some embodiments, the plasmid is replicated. In some embodiments, the plasmid contains plasmid 1 (sequence number 19). In some embodiments, the plasmid contains a sequence having at least 70% identity to sequence number 19.

[0091] In some embodiments, the plasmid includes an origin of replication (ori). In some embodiments, the plasmid includes an ori containing a sequence that has at least 70% identity to sequence number 16. In some embodiments, the plasmid includes an ori containing the sequence of sequence number 16. In some embodiments, the ori includes at least one mutation. In some embodiments, the ori mutation includes at least one of the following ori1 to ori16. In some embodiments, the ori includes a sequence that has at least 70% identity to any one of sequence numbers 1 to 15. In some embodiments, the ori includes a sequence that has at least 70% identity to sequence number 10. In some embodiments, the ori includes a sequence that has at least 70% identity to sequence number 11. In some embodiments, the ori includes a sequence that has at least 70% identity to any one of sequence numbers 1 to 15. In some embodiments, the ori includes the sequence of sequence number 10. In some embodiments, the ori includes the sequence of sequence number 11.

[0092] Nucleic acids may also contain one or more elements from other vectors. For example, other vectors include phages, cosmids, fasmids, fosmids, bacterial artificial chromosomes, yeast artificial chromosomes, viruses and retroviruses (e.g., vaccinia, adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, Epstein-Barr virus, fowlpox virus, pseudorabies virus, baculovirus) and vectors derived therefrom. In other embodiments, the nucleic acids described herein do not contain any elements from any one or more other vectors.

[0093] When applied to nucleic acid sequences, the term “isolated” in the context of this invention means that a polynucleotide sequence has been removed from its natural genetic environment and is therefore free from other exogenous or undesirable coding sequences (although it may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators) and is in a form suitable for use in a genetically engineered protein production system. Such an isolated molecule is a molecule that has been separated from its natural environment.

[0094] Therefore, in some embodiments, the nucleic acid vector has the nucleic acid sequence of SEQ ID NO: 21. In other embodiments, the nucleic acid vector of the present invention has a nucleic acid sequence having at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99% sequence identity with respect to SEQ ID NO: 22.

[0095] A nucleic acid sequence or fragment thereof is "substantially homologous" or "substantially identical" to a reference sequence if, when optimally aligned with another nucleic acid (or its complementary strand) (with appropriate nucleotide insertions or deletions), it has sequence identity in at least about 70%, 75%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, or 99% of its nucleotide bases. Methods for determining sequence identity of nucleic acid sequences are known in the art.

[0096] A "variant" nucleic acid sequence is substantially homologous (or substantially identical) to the reference sequence (or a fragment thereof) if the "variant" sequence and the reference sequence can hybridize under stringent (e.g., highly stringent) hybridization conditions. Nucleic acid sequence hybridization is influenced by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvent, in addition to base composition, complementary strand length, and the number of base mismatches between the nucleic acids being hybridized, as will be readily apparent to those skilled in the art. Stringent temperature conditions are preferred, generally including temperatures above 30°C, typically above 37°C, and preferably above 45°C. Stringent salt conditions are usually less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. pH is typically between 7.0 and 8.3. Combinations of parameters may be more important than any single parameter.

[0097] Numerous algorithms are available for aligning two nucleic acid sequences. Typically, one sequence acts as a reference sequence, which can be compared to the test sequence. Sequence comparison algorithms calculate the sequence identity percentage of the test sequence(s) to the reference sequence based on specified program parameters. Algorithmization of nucleic acid sequences for comparison can be performed using algorithms performed on a computer (e.g., GAP, BESTFIT, FASTA, or TFASTA), or by the BLAST and BLAST2.0 algorithms.

[0098] In sequence identity comparison, identity can exist over a region of the sequence that is at least 10 nucleic acid residues long, for example, at least 15, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 685 nucleotides long, up to, for example, the full length of the reference sequence.

[0099] Substantially homologous or substantially identical nucleic acids have one or more nucleotide substitutions, deletions, or additions. In many embodiments, these changes are of a minor nature, including only conservative nucleic acid substitutions, such as those involving the same amino acid encoded during translation, or different but conserved amino acid substitutions. Conservative amino acid substitutions are those made by replacing one amino acid with another within the following groups: basic: arginine, lysine, histidine; acidic: glutamic acid, aspartic acid; polar: glutamine, asparagine; hydrophobic: leucine, isoleucine, valine; aromatic: phenylalanine, tryptophan, tyrosine; minor: glycine, alanine, serine, threonine, methionine; substantially homologous nucleic acids also include those containing other substitutions that do not significantly affect the folding or activity of the translation product.

[0100] The nucleic acid vector of the present invention may be an empty vector or may contain an insertion that may be an expression cassette or an open reading frame (ORF). An "open reading frame" is a continuous extension of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a protein or peptide. An expression cassette encodes RNA comprising at least the following elements: a 5' untranslated region, an open reading frame region encoding mRNA, a 3' untranslated region, and a poly-A tail. An open reading frame may encode any mRNA.

[0101] The "5' untranslated region (UTR)" refers to the region of mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) that does not code for a protein or peptide.

[0102] The "3' untranslated region (UTR)" refers to the region of mRNA immediately downstream (i.e., 3') of a stop codon that does not code for a protein or peptide (i.e., the codon of the mRNA transcript that signals the end of translation).

[0103] A "poly-A tail" is a region of mRNA located downstream of the 3' UTR, for example, immediately downstream (i.e., 3'), that contains multiple consecutive adenosine monophosphates. A poly-A tail can contain 10 to 300 adenosine monophosphates. For example, a poly-A tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains 50 to 250 adenosine monophosphates. In relevant biological environments (e.g., intracellular, in vivo), poly(A)tails function to protect mRNA from enzymatic degradation (e.g., in the cytoplasm) and to assist in transcription termination, mRNA transport from the nucleus, and translation.

[0104] Those skilled in the art will understand that different species exhibit “preferred codon use.” As used herein, the term “preferred codon use” refers to the codon most frequently used in cells of a particular species, and therefore favors one or more representative codons that may code for each amino acid. For example, the amino acid threonine (Thr) can be coded by ACA, ACC, ACG, or ACT, but in mammalian host cells, ACC is the most commonly used codon. In other species, different Thr codons may be preferred. Preferred codons for a particular host cell species can be introduced into the polynucleotides of the present invention by various methods known in the art. Alternatively, unfavorable codons may be used. In some embodiments of the present invention, nucleic acid sequences are codon-optimized.

[0105] A “fragment” of the polynucleotide of interest comprises a sequence of consecutive nucleotides from the sequence of the full-length polynucleotide. For example, a “fragment” of the polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of the polynucleotide (e.g., at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 consecutive nucleic acid residues of the polynucleotide).

[0106] A “nucleic acid vector” is a polynucleotide that carries at least one foreign or heterologous nucleic acid fragment. A nucleic acid vector can function like a “molecular carrier,” delivering the nucleic acid fragment, the polynucleotide, to a host cell, or functioning as an IVT template. As used herein, “in vitro transcription (IVT) template” refers to deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some embodiments, the IVT template encodes a 5' untranslated region, includes an open reading frame, and encodes a 3' untranslated region and a poly-A tail. The specific nucleotide sequence composition and length of the IVT template depend on the target mRNA encoded by the template.

[0107] In some embodiments, the nucleic acid vector according to the present invention is a cyclic nucleic acid such as a plasmid. In other embodiments, it is a linearized nucleic acid. According to one embodiment, the nucleic acid vector includes predetermined restriction sites that can be used for linearization of the vector. Intelligent placement of the linearization restriction sites is important because the restriction sites determine where the vector nucleic acid is opened / linearized. It is preferable that the restriction enzyme selected for linearization does not cleave within critical components of the vector.

[0108] The terms 5' and 3' are used herein to describe the characteristics of nucleic acid sequences relating to the position of gene elements and / or the direction of events (5' to 3'), such as transcription by RNA polymerase or translation by ribosomes proceeding in the 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Traditionally, DNA sequences, gene maps, vector cards, and RNA sequences are drawn from left to right in the 5' to 3' direction, or the 5' to 3' direction is indicated by an arrow pointing in the 3' direction. Thus, following this convention, 5' (upstream) indicates gene elements positioned towards the left-handed side, and 3' (downstream) indicates gene elements positioned towards the right-handed side. [Examples]

[0109] Example 1: Modification of the host strain alters plasmid production Introduction: Purpose and Significance E. coli is a microorganism that has been used for cloning purposes and plasmid DNA production. High-yielding strains, especially those that produce plasmids on a large scale, are valuable. Methods for increasing plasmid DNA yield from E. coli using various metabolic engineering techniques are disclosed herein. In some cases, the endogenous DNA restriction system EcoKI is removed, improving the cloning efficiency of unmethylated plasmids.

[0110] E. coli strains currently available for cloning plasmid DNA E. coli K12 derivatives using DH5α, JM108, DH10β, etc., have been used in plasmid DNA cloning and production. These primarily result in the inactivation of genes encoding nucleases, recombinases, and other enzymes that reduce the stability, purity, and cloning efficiency of strain DNA. Here, we demonstrate the inactivation of all or part of the EcoKI restriction system, enabling the cloning of eukaryotic or unmethylated DNA. If left untreated, the EcoKI system recognizes unmethylated DNA as foreign and degrades it if it also possesses its own EcoKI recognition site. While inactivating the EcoKI system from E. coli is not essential for plasmid DNA cloning, it significantly improves cloning and transformation efficiency if the desired plasmid DNA contains the EcoKI recognition site (Table 1). [Table 1]

[0111] Nucleotide biosynthesis in E. coli to increase the flux through the pathway Nucleotide biosynthesis is a carbon, energy, and redox-intensive process; therefore, the expression of cellular nucleotide biosynthetic pathways is strictly controlled by transcriptional repression, and furthermore, several key enzymes in these pathways are allosterically regulated by downstream metabolites and / or cofactors that indicate the cell's low-energy state. Briefly, pyrimidines and purines are produced using 5-carbon precursor, 5-phospho-α-D-ribose 1-diphosphate (PRPP), which functions as the main building block of nucleotides. This metabolite is produced by ribose phosphate diphosphokinase (PrsA) from D-ribose 5-phosphate (R5P), an intermediate in the pentose phosphate pathway. Because PRPP synthesis involves carbon in the energy-intensive nucleotide biosynthesis pathway, cells strictly regulate this step by controlling the expression of the prsA gene and by regulating the activity of the PrsA enzyme through allosteric repression by ADP. E. coli also possesses PurR, a key transcriptional regulator of the pyrimidine and purine biosynthesis pathways, which itself is regulated by inosine and guanine, products of the purine pathway. Increased intracellular concentrations of inosine and / or guanine cause the metabolites to associate with PurR enzymes, inducing the binding of regulons to the promoters of 32 PurR genes, thereby repressing the expression of nucleotide biosynthesis pathways. Indeed, knockout of the purR gene from the E. coli genome significantly increases the transcription of genes normally repressed by PurR. No examples of metabolic manipulation of the nucleotide biosynthesis pathway in E. coli to improve plasmid DNA productivity are known.

[0112] In this process, E. coli strain 1 was created, and subsequently, offspring of strain 1 were created with improved plasmid DNA yield (pDNA mg / biomass mg or plasmid copy number) and higher cloning efficiency by increasing the activity of the purine and pyrimidine biosynthesis pathways and by removing the EcoKI restriction system, respectively.

[0113] method [Table 2] [Table 3] Measurement of plasmid yield of E. coli strain in a shaking flask Each strain of E. coli was transformed with plasmids as specified in the data. The cultures were incubated in a 500 ml shaking flask containing 60 ml of TB animal-free (TBAF) broth (Teknova, cat#T7660) with 50 mM MOPS (Teknova, cat#M8405) and 50 μg / ml kanamycin (Teknova, cat#K2125) from colonies or glycerol stocks as specified, at 37°C and 300 rpm. Growth was measured using absorbance at 600 nm, and plasmid yield was obtained by alkaline lysis and ULC analysis of cell pellets from each culture.

[0114] Measurement of E. coli strain plasmid productivity in an Ambr250 bioreactor Seed Fermentation: For seed fermentation, the culture medium was prepared by adding 1 mL of 50 mg / ml kanamycin stock and 100 μL of 10% antifoaming agent 204 per liter of TBAF medium. 18.75 mL of the seed medium was aseptically added to a 125 mL baffled shaking flask, and 94 μL of thawed seed culture from glycerol stock was seeded. The seed flask was incubated in a stirring incubator at 37°C at 250 RPM (orbital diameter 1 inch) for 4–5 hours until an OD600 of 0.6–0.8 was reached (targeting intermediate logarithmic growth). This seed culture was transferred to AMBR vessels for seeding at 0.1% (v / v) bacterial species.

[0115] Production Fermentation: The basal medium for fermentation was TBAF containing 50 mg / ml kanamycin at a concentration of 1 ml / L. 160 mL of this medium was added sterile to each AMBR vessel and batched with 16 mL of 50% sterile glycerol (60 g / L glycerol batch) and 1 mL of 10% sterile defoamer 204. The pH during fermentation was maintained at 7.3 ± 0.1 using 15% ammonium hydroxide and 50% (v / v) glycerol (pH stat carbon source supply). The temperature was maintained at 37 ± 0.5°C during fermentation. Dissolved oxygen (DO) was maintained at 30% saturation using a stirring lamp at 700–3000 RPM, followed by oxygen concentration of 21–40%. The airflow was maintained constant at 1.0 VVM throughout the fermentation. TBAF supply was started at 2 ml / hour during a 12-hour EFT. Samples were taken from each container at regular intervals for plasmid DNA measurement (using minipreps followed by nanodrops), biomass measurement (OD600 and g / l wet cell weight (WCW)), and residual metabolite analysis (glycerol, acetate, phosphate, and ammonia).

[0116] Measurement of plasmid copy number in E. coli strains possessing one or more plasmids for production. Plasmid copy number (PCN) was determined using a TaqMan-based (Life Technologies) quantitative PCR (qPCR) method as follows: In short, E. coli cultures were centrifuged, resuspended in water, and diluted (10 -1 →10 -7 After dilution, the sample was heated at 98°C for 10 minutes to lyse the cells, and then transferred to a qPCR plate containing the enzyme mix, primers, and probe. PCN was determined by the ΔΔCt method (difference in Ct values ​​of plasmid DNA and genomic DNA at a given dilution) and by calculating the relative plasmid:genomic DNA ratio using standard curves for plasmid DNA and E. coli DNA.

[0117] Construction of a knockout cassette The knockout cassette for strain manipulation contained a DNA cassette encoding a kanamycin resistance marker (kan) in addition to sacB (encoding the enzyme levansculas) for negative selection. To enable the integration of this kan-sacB knockout cassette into the correct position in the genome, small 45 bp homologous regions upstream and downstream were added to the knockout cassette using PCR (Figure 2) and Hercules II DNA polymerase (Agilent, Cat#600697). The knockout cassette was amplified from plasmid 5, which was generated internally and contained the kan-sacB cassette (Figure 3).

[0118] Introduction of genome deletion that leaves no trace of E. coli The genetically modified strains were first transformed with plasmid 6 (Figure 3), and transformants were selected by seeding onto LB animal-free (LBAF) agar containing 100 μg / ml carbenicillin (Teknova, Cat#L1092). Next, a single transformant was cultured for 16 hours at 30°C in LBAF broth (Teknova cat#L8900-06) containing 100 μg / ml carbenicillin. Subsequently, 30 μl of this overnight culture was transferred to a test tube containing 3 ml of LBAF broth with 100 μg / ml carbenicillin (Teknova, Cat#C2135), and incubated at 30°C at 250 rpm for 2 hours. After a 2-hour incubation, expression of the gene encoding λ-RED and codon-optimized E. colirecA was induced using 100 ng / ml anhydrotetracycline (aTc, Fisher#AC233131000) and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Millipore cat#70527-3), respectively. Further 2-3 hours of shaking incubation at 30°C were followed by OD (Oral Dissociation). 600When the saturation was approximately 0.6–1.0, 1 ml of culture was harvested and 0.1 ml of electrocompetent cells were prepared. 50 μl of electrocompetent cells were mixed with 1 μg of purified knockout cassette and electroporated at 1800 volts in a 1 mm gap cuvette. Transformants were rescued in 1 ml of SOC medium (NEB cat#B9020S) at 30°C at 300 rpm for 2 hours, then seeded on LBAF agar containing 50 μg / ml kanamycin and 100 μg / ml carbenicillin (Teknova, cat#L3819) and incubated overnight at 30°C. Next, colony PCR (cPCR) using LongAmp Taq DNA polymerase (NEB, cat#M0287L) was used to screen for primary integrations using kanamycin resistance genes, universal primers that bind to kan, and site-specific primers that bind upstream of the genes targeted for knockout. In parallel with cPCR, the same clones were spotted onto LBAF agar containing 35 μg / ml kanamycin and 100 μg / ml carbenicillin, and onto LB agar containing 60 g / l sucrose (Teknova, cat#L1143). These plates were incubated overnight at 30°C. After confirmation of the primary integration by cPCR, sucrose sensitivity was confirmed by visually checking for the "no growth" phenotype with the clone spotted on LBAF agar containing 60 g / l sucrose. Once the primary integration clone was confirmed by cPCR and sucrose sensitivity was also confirmed, the knockout cassette was removed using a similar method described below.

[0119] To remove the designated knockout cassette and obtain a traceless deletion, linear dsDNA fragments containing only the UHR and DHR regions ("pop-out cassettes") were amplified from gBlock (IDT) and primers. The confirmed primary integrations were incubated at 30°C for 16 hours in LBAF broth containing 100 μg / ml carbenicillin and 50 μg / ml kanamycin. Subsequently, 30 μl of this overnight culture was transferred to a test tube containing 3 ml of LBAF broth containing 100 μg / ml carbenicillin and 50 μg / ml kanamycin, and incubated at 30°C at 250 rpm for 2 hours. After 2 hours of incubation, expression of the gene encoding λ-red and codon-optimized E. colirecA was induced using 100 ng / ml aTc and 1 mM IPTG, respectively. After further 2-3 hours of shaking incubation at 30°C, OD was performed. 600 When the saturation was approximately 0.6–1.0, 1 ml of culture was collected and 0.1 ml of electrocompetent cells were prepared. 50 μl of electrocompetent cells were mixed with 1 μg of purified pop-out cassette and electroporated at 1800 volts in a 1 mm gap cuvette. Transformants were rescued in 1 ml of SOC medium at 30°C at 300 rpm for 2 hours, and then transferred to a 125 ml shaking flask containing 9 ml of LBAF broth. Next, this diluted culture was grown at 30°C at 300 rpm for 5-16 hours. Subsequently, 50 µl of the culture was transferred to a test tube containing 5 ml of LBAF unsalted broth containing sucrose (10 g / l Soyton (BD Biosciences, cat#243620)), 5 g / l yeast extract (Fisher Scientific, cat#DF210929), and 60 g / l sucrose (Fisher Scientific, cat#S5-500). Next, the diluted culture was filter-sterilized using a 0.2 µM filter (Corning #430769). Then, this sucrose-containing culture was incubated overnight (approximately 16 hours) at 30°C at 250 rpm, and 10 ml was added to sterile LBAF broth. -6 The mixture was diluted and seeded onto LBAF agar medium (200 μl seeding), and incubated overnight at 37°C.

[0120] Once isolated colonies were obtained on LBAF agar plates, clones were screened using cPCR and primers that bind upstream and downstream of the knockout gene(s), successfully removing the knockout cassette (kan-sacB). In parallel, clones were spotted onto LBAF agar and LBAF containing 100 μg / ml carbenicillin. These plates were incubated overnight (16 hours) at 30°C to confirm the loss of plasmid 6, a temperature-sensitive plasmid necessary for genome editing. To construct strain 3, a linear "popout cassette" containing UHR_P(J23119) → prsA_D128A_DHR (UHR and DHR are specific to the region adjacent to the mrr-hsdRMS-symE-mcrBC locus) was used to simultaneously remove the kan-sacB knockout cassette from strain 5, enabling constitutive expression of prsA_D128A(prsA*).

[0121] Determination of the stability of the poly-A tail in strain 4. To determine the stability of the poly(A) tail after transformation, 50 μl of chemically competent cells from strain 4 or a control strain were transformed with the circular plasmids plasmids 1 and 2. Transformants were rescued in 1 ml of SOC at 30°C at 300 rpm for 1 hour and seeded on LBAF agar containing 50 μg / ml kanamycin. 96 colonies were collected from each transformant in 500 μl of TBAF + 50 μg / ml kanamycin and grown at 37°C at 300 rpm for 16 hours. Plasmid DNA was isolated and sent for Sanger sequencing of the poly(A) tail. The sequences were then analyzed using CNN analysis (developed in-house) to quantify the percentage of clones likely to possess the poly(A) tail.

[0122] To determine the stability of the poly(A) tail over multiple generations of growth, colonies were taken from strain 4, strain 1, and a control strain containing plasmid 2 (SEQ ID NO: 20), placed in test tubes containing 5 ml of TBAF with 50 μg / ml kanamycin, and incubated at 37°C at 300 rpm for 16-24 hours. The following day, the cultures were OD (Oxygen-Dose).600 Sampling was performed for 600 , and plasmid DNA was isolated. Next, 1 μl of each culture was used to inoculate a separate set of test tubes containing 5 ml of LBAF with 50 μg / ml kanamycin. This process was repeated for 6 days. Plasmid DNA from each strain was isolated by miniprep (Qiagen), and samples from each time point were sent for poly-A tail sequencing. The length of the poly-A tail was determined using Sanger sequencing and was 5 bases or less with a CV score < 30.

[0123] Preparation of glycerol stocks and competent cell banks To create glycerol stocks for long-term storage, strains 3 and 4 were streaked from glycerol stocks onto LBAF agar plates and incubated overnight at 30 °C. One colony of each strain was inoculated into 3 ml of TBAF broth in a 1-liter baffled shake flask and incubated at 30 °C at 250 rpm for 16 hours. The next day, 100 ml of TBAF broth in a 1-liter baffled shake flask was inoculated to an OD 600 = 0.05, and incubated at 30 °C at 250 rpm for 4 - 6 hours until an OD 600 of approximately 0.6 was reached. At this target OD 600 , 50 ml of sterile 50% glycerol was added to each culture, mixed, 700 μl was aliquoted into 1 ml FluidX tubes, and stored at -80 °C. The viability was determined by thawing one tube from each lot, inoculating the dilution onto LBAF agar plates, and incubating overnight at 30 °C.

[0124] To create competent cell banks, strains 3 and 4 were streaked from glycerol stocks onto LBAF agar plates and incubated overnight at 30 °C. One colony of each strain was inoculated into 100 ml of animal-free SOB broth (Teknova, cat# S2615) in a 1-liter baffled shake flask and incubated at 18 °C at 250 rpm for 30 hours. When a target OD 600 of approximately 0.2 was achieved, the cells were harvested, washed, and aliquoted into sterile FluidX tubes (50 μl per tube).

[0125] Transformation efficiency was determined by the average transformation efficiency obtained when 10 ng of plasmid 1 (SEQ ID NO: 19) was transformed into 50 μl of competent cells (n=2) at 42°C for 30 seconds, followed by incubation at 4°C for 2 minutes. 0.95 ml of SOC was added to the cells, and the vial was incubated at 30°C at 250 rpm for 1 hour, after which the cells were seeded on LBAF agar containing 50 μg / ml kanamycin.

[0126] Culture purity was determined by spreading 75 μL of each competent cell line, strain 3 and strain 4, onto both 1× trypsin soy agar (TSA) plates and 1× Sabouraud dextrose agar (SDA) plates, incubating the TSA at 30°C and the SDA at 22°C for 3–5 days, and then visually inspecting the plates for any accidental microbial growth. After 76 hours of incubation, no visible contaminant growth was observed on any of the plates.

[0127] result Construction of Stock 2, Stock 3, and Stock 4 Using strain 1 (Escherichia coli MG1655 ΔendA ΔrecA) as the parent strain, strains 2, 3, and 4 were created as shown in Figure 3. All necessary genetic modifications to the genome were performed as described in the Methods section and confirmed by PCR. All final strains were confirmed to be kanamycin-sensitive, carbenicillin-sensitive, and sucrose-insensitive. Furthermore, Sanger sequencing was performed on the PCR products produced to confirm the new genotypes. All strains were confirmed to have the correct intended DNA sequences at the modified genomic loci.

[0128] Removing the EcoKI restriction system improves transformation efficiency - strain 2 Wild-type E. coli K12 strain (e.g., parent of strain 1) possesses a native restriction endonuclease system (EcoKI) that degrades unmethylated DNA at its own EcoKI restriction site(s). We successfully removed the EcoKI restriction system from strain 1 to obtain strain 2. Upon completion, we confirmed the acquisition of the desired phenotype by attempting to transform strains 1 and 2 with methylated and unmethylated plasmids containing three EcoKI sites. Transformation of strain 1 with the methylated plasmid resulted in a bacterial flora, but transformation with the same unmethylated plasmid yielded no colonies, suggesting that the EcoKI system may have a significant negative impact on transformation efficiency. In contrast to strain 1, strain 2, due to the removal of EcoKI, exhibits similar transformation efficiency with either methylated or unmethylated plasmids. This allows strain 2 and its progeny to be used in cell banking workflows and higher-throughput cloning platforms such as preclinical DNA. PVU strain 2 can accept plasmid DNA from a methylation-deficient host (such as a control strain), or DNA cloned using gBlock or PCR products (unmethylated DNA fragments).

[0129] Overexpression of PrsA* in strain 1 increases plasmid yield in shaking flasks. Genetic targets were identified for overexpression resulting in increased plasmid DNA yield. A panel of single-copy overexpression plasmids, each carrying a unique codon-optimization gene as shown in Figures 5A–C, was tested using strain 1, which carries plasmid 1 (SEQ ID NO: 19), as the host to determine if synthetic overexpression of any of the tested genes could increase the copy number of a representative production plasmid. Growth and plasmid DNA yield were tested as shown in Figures 5A–C, and overexpression of prsA* significantly increased plasmid DNA yield (Figures 5A–C) and copy number (Figure 6). This variant enzyme possesses a mutation that removes ADP-mediated feedback repression, thus de-regulating a crucial step in providing PRPP, a metabolite of purine and pyrimidine synthesis. To create a plasmid-free strain that stably expresses this prsA variant, the intermediate strain 1Δ(mrr-hsdRMS-symE-mcrBC)::kan-sacB, created during the production of strain 2, was used to incorporate a constitutive expression cassette in place of the EcoKI system. The resulting strain, strain 3, is a descendant of strain 1 that possesses the locus encoding its EcoKI restriction, which has been replaced from the genome with constitutive expression of prsA*. The growth and plasmid productivity of strain 3 compared to strain 1 and the NEB stable type were assayed in a shaking flask. Plasmid DNA yield was significantly increased in strain 3 compared to its parent strain 1, similar to what was observed when prsA* was expressed from a single-copy plasmid (Figure 7A-B).

[0130] Inactivation of purR and overexpression of prsA* further improve plasmid yield in shaking flasks. The objective was to release the repression of 32 genes encoding enzymes for nucleotide biosynthesis by removing the transcriptional repressor PurR (Figure 1A-B) from strain 3. The structural changes that occur when PurR binds to guanine and hypoxanthine (products of the purine synthesis pathway) allow the enzymes to bind to the promoter of their regulons, resulting in transcriptional repression. The resulting strain 4, lacking PurR, has a higher carbon flux capacity for nucleotide synthesis than its parent strain 3. Flask experiments performed with strains 1, 3, and 4 indeed showed higher plasmid DNA yields for the most recent strain (Figure 8A-B) (strain 1 < strain 3 < strain 4). All strains tested grew well and produced similar final culture densities.

[0131] Strains 3 and 4 show higher plasmid DNA yields compared to strain 1 in the Ambr250 bioreactor. Figure 9A shows the dynamic profile of pDNA production. A statistical analysis of pDNA produced at 22 hours of EFT is shown in Figure 9B. Figure 9B shows that strain 3 is statistically higher than strain 1 with a 95% confidence interval (the two strains were compared using a controlled Dunnett test to compare statistics). Both strains produced comparable biomass. Therefore, for strain 3, the specific productivity (measured as WCW g / L), calculated as pDNA produced per gram of biomass (mg / L), was higher than that of strain 1 (up to approximately 1.2 × higher) (Figure 10). Fermentation results comparing the pDNA productivity of strains 4 and 1 (Figures 11A-B) showed that strain 4 produced more pDNA than strain 1 at all time points after 16 hours of EFT. Both strains 4 and 1 produced comparable biomass. Therefore, the specific productivity of strain 4, calculated as pDNA produced per gram of biomass (mg / L) (measured as WCW g / L), was significantly higher than that of strain 1 (up to approximately 1.8 × higher) (Figure 12). In summary, both strains 3 and 4 showed significantly higher specific plasmid DNA productivity compared to their parent strain 1 in the Ambr250 bioreactor, with strain 4 being the most productive (strain 1 < strain 3 < strain 4). Strain 4, when transformed with a circular plasmid, exhibits improved poly(A) tail stability compared to the NEB-stable strain.

[0132] To confirm that strain 4 maintains a poly(A) tail within the desired length specification (95–105 bp), two different plasmids containing high-quality poly(A) tails were transformed into strain 4 and the NEB stable form as a control. After growing 96 colonies from each transformation, plasmid DNA was isolated and the poly(A) tails were sequenced. To analyze the Sanger sequencing data produced in this experiment, an algorithm was used to determine clones that were likely to pass through the tail (a tail within the length and purity specifications). As shown below, clones taken from strain 4 were significantly more likely to pass through the poly(A) tail compared to the NEB stable form. [Table 4]

[0133] Strain 4 maintains the stability of the poly-A tail over multiple generations of propagation. In addition to maintaining the poly(A) tail after transformation, the long-term poly(A) stability of strain 4 was characterized compared to the NEB-stable strain. For strain 4, deletions of 3–5 base pairs in the poly(A) tail were observed after approximately 69 generations (Table 5, Figures 13A–C). These results are similar to those historically obtained for the NEB-stable strain and strain 1, indicating comparable long-term tail stability in strains 4, 1, and the NEB-stable strain. Importantly, no tail heterogeneity was observed after 69 generations of growth. Approximately 50 generations of growth in a large-scale pDNA production process on a 30-liter or 300-liter fermentation scale (Figure 13C), these data support the use of strain 4 as a host for pDNA production. [Table 5]

[0134] Growth profile of strain 4 for large plasmid DNA Strains 4 and 1, each possessing the indicated plasmid, were seeded in shaking flasks, and their growth profiles were evaluated compared to those of strain 1. As shown in Figure 14, strain 4 showed a longer growth delay, but the difference was slight.

[0135] Creation of competent cell banks for strains 3 and 4 Strains 3 and 4 were grown from colonies under sterile conditions in LBAF broth, and each strain was packed into 96 1 ml FluidX tubes (1 lot) and stored at -80°C. Furthermore, many chemically competent cells were prepared for each strain as described in the Methods section. These lots were QC tested for the presence of phages using a mitomycin C induction assay and tested to confirm the purity of the strains. No phages were detected, and the purity of each lot tested was confirmed (Table 6). The transformation efficiency obtained was sufficient for use and was equivalent to that obtained using strain 1. [Table 6]

[0136] conclusion A new strain of E. coli was created with improved cloning efficiency and plasmid DNA yield for use in high-throughput cloning processes. The EcoKI restriction system was removed from strain 1, thereby enabling efficient transformation with unmethylated DNA (e.g., gBlock, PCR products, and circular plasmids isolated from the NEB stable form). Several additional genomic modifications were then introduced, resulting in an increase in nucleotide biosynthesis pathways. The final strain, strain 4, readily accepts unmethylated plasmid DNA isolated from the NEB stable form, or DNA from Gibson assembly reactions using synthesized or PCR gene fragments. As shown herein, strain 4 also exhibits significantly higher plasmid DNA productivity (1.8 × ~ 2 ×) compared to the parent strain, strain 1, in shaking flasks and an Ambr250 bioreactor mimicking a large-scale GMP fermentation process. Further characterization of strain 4 also shows that the strain exhibits improved poly(A) tail stability compared to the NEB stable form during transformation events and maintains poly(A) tail purity across multiple generations of growth.

[0137] Example 2: Mutations in the plasmid replication mechanism affect plasmid production and replication efficiency. Mutations were introduced into the pUC origin (SEQ ID NO: 16) of the control plasmid PL_007984 (SEQ ID NO: 19) to increase plasmid titer. The manipulated segment of the pUC origin was synthesized as a double-stranded DNA fragment (IDT gBlock). The parent plasmid was digested with BssHII and ApaLI, and the gBlocks were cloned into the plasmid by Gibson assembly. The new variant plasmids were sequenced. The mutations created and tested were produced by introducing specific sequences of partial RNAII / I, which are shown herein as SEQ ID NOs: 1–15.

[0138] Replication was assessed by measuring plasmid production as mg / liter of pDNA. Modification 9 (Ori10, SEQ ID NO: 10) contains an early deletion of the RNAII transcription region. Modifications 9 and 10 (SEQ ID NOs: 10-11, respectively) showed a significant increase in titer (56% / 60% increase, respectively, Figure 18A). Strains containing modifications 9 and 10 (SEQ ID NOs: 10-11, respectively) also showed improved productivity. As shown in Figure 18B, modifications 9 and 10 (SEQ ID NOs: 10-11, respectively) had a greater weight of pDNA per gram of wet cell weight than the parent plasmid (Plasmid 1, SEQ ID NO: 19). [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11] [Table 7-12] [Table 7-13] [Table 7-14] [Table 7-15] [Table 7-16] [Table 7-17] [Table 7-18] [Table 7-19] [Table 7-20] [Table 7-21] [Table 7-22] [Table 7-23]

[0139] All references cited herein are incorporated entirely by reference. While several aspects of at least one embodiment of the present invention have been described, it should be understood that various changes, modifications, and improvements will readily come to mind for those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and within the spirit and scope of the invention. Therefore, the foregoing description and drawings are merely examples.

[0140] Other Embodiments Embodiment 1. An engineered nucleic acid vector comprising a stationary-phase-inducible promoter and a primosome assembly site (PAS).

[0141] Embodiment 2. The engineered nucleic acid vector according to Embodiment 1, further comprising a point mutation on RNAII that causes the formation of an important stem-loop, SL4.

[0142] Embodiment 3. An engineered nucleic acid vector according to Embodiment 1 or 2, wherein the natural promoter of RNAII is disrupted.

[0143] Embodiment 4. An engineered nucleic acid vector according to Embodiment 1 or 2, wherein the natural promoter of RNAII is deleted.

[0144] Embodiment 5. The manipulated nucleic acid vector according to Embodiment 1 or any one of Embodiments 2 to 4, wherein the stationary phase induction promoter is P(osmY).

[0145] Embodiment 6. The manipulated nucleic acid vector according to Embodiment 5, wherein P(osmY) has the sequence of SEQ ID NO: 27.

[0146] Embodiment 7. The manipulated nucleic acid vector according to any one of Embodiments 1 to 6, wherein the PAS has the sequence of Sequence ID No. 28.

[0147] Embodiment 8. The manipulated nucleic acid vector according to Embodiment 2 or any one of Embodiments 3 to 7, wherein SL4 has the sequence of Sequence ID No. 29.

[0148] Embodiment 9. The manipulated nucleic acid vector according to Embodiment 8, wherein the vector is plasmid 1 (+PAS+P(osmY)).

[0149] Embodiment 10. The engineered nucleic acid vector according to Embodiment 8 or Embodiment 9, wherein the vector is plasmid 2 (+PAS+P(osmY)+SL4).

[0150] Embodiment 11. The manipulated nucleic acid vector according to Embodiment 1, wherein the vector has a sequence with at least 70% sequence identity with respect to sequence number 19.

[0151] Embodiment 12. The manipulated nucleic acid vector according to Embodiment 1, wherein the vector has a sequence with at least 70% sequence identity with respect to SEQ ID NO: 20.

[0152] Embodiment 13. An engineered nucleic acid vector according to any one of Embodiments 1 to 12, comprising the following 5' to 3' arrangement: (a) a replication origin, (b) a promoter, and (c) an antibiotic resistance gene.

[0153] Embodiment 14. An engineered nucleic acid vector according to any one of Embodiments 1 to 13, further comprising an open reading frame (ORF) encoding the mRNA of interest.

[0154] Embodiment 15. Genotype: | <repA|ori_ts|<recA|<bla|<tetR|<P(tetR)|P(tet)> Recombinant plasmids containing |gamma>|beta>|exo>|a>|.

[0155] Embodiment 16. A recombinant plasmid comprising a nucleic acid sequence having at least 70% identity with SEQ ID NO: 19.

[0156] Embodiment 17. A recombinant plasmid comprising a nucleic acid sequence having at least 70% identity with SEQ ID NO: 20.

[0157] Embodiment 18. A method for carrying out an in vitro transcription reaction using an engineered nucleic acid vector described in any one of Embodiments 1 to 17.

[0158] Embodiment 19. Nucleic acid containing the prsA variant.

[0159] Embodiment 20. The nucleic acid according to Embodiment 19, wherein the nucleic acid has 70% to 99% sequence identity with respect to prsA* (SEQ ID NO: 23).

[0160] Embodiment 21. The nucleic acid according to Embodiment 19, wherein the nucleic acid has at least 70% sequence identity with respect to prsA* (SEQ ID NO: 23).

[0161] Embodiment 22. The nucleic acid according to Embodiment 19, wherein the nucleic acid has at least 80%, 90%, or 95% sequence identity with respect to prsA* (SEQ ID NO: 23).

[0162] Embodiment 23. The nucleic acid according to Embodiment 19, wherein the nucleic acid encodes a protein having at least 95% sequence identity with respect to prsA* (SEQ ID NO: 24).

[0163] Embodiment 24. The nucleic acid according to Embodiment 19, wherein the nucleic acid encodes a protein having 100% sequence identity with SEQ ID NO: 23 or 100% sequence identity with SEQ ID NO: 24.

[0164] Embodiment 25. A genetically modified microorganism containing the prsA variant, wherein the genetically modified microorganism has a genome in which the repressor gene purR is disrupted.

[0165] Embodiment 26. The genetically modified microorganism according to Embodiment 25, wherein the prsA variant has 70% to 99% sequence identity with respect to prsA.

[0166] Embodiment 27. The genetically modified microorganism according to Embodiment 25, wherein the prsA variant has 90% sequence identity with prsA* (SEQ ID NO: 23).

[0167] Embodiment 28. The genetically modified microorganism according to Embodiment 25, wherein the prsA variant includes the sequence of SEQ ID NO: 23.

[0168] Embodiment 29. A genetically modified microorganism according to any one of Embodiments 25 to 28, wherein the purR is deleted.

[0169] Embodiment 30. The genetically modified microorganism according to Embodiment 29, wherein purR includes the sequence of SEQ ID NO: 25.

[0170] Embodiment 31. A genetically modified microorganism according to any one of Embodiments 25 to 30, wherein the EcoKI restriction system is deleted from the genome.

[0171] Embodiment 32. A genetically modified microorganism according to any one of Embodiments 25 to 31, wherein endA is deleted from the genome.

[0172] Embodiment 33. A genetically modified microorganism according to any one of Embodiments 25 to 32, wherein recA is deleted from the genome.

[0173] Embodiment 34. The genetically modified microorganism is a recombinant strain of Escherichia coli (E. coli), as described in any one of Embodiments 25 to 33.

[0174] Embodiment 35. A recombinant strain of Escherichia coli (E. coli) comprising an E. coli genome having at least the following gene deletions: endA (ΔendA) and recA (ΔrecA).

[0175] Embodiment 36. The recombinant strain according to Embodiment 35, wherein the E. coli is derived from MG1655.

[0176] Embodiment 37. The recombinant strain according to Embodiment 35 or Embodiment 36, wherein the E. coli genome comprises the nucleic acid sequence of the MG1655 genome, which includes at least the following gene deletions with respect to MG1655: endA (ΔendA) and recA (ΔrecA).

[0177] Embodiment 38. The recombinant strain according to Embodiment 35 or any one of Embodiments 36-37, wherein the E. coli genome comprises a nucleic acid sequence with at least 95% sequence identity with the MG1655 genome.

[0178] Embodiment 39. The EcoKI restriction system is a recombinant strain according to any one of Embodiments 35 to 38, which is deleted from the genome of E. coli.

[0179] Embodiment 40. The recombinant strain according to Embodiment 39, wherein the E. coli genome comprises a nucleic acid sequence having at least 80% identity with the MG1655 genome.

[0180] Embodiment 41. The recombinant strain according to Embodiment 39 or Embodiment 40, wherein the E. coli genome comprises a nucleic acid sequence that includes the nucleic acid sequence of the MG1655 genome, which includes the EcoKI restriction deletion with respect to the MG1655 genome.

[0181] Embodiment 42. The recombinant strain according to any one of Embodiments 35 to 41, wherein the E. coli comprises the prsA variant.

[0182] Embodiment 43. The recombinant strain according to Embodiment 42, wherein the E. coli genome comprises a nucleic acid sequence having at least 80% identity with the MG1655 genome.

[0183] Embodiment 44. The recombinant strain according to Embodiment 43, wherein the E. coli genome includes the nucleic acid sequence of SEQ ID NO: 23.

[0184] Embodiment 45. A recombinant strain according to any one of Embodiments 35 to 44, wherein the purR sequence is deleted from the genome of E. coli.

[0185] Embodiment 46. The recombinant strain according to Embodiment 45, wherein the E. coli genome comprises a nucleic acid sequence having at least 80% identity with the MG1655 genome.

[0186] Embodiment 47. The recombinant strain according to Embodiment 46, wherein the E. coli genome has the nucleic acid sequence of Sequence ID No. 25 deleted with respect to the MG1655 genome.

[0187] Embodiment 48. The recombinant strain according to any one of Embodiments 35 to 47, wherein the E. coli genome further comprises at least one gene deletion selected from the group including mrr, hsdR, hsdM, hsdS, symE, and mcrBC.

[0188] Embodiment 49. The E. coli genome is a recombinant strain according to any one of Embodiments 35 to 48, derived from the MG strain or the KS strain.

[0189] Embodiment 50. A genetically modified microorganism containing strain 3.

[0190] Embodiment 51. A genetically modified microorganism containing strain 4.

[0191] Embodiment 52. An engineered nucleic acid vector comprising a nucleic acid having at least 70% sequence identity with respect to SEQ ID NO: 21.

[0192] Embodiment 53. An engineered nucleic acid vector comprising a nucleic acid having at least 80% sequence identity with respect to SEQ ID NO: 21.

[0193] Embodiment 54. An engineered nucleic acid vector comprising a nucleic acid having at least 90% sequence identity with respect to SEQ ID NO: 21.

[0194] Embodiment 55. An engineered nucleic acid vector comprising a nucleic acid having at least 95% sequence identity with respect to SEQ ID NO: 21.

[0195] Embodiment 56. An engineered nucleic acid vector comprising a nucleic acid having SEQ ID NO: 21.

[0196] Embodiment 57. An engineered nucleic acid vector comprising a nucleic acid having at least 70% sequence identity with respect to SEQ ID NO: 22.

[0197] Embodiment 58. An engineered nucleic acid vector comprising a nucleic acid having at least 80% sequence identity with respect to SEQ ID NO: 22.

[0198] Embodiment 59. An engineered nucleic acid vector comprising a nucleic acid having at least 90% sequence identity with respect to SEQ ID NO: 22.

[0199] Embodiment 60. An engineered nucleic acid vector comprising a nucleic acid having at least 95% sequence identity with respect to SEQ ID NO: 22.

[0200] Embodiment 61. An engineered nucleic acid vector comprising a nucleic acid having sequence number 22.

[0201] Embodiment 62. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 70% sequence identity with respect to any one of sequence numbers 1 to 15.

[0202] Embodiment 63. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 80% sequence identity with respect to any one of sequence numbers 1 to 15.

[0203] Embodiment 64. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 90% sequence identity with respect to any one of sequence numbers 1 to 15.

[0204] Embodiment 65. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 95% sequence identity with respect to any one of sequence numbers 1 to 15.

[0205] Embodiment 66. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 99% sequence identity with respect to any one of sequence numbers 1 to 15.

[0206] Embodiment 67. An engineered nucleic acid vector comprising one of the nucleic acid sequences SEQ ID NOs: 1 to 15.

[0207] Embodiment 68. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 10.

[0208] Embodiment 69. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 70% sequence identity with respect to SEQ ID NO: 11.

[0209] Embodiment 70. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 10.

[0210] Embodiment 71. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 95% sequence identity with respect to SEQ ID NO: 11.

[0211] Embodiment 72. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 10.

[0212] Embodiment 73. An engineered nucleic acid vector comprising a nucleic acid sequence having at least 99% sequence identity with respect to SEQ ID NO: 11.

[0213] Embodiment 74. An engineered nucleic acid vector comprising the nucleic acid sequence of SEQ ID NO: 10.

[0214] Embodiment 75. An engineered nucleic acid vector comprising the nucleic acid sequence of SEQ ID NO: 11.

[0215] In addition to the embodiments expressly described herein, it should be understood that all features disclosed herein can be combined in any combination (e.g., rearrangements, combinations). Each element disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly indicated, each disclosed feature is merely an example of a general set of equivalent or similar features.

[0216] From the above description, those skilled in the art will be able to easily identify the essential features of the present invention and make various changes and modifications to the invention to suit various uses and circumstances without departing from its spirit and scope. Therefore, other embodiments are also within the scope of the claims.

Claims

1. An engineered nucleic acid vector comprising a stationary-phase induction promoter P(osmY) having the sequence of SEQ ID NO: 27 and a primosome assembly site (PAS) having the sequence of SEQ ID NO: 28, wherein the native promoter of RNAII is substituted with the stationary-phase induction promoter P(osmY), and the vector contains at least 90% sequence-identical sequences to SEQ ID NO: 21 or SEQ ID NO:

22.

2. The manipulated nucleic acid vector according to claim 1, further comprising four point mutations contained in Sequence ID No. 29 that cause the formation of a stem-loop 4 (SL4).

3. The manipulated nucleic acid vector according to claim 1 or 2, wherein SL4 has the sequence of sequence number 29.

4. The manipulated nucleic acid vector according to claim 1, wherein the vector comprises the sequence of sequence number 21 or sequence number 22.

5. The following arrangement from 5' to 3': (a) origin of replication; (b) Stationary-phase induction promoter P(osmY), and (c) Antibiotic resistance gene A manipulated nucleic acid vector according to any one of claims 1 to 4, comprising:

6. An engineered nucleic acid vector according to any one of claims 1 to 5, further comprising an open reading frame (ORF) encoding the target mRNA.

7. A method for carrying out an in vitro transcription reaction using an engineered nucleic acid vector according to any one of claims 1 to 6.

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