WO2022250068A1 - プラスミドの製造方法及びプラスミド - Google Patents

プラスミドの製造方法及びプラスミド Download PDF

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WO2022250068A1
WO2022250068A1 PCT/JP2022/021313 JP2022021313W WO2022250068A1 WO 2022250068 A1 WO2022250068 A1 WO 2022250068A1 JP 2022021313 W JP2022021313 W JP 2022021313W WO 2022250068 A1 WO2022250068 A1 WO 2022250068A1
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plasmid
dna
multimodular
biosynthetic enzyme
producing
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デイビッド リップス
ハイシュ ベルクレイ
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Spiber Inc
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Spiber Inc
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Priority to CN202280034051.9A priority Critical patent/CN117280036A/zh
Priority to EP22811338.7A priority patent/EP4349988A4/en
Priority to JP2023523494A priority patent/JP7370121B2/ja
Priority to US18/290,394 priority patent/US20250283057A1/en
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2800/101—Plasmid DNA for bacteria
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    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/10—Vectors comprising a special origin of replication system multiple origins of replication

Definitions

  • the present invention relates to a plasmid production method and a plasmid using a DNA integration method using a Bacillus subtilis plasmid transformation system.
  • Natural compounds produced by microorganisms such as actinomycetes and filamentous fungi are known to be useful substances with a wide variety of structures and biological activities. At present, it is easy to identify biosynthetic gene clusters by deciphering the genome of producing bacteria. It has also become clear that there are many useful substance gene clusters that have not been used by humans. Among microbial secondary metabolites, studies on biosynthetic gene clusters have focused on industrially important polyketide compounds and peptide compounds.
  • PKS polyketide synthase
  • the plasmids that are required to be constructed have the problem of compatibility between host cells and compatibility due to synthesis methods, and new methods are still required for heterologous expression of genes.
  • the object of the present invention is to provide a novel plasmid production method and plasmid for heterologous gene expression.
  • the present inventors used a DNA integration method using a plasmid transformation system of Bacillus subtilis to adjust a first gene cluster containing a plurality of genes, and The present inventors have found that by using a plasmid in which a second gene cluster containing a junction initiation sequence and the first gene cluster are linked, it becomes unnecessary to modify the plasmid for each host cell.
  • the present invention is based on this new finding.
  • the present invention provides, for example, the following inventions.
  • a method for producing a plasmid containing a plurality of genes encoding a multimodular biosynthetic enzyme wherein a first gene cluster containing the plurality of genes is adjusted by a DNA integration method using Bacillus subtilis; a second gene cluster comprising a replication origin of E. coli, a replication origin of E. coli and a junction initiation sequence for actinomycetes, and the first gene cluster.
  • [5] A method for producing a multimodular biosynthetic enzyme by causing a host cell containing the plasmid produced by the method of [1] or [2] to produce a multimodular biosynthetic enzyme.
  • [6] The method for producing a multimodular biosynthetic enzyme according to [5], wherein the host cell is an actinomycete.
  • PKS type I polyketide synthase
  • the method for producing a plasmid of the present invention comprises a first gene cluster containing a plurality of genes encoding multimodular biosynthetic enzymes, a Bacillus subtilis replication origin, an E. coli replication origin, and a conjugation initiation sequence for actinomycetes.
  • the advantage of using a plasmid linked to a second gene cluster is that it is not necessary to modify the plasmid for each host cell for expression in heterologous expression host cells.
  • FIG. 1 is an explanatory diagram showing a plasmid map.
  • FIG. 2 is an explanatory diagram showing the flow of gene expression in a plasmid production method and a heterologous expression host cell.
  • multimodular biosynthetic enzymes include type I polyketide synthase (PKS) and non-ribosomal peptide synthetase.
  • the DNA encoding the PKS or non-ribosomal peptide synthetase contained in the plasmid of the present invention may be of a natural type or may be of modified codon usage, and may contain one or more amino acids. It may be modified.
  • the Streptomyces PKS contains the products of three open reading frames (ORF1, ORF2, ORF3). PKS contains three domains, a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP), and these three domains are capable of elongating polyketide chains.
  • PKS may further have domains involved in main chain modification, such as ketoreductase (KR) domain, dehydratase (DH) domain, and enoyl reductase (ER) domain.
  • KR ketoreductase
  • DH dehydratase
  • ER enoyl reductase
  • Compounds prepared by PKS include 6-deoxyerythronolide B (6-dEB), frenolicin, granaticine, tetracenomycin, 6-methylsalicylic acid, oxytetracycline, tetracycline, erythromycin, griseucin, nanaomycin, medermycin, Daunorubicin, tyrosine, carbomycin, spiramycin, avermectin, monensin, nonactin, kramycin, lipomycin, rifamycin, candicidin.
  • 6-dEB 6-deoxyerythronolide B
  • frenolicin gran
  • PKS A type I polyketide synthase (PKS), which is not particularly limited, but includes, for example, a PKS encoded by the DNA sequence represented by SEQ ID NO: 2, which has a homology of 80% or more to SEQ ID NO: 2, It may be 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more.
  • Non-ribosomal peptides refer to, but are not limited to, a class of peptides belonging to, for example, a family of complex natural products composed of simple amino acid monomers. It is synthesized in many bacteria or fungi by large multifunctional proteins called non-ribosomal peptide synthetases (NRPS).
  • NRPS non-ribosomal peptide synthetases
  • a feature of the NRPS system is its ability to synthesize peptides containing proteinogenic and non-proteinogenic amino acids.
  • Non-ribosomal peptide synthetase refers to, but is not limited to, large multifunctional proteins that are organized into cooperative groups of active sites called modules, where each module is responsible for peptide elongation and functionalization. Required to catalyze one cycle of group modification.
  • modules large multifunctional proteins that are organized into cooperative groups of active sites called modules, where each module is responsible for peptide elongation and functionalization. Required to catalyze one cycle of group modification.
  • the number and order of modules, as well as the types of domains present within the modules on each NRPS dictate the number, order, and selection of amino acids incorporated, and modifications associated with specific types of extensions, to obtain peptides. Determine the structural variation of the product.
  • a plasmid contains a control sequence operably linked to DNA encoding a desired multimodular biosynthetic enzyme, such as PKS.
  • Suitable expression systems for use in the present invention include systems that function in eukaryotic and prokaryotic host cells. However, as explained above, prokaryotic systems are preferred and, in particular, systems compatible with Streptomyces are of particular interest. Control sequences for use in such systems include promoters, ribosome binding sites, terminators, enhancers and the like. Useful promoters are those that function in Streptomyces host cells and include, but are not limited to, pGapdh, pErmE, pKasO, and the like.
  • a selectable marker can also be included in the plasmid.
  • markers are known, including genes that are useful in the selection of transformed cell lines and generally whose expression confers a selectable phenotype on transformed cells when the cells are grown in an appropriate selective medium. be.
  • markers include, for example, genes that confer antibiotic resistance or sensitivity to the plasmid.
  • some polyketides are naturally colored, a feature that provides a built-in marker for selecting cells that have been successfully transformed with the constructs of the invention.
  • plasmids of the invention are known to those of skill in the art and typically involve the use of CaCl2 or divalent cations and other agents such as DMSO.
  • DNA can also be introduced into bacterial cells by electroporation. Once the multimodular biosynthetic enzyme PKS is expressed, polyketide-producing colonies can be identified and isolated using known techniques.
  • the plasmids of the invention may be introduced into host cells using conjugative transfer between bacteria.
  • the PKS-encoding base region is transferred to an E. coli plasmid and transferred from E. coli to actinomycetes by conjugation.
  • the PKS-encoding DNA is thereby integrated into the genome of a host cell, such as an actinomycete. If the host cell is an actinomycete, the genus Streptomyces is preferred.
  • a plasmid containing multiple genes encoding a multimodular biosynthetic enzyme of the present invention contains DNA encoding a domain contained in the multimodular biosynthetic enzyme, and its type and size are not particularly limited.
  • the type of DNA encoding the domain contained in the multimodular biosynthetic enzyme is not particularly limited, and may be not only naturally-derived sequences such as microorganisms, but also artificially-designed sequences. Preferable examples include gene clusters that constitute PKS or NRPS. For naturally occurring DNA sequences, within a given organism of origin, predominantly one codon is used by the organism to express the corresponding amino acid, but for heterologous expression the codon usage of the host must be matched.
  • GC content base guanine and cytosine content within the sequence
  • repetitive sequences and the like.
  • Repeated sequences reduce genetic stability, create a risk of incorrect hybridization, and inhibit synthesis of repeated segments. Therefore, synthetic genes need to be optimized with respect to codon usage and GC content. However, these requirements are usually difficult to optimally meet simultaneously. For example, codon optimization can result in highly repetitive DNA sequences or high GC content.
  • the GC content is 30-70%. It is preferably 70% or less, 68% or less, 65% or less, or 60% or less.
  • a target plasmid can be synthesized with high efficiency even if the GC content is 50% or more, 52% or more, 55% or more, 58% or more, or 60% or more.
  • codons are optimized so that repeats of base sequences of 20 bp or more do not occur. It is preferable to avoid extreme differences in GC content within genes. For example, it is preferred that the difference in GC content between the highest and lowest 50bp stretches is 52% or less. It is preferred to have as little homopolymer as possible. It is preferred to minimize the number/length of small repeats interspersed in the DNA sequence as much as possible.
  • the method for accumulating DNA using Bacillus subtilis is not particularly limited as long as it is a method for accumulating DNA using Bacillus subtilis. 253462, Japanese Patent Application Laid-Open No. 2007-135533, and the like.
  • the OGAB method (Ordered Gene Assembly in Bacillus subtilis method) is a method for integrating multiple DNA fragments using a Bacillus subtilis plasmid transformation system. Specifically, the DNA fragment to be integrated and the plasmid vector for integration are prepared to have a specific overhang of 3 to 4 bases, and the order and orientation of the DNA fragment to be ligated are specified using this complementarity. It is a method of concatenation.
  • a plurality of DNA fragments are formed in a certain order and orientation by using the DNA uptake ability and homologous recombination ability of microorganisms such as bacteria of the genus Bacillus.
  • Each end of the vector fragment was prepared by designing SfiI cleavage sites by generating ends that allow each fragment to be sequentially ligated once in one DNA integration unit, and these SfiI fragments were combined in equimolar amounts. After mixing and adjusting the concentration so that , a ligation reaction is carried out in the presence of polyethylene glycol and salt to generate a linear high-molecular-weight DNA having a structure in which this DNA linking unit is repeatedly repeated. Transformation into B.
  • subtilis competent cells allows the DNA to be ligated into the B. subtilis plasmid in the desired order and orientation.
  • Bacillus subtilis competent cells in which a sequence common to the sequence in the plasmid is inserted into the genomic DNA are co-cultivated with linear high-molecular-weight DNA having a structure in which the DNA linking unit obtained above is repeated multiple times. allows the DNA to be ligated into the Bacillus subtilis genomic DNA in the desired order and orientation.
  • the combi-OGAB method is a method described in International Publication No. WO2020/203496.
  • OGAB method gene integration method using a plasmid transformation system of Bacillus subtilis
  • all of the methods used for the integration of combinatorial libraries are This is a method in which the molar concentration ratio of DNA fragments approaches 1 as much as possible.
  • a seed plasmid is constructed by ligating all of the option gene fragments to be combinatorialized.
  • seed plasmids of the same number as the maximum number of options are prepared by separately constructing seed plasmids for other option gene fragments.
  • a solution in which gene fragments are mixed in an equimolar amount is obtained.
  • This solution remains equimolar when mixed with other seed plasmids.
  • various gene fragments contained in these solutions are linearly ligated to obtain a pseudo-tandem repeat high-molecular-weight DNA in which the plasmid vector portion periodically appears, and this is used to transform Bacillus subtilis.
  • a combinatorial library is efficiently constructed by circularization using the homology of the plasmid vector portion in Bacillus subtilis. According to this method, equimolar concentrations of gene fragments required for constructing a combinatorial library can be prepared very simply and reliably, and the scale of library construction can be increased to an unprecedented scale.
  • a first gene cluster of the present invention is a gene cluster comprising a plurality of genes encoding multimodular biosynthetic enzymes.
  • the second gene cluster of the present invention is a gene cluster comprising a Bacillus subtilis origin of replication, an E. coli replication origin and an actinomycete junction initiation sequence.
  • the replication origin of Bacillus subtilis of the present invention is not particularly limited as long as it can exhibit its function.
  • the E. coli replication origin of the present invention is not particularly limited as long as it can exhibit its function, and examples thereof include RepA.
  • conjugation initiation sequence to actinomycetes The conjugation initiation sequence to the actinomycete of the present invention is not particularly limited as long as it can exhibit its function.
  • the plasmid of the present invention contains a Bacillus subtilis origin of replication, an E. coli origin of replication and a junction initiation sequence for actinomycetes, a prokaryotic F-factor partitioning system for single-copy maintenance in E. coli, a recipe at a defined location. It may also contain a site-specific recombination system that allows the vector to integrate into the genome of the host host, one or more selectable markers that function in Bacillus subtilis, E. coli, and Streptomyces expression hosts, and the like.
  • the replication origin of Bacillus subtilis is not particularly limited, it includes, for example, the one represented by SEQ ID NO: 3, and the homology with SEQ ID NO: 3 is 80% or more, 85% or more, 90% or more, 93% or more, 95% or more. % or more, 97% or more, or 99% or more.
  • the origin of replication in E. coli is not particularly limited. % or more, 95% or more, 97% or more, or 99% or more.
  • the conjugation initiation sequence to actinomycetes is not particularly limited, but includes those shown in SEQ ID NO: 6, and the homology with SEQ ID NO: 6 is 80% or more, 85% or more, 90% or more, 93% or more, 95% % or more, 97% or more, or 99% or more.
  • the prokaryotic F factor partitioning system for single copy maintenance in E. coli is not particularly limited, but includes, for example, the one shown in SEQ ID NO: 5, which has 80% or more, 85% or more homology with SEQ ID NO: 5. , 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more.
  • Site-specific recombination systems that allow the integration of the vector into the genome of the recipient host at a defined location include, but are not limited to, those shown in SEQ ID NO:7, which have homology to SEQ ID NO:7. However, it may be 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more.
  • One or more selectable markers that function in Bacillus subtilis, E. coli, and actinomycete expression hosts include, but are not limited to, those shown in SEQ ID NO: 8, which have 80% or more homology with SEQ ID NO: 8. , 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, or 99% or more.
  • OGAB vector 1.0 (SEQ ID NO: 22), OGAB vector 2.0 (SEQ ID NO: 23), OGAB vector 2.1 (SEQ ID NO: 24), OGAB vector 2.2 (SEQ ID NO: 25). 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 99% or more Anything is fine.
  • a method for producing a multimodular biosynthetic enzyme of the present invention is a method for producing a plasmid containing a plurality of genes encoding a multimodular biosynthetic enzyme, wherein the DNA is accumulated using Bacillus subtilis. adjusting a first gene cluster containing genes, and linking the first gene cluster with a second gene cluster containing a Bacillus subtilis replication origin, an E. coli replication origin, and a junction initiation sequence for actinomycetes.
  • a plasmid produced by the OGAB method can be used as the DNA integration method using the Bacillus subtilis.
  • a multimodular biosynthetic enzyme can be produced by a known method using a plasmid in a host cell. Multimodular biosynthetic enzymes can be obtained. "Culture” means either culture supernatant, cultured cells, cultured cells, or disrupted cells or cells. A method for culturing the transformant of the present invention can be carried out according to a conventional method used for culturing a host.
  • the medium for culturing the transformant of the present invention contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the host, and is a natural medium as long as it is a medium capable of efficiently culturing the transformant.
  • synthetic media may be used.
  • Carbon sources include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol.
  • Nitrogen sources include ammonium salts of inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, or other nitrogen-containing compounds.
  • inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
  • Cultivation is usually carried out at 28-38°C under aerobic conditions such as shaking culture or aeration stirring culture. Adjustment of pH is performed using an inorganic or organic acid, an alkaline solution, or the like.
  • the expression products can be collected by disrupting the cells or cells by subjecting them to a homogenizer treatment or the like.
  • the culture medium is used as it is, or the bacterial bodies or cells are removed by centrifugation or the like. After that, the expression product is collected from the culture by extraction by ammonium sulfate precipitation or the like, and if necessary, further isolated and purified using various chromatography or the like.
  • the host cell for producing the multimodular biosynthetic enzyme is not particularly limited, but it is preferable to use actinomycetes.
  • the host cell is preferably an actinomycete, more preferably of the genus Streptomyces.
  • the greatest advantage of using host cells of the genus Streptomyces is the high production titer compared to heterologous expression production using E. coli, and the presence of a post-translational modification system essential for the expression of type I PKS activity. mentioned. Specifically, S. albus, S. ambofaciens, S. avermitilis, S. azureus, S. cinnamonensis, S.
  • a method for introducing a plasmid containing multiple genes encoding a multimodular biosynthetic enzyme into an actinomycete can be performed by a known method, and is not particularly limited. methods and the like.
  • a multimodular biosynthetic enzyme is produced when the plasmid containing multiple genes encoding the multimodular biosynthetic enzyme is further a plasmid containing a Bacillus subtilis origin of replication, an E. coli replication origin and a conjugation initiation sequence for actinomycetes.
  • actinomycetes as the host cells, multimodular biosynthetic enzymes can be produced without modifying the plasmids prepared by the DNA integration method using Bacillus subtilis into structures suitable for Escherichia coli and actinomycetes. It is advantageous and can reduce the steps required to produce multimodular biosynthetic enzymes.
  • the produced multimodular biosynthetic enzyme can be usefully utilized in various fields.
  • Kitasatospora aureofaciens from the MiBig Repository of Known Biosynthetic Gene Clusters (https://mibig.secondarymetabolites.org/repository/BGC0001003/index.html#r1c1) for reproducibility such as codon optimization.
  • the design was performed and synthesized by outsourced (Twist Bioscience) with all DNA fragments (SEQ ID NOs: 9-21) of the targeted PKS cluster constructs, amplified in E. coli and re-extracted.
  • the concentrations of all DNA fragments were measured with a UV spectrophotometer (Thermofisher Nanodrop) and adjusted to generate an equimolar fragment mixture.
  • Phenol-chloroform treatment, butanol treatment, and ethanol precipitation were performed to purify the treated DNA fragments.
  • the treated DNA fragment was treated with OGAB vector 2.0 (SEQ ID NO: 23), 1 ⁇ l of T4 DNA ligase (TAKARA BIO), and ligated. The ligation was completed by mixing with buffer and incubating at 37°C for 3 hours.
  • the DNA ligation solution containing the DNA construct was mixed with Bacillus subtilis competent cells and the cells were mixed at 37°C for 90 minutes. After the incubation period, the cells were inoculated onto tetracycline selection plates.
  • transformants were picked from the plates and cultured overnight at 37°C in 2 ml of LB medium.
  • Plasmid extraction was performed according to a known procedure, and it was confirmed that the resulting DNA was assembled as expected.
  • a successfully assembled OGAB shuttle vector construct containing the target PKS cluster was transformed into competent cells of E. coli ET12567/pUZ8002, cultured on plates containing apramycin, and selected.
  • Transformants were selected and cultured overnight in 10 ml of LB medium containing chloramphenicol, kanamycin, and apramycin.
  • the culture was diluted 1:100 with fresh LB medium and antibiotics (chloramphenicol, kanamycin, and apramycin) and grown to an OD600 of 0.4〜0.6.
  • E. coli cells were washed twice with an equal volume of LB medium to remove all antibiotics that may inhibit actinomycetes, and resuspended in 0.1 volume of LB medium.
  • a plate of S. albus J1074 spores was prepared individually and extracted with water to create a spore solution of Streptomyces. 500 ⁇ L of YT medium was added to 500 ⁇ l of this spore solution. The resulting spore solution mixture was heat-shocked at 50°C for 10 minutes and allowed to cool.
  • the resulting mixture was plated on MS agar plates + 10 mM MgCl and incubated at 30°C for 16-20 hours.
  • the plate was covered with 1 ml of water containing 0.5 naradixic acid and 1 mg apramycin, and incubation was continued at 30°C for about 4 days.
  • a potential zygote was confirmed in a selective medium containing nalidixic acid.

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