WO2018179834A1 - Method for producing rna - Google Patents
Method for producing rna Download PDFInfo
- Publication number
- WO2018179834A1 WO2018179834A1 PCT/JP2018/003692 JP2018003692W WO2018179834A1 WO 2018179834 A1 WO2018179834 A1 WO 2018179834A1 JP 2018003692 W JP2018003692 W JP 2018003692W WO 2018179834 A1 WO2018179834 A1 WO 2018179834A1
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- WO
- WIPO (PCT)
- Prior art keywords
- rna
- gene
- promoter
- objective
- bacterium
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/05—Alcaligenes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/13—Brevibacterium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/15—Corynebacterium
Definitions
- the present invention relates to a method for producing RNA (ribonucleic acid) by fermentation using a microorganism.
- RNA by using a microorganism there have been known, for example, a method of using Escherichia coli deficient in ribonuclease III (RNaseIII) as a host to accumulate RNA in bacterial cells (Non-patent document 1), a method of using Pseudomonas syringae as a host in combination with the function of phi 6 bacteriophage to accumulate RNA in capsids (Non-patent document 2), a method of using Escherichia coli as a host to accumulate RNA fused with a partial sequence of tRNA in bacterial cells (Non-patent document 3), a method of using Escherichia coli as a host to produce siRNA while allowing the siRNA to form a complex with a protein that binds to the siRNA (Non-patent document 4), a method of using a bacterium of the genus Rhodovulum as a host to produce RNA by secretor
- Patent document 1 WO2009/063969
- Patent document 2 WO2010/084371
- Non-patent document 1 Tenllado F, et. al., Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections. BMC Biotechnol. 2003 Mar 20;3:3.
- Non-patent document 2 Aalto AP, et. al., Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA. 2007 Mar;13(3):422-9.
- Non-patent document 3 Ponchon L, et. al., A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc. 2009;4(6):947-59.
- Non-patent document 4 Huang L, et. al., Efficient and specific gene knockdown by small interfering RNAs produced in bacteria. Nat Biotechnol. 2013 Apr;31(4):350-6.
- An object of the present invention is to provide a method for efficiently producing RNA.
- RNA can be efficiently produced by using a coryneform bacterium deficient in ribonuclease III (RNaseIII) as a host, and accomplished the present invention.
- RNaseIII coryneform bacterium deficient in ribonuclease III
- a method for producing objective RNA comprising: culturing a coryneform bacterium having an expression unit for the objective RNA in a medium, to express the objective RNA and accumulate the objective RNA in cells of the bacterium; and collecting the objective RNA from the cells, wherein the bacterium has been modified so that the activity of ribonuclease III is reduced as compared with a non-modified strain.
- ribonuclease III is a protein defined in (a), (b), or (c) mentioned below: (a) a protein comprising the amino acid sequence of SEQ ID NO: 52; (b) a protein comprising the amino acid sequence of SEQ ID NO: 52, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having ribonuclease III activity; (c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 52, and having ribonuclease III activity.
- the expression unit contains a promoter sequence that functions in the coryneform bacterium and a nucleotide sequence encoding the objective RNA in the direction from 5' to 3'.
- the promoter sequence is a promoter derived from a phage.
- the promoter sequence is F1 promoter or T7 promoter.
- the promoter sequence is a promoter defined in (a) or (b) mentioned below: (a) a promoter comprising the nucleotide sequence of SEQ ID NO: 13 or 78; (b) a promoter comprising an nucleotide sequence showing an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 13 or 78.
- the bacterium is a bacterium belonging to the genus Corynebacterium.
- the method mentioned above, wherein the bacterium is Corynebacterium glutamicum.
- Diagrams showing construction schemes of plasmids. (A) pVC7-Pf1-Hv-iap. (B) pVC7-Pf1-Hv-iap-Pf1rev. Agarose gel electrophoretograms (photographs) showing an improved effect in RNA production due to deletion of rnc gene.
- Agarose gel electrophoretograms showing an improved effect in RNA production due to high copy number variations of RNA expression plasmid.
- Agarose gel electrophoretograms showing comparison result between RNA production amounts observed for F1-promoter-expression system in C. glutamicum and those observed for T7-promoter-induced-expression system in E. coli.
- a diagram showing a construction scheme of plasmid pPK-T7lac A diagram showing a construction scheme of plasmid pPK-T7lac-vd-antiOlac.
- a diagram showing a construction scheme of plasmid pVC54 A diagram showing a construction scheme of plasmid pBS5t-ptrB*.
- a diagram showing a construction scheme of plasmid pBS5t-ptrB*-T7pol A diagram showing a construction scheme of plasmid pVC54-T7pol.
- An agarose gel electrophoretogram showing RNA production by using T7-promoter-induced-expression system in C. glutamicum. Lanes 1-2 and 6-7, clone A; Lanes 3-4 and 8-9, clone B; and Lane 5, Century-Plus RNA Marker (Ambion AM7145).
- An agarose gel electrophoretogram showing RNA production by using T7-promoter-induced-expression system in C. glutamicum.
- An agarose gel electrophoretogram showing RNA production by using high copy number plasmid pPK4H1.
- the method of the present invention is a method for producing objective RNA, the method comprising culturing a coryneform bacterium having an expression unit for the objective RNA in a medium, and collecting the transcribed objective RNA, wherein the bacterium has been modified so that the activity of ribonuclease III is reduced.
- the coryneform bacterium used for this method is also referred to as "bacterium of the present invention”.
- Bacterium of the present invention is a coryneform bacterium having an expression unit for objective RNA, which has been modified so that the activity of ribonuclease III is reduced.
- the bacterium of the present invention can be obtained by applying introduction of the expression unit for the objective RNA and reduction in the activity of ribonuclease III to a coryneform bacterium.
- modifications for constructing the bacterium of the present invention can be performed in an arbitrary order. That is, the bacterium of the present invention can be obtained by, for example, introducing the expression unit for the objective RNA to a coryneform bacterium, and then modifying the coryneform bacterium so that the activity of ribonuclease III is reduced.
- the bacterium of the present invention can also be obtained by, for example, modifying a coryneform bacterium so that the activity of ribonuclease III is reduced, and then introducing the expression unit for the objective RNA to the coryneform bacterium.
- the bacterium of the present invention or a bacterium from which the bacterium of the present invention is constructed is also referred to as "host".
- the bacterium of the present invention has an ability to produce the objective RNA (objective RNA-producing ability). Specifically, the bacterium of the present invention has the objective RNA-producing ability at least due to that the bacterium has the expression unit for the objective RNA.
- the bacterium of the present invention may be, for example, a bacterium that has acquired the objective RNA-producing ability due to introduction of the expression unit for the objective RNA, or due to a combination of introduction of the expression unit for the objective RNA and reduction in the activity of ribonuclease III.
- a strain to be used for constructing the bacterium of the present invention and before being modified so that the activity of ribonuclease III is reduced may or may not be able to produce the objective RNA, on the assumption that the strain has the expression unit for the objective RNA.
- the bacterium of the present invention may have any characteristics so long as the bacterium has the objective RNA-producing ability.
- the bacterium of the present invention may or may not have a vector such as plasmid, other than a vector containing the expression unit for the objective RNA. That is, for example, when the bacterium of the present invention inherently has a plasmid, the plasmid may be cured (removed).
- the term "bacterium having an objective RNA-producing ability” refers to a bacterium having an ability to express and accumulate the objective RNA in cells of the bacterium in such a degree that the objective RNA can be collected, when the bacterium is cultured in a medium.
- the bacterium having the objective RNA-producing ability may be a bacterium that is able to accumulate the objective RNA in cells of the bacterium in an amount larger than that obtainable with a non-modified strain.
- non-modified strain refers to a control strain that has not been modified so that the activity of ribonuclease III is reduced.
- examples of the non-modified strain include a wild-type strain and parental strain.
- the bacterium having the objective RNA-producing ability may also be a bacterium that is able to accumulate the objective RNA in cells of the bacterium in an amount of 1 mg/L-culture or more, 2 mg/L-culture or more, 5 mg/L-culture or more, 10 mg/L-culture or more, 20 mg/L-culture or more, 50 mg/L-culture or more, or 100 mg/L-culture or more.
- a coryneform bacterium is used as a host.
- the coryneform bacterium include bacteria belonging to the genus Corynebacterium, Brevibacterium, Mycobacterium, Microbacterium, or the like.
- coryneform bacteria include the following species. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium crenatum Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerin
- coryneform bacteria include the following strains. Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium crenatum AS1.542 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC
- Corynebacterium bacteria include bacteria that had previously been classified into the genus Brevibacterium, but are presently united into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)).
- Corynebacterium stationis includes bacteria that had previously been classified as Corynebacterium ammoniagenes, but are presently re-classified into Corynebacterium stationis on the basis of nucleotide sequence analysis of 16S rRNA etc. (Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)).
- strains are available from, for example, the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Maryland 20852, P.O. Box 1549, Manassas, VA 20108, United States of America). That is, registration numbers are given to the respective strains, and the strains can be ordered by using these registration numbers (refer to http://www.atcc.org/). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection. These strains can also be obtained from, for example, the depositories at which the strains were deposited.
- the bacterium of the present invention has the expression unit for the objective RNA.
- a coryneform bacterium having the expression unit for the objective RNA can be obtained by introducing the expression unit for the objective RNA to a coryneform bacterium.
- object RNA refers to RNA to be produced according to the method of the present invention.
- one kind of objective RNA may be produced, or two or more kinds of objective RNAs may be produced.
- the objective RNA is not particularly limited so long as it is RNA exogenous to the host, i.e. so long as it is RNA other than the RNAs of the host.
- the objective RNA can be appropriately selected according to various conditions such as the purpose of use of the objective RNA.
- the objective RNA may be, for example, naturally existing RNA, modified RNA thereof, or artificially designed RNA.
- the objective RNA may be, for example, RNA derived from a microorganism, RNA derived from a plant, RNA derived from an animal, or RNA derived from a virus.
- the objective RNA may be, for example, mRNA (messenger RNA), or non-coding RNA such as rRNA (ribosomal RNA), tRNA (transfer RNA), miRNA (micro RNA), siRNA (small interfering RNA), ribozyme, and RNA aptamer.
- the mRNA may be, for example, one encoding a protein having some function such as enzyme, receptor, transporter, antibody, structural protein, and regulator, or one encoding a protein having no function per se.
- protein referred to herein includes so-called peptides such as oligopeptide and polypeptide.
- the objective RNA may be, for example, RNA having any of the nucleotide sequences of such RNAs as mentioned above.
- the objective RNA may also be, for example, RNA having a partial sequence of any of the nucleotide sequences of such RNAs as mentioned above.
- the objective RNA may also be, for example, RNA having a complementary sequence of any of the nucleotide sequences of such RNAs as mentioned above or partial sequences thereof.
- the objective RNA may also be, for example, RNA having a variant sequence of any of the nucleotide sequences of such RNAs as mentioned above, partial sequences thereof, or complementary sequences thereof.
- the descriptions concerning variants of the ribonuclease III gene mentioned later can be applied mutatis mutandis to such a variant sequence.
- the objective RNA may also have, for example, a combination of two or more nucleotide sequences selected from the nucleotide sequences of such RNAs as mentioned above, partial sequences thereof, complementary sequences thereof, and variant sequences thereof.
- the objective RNA include a partial sequence of mRNA of inhibitor of apoptosis protein of Henosepilachna vigintioctopunctata, and a partial sequence of mRNA of subunits A and E constituting ATPase in a vacuole of Colorado potato beetle (Leptinotarsa decemlineata).
- the objective RNA may be, for example, single-stranded RNA (RNA consisting of one molecule of RNA chain), or double-stranded RNA (RNA consisting of two molecules of RNA chain).
- the double-stranded RNA may be a double strand consisting of a single kind of RNA molecule (homo-double strand), or a double strand consisting of two different kinds of RNA molecules (hetero-double strand).
- Specific examples of the double-stranded RNA include, for example, double-stranded RNA consisting of an RNA strand and a complementary strand thereof.
- the objective RNA may also be, for example, a double strand consisting of one molecule of RNA chain and one molecule of DNA chain.
- the objective RNA may contain both a single-stranded region and a double-stranded region. That is, for example, the single-stranded RNA may partially form a double-stranded structure, such as stem-loop structure, within a molecule. Also, for example, the double-stranded RNA may partially contain a single-stranded structure.
- the length of the objective RNA is not particularly limited.
- the length of the objective RNA may be 10 residues or more, 20 residues or more, 50 residues or more, or 100 residues or more, or may be 10000 residues or less, 5000 residues or less, 2000 residues or less, 1000 residues or less, or 500 residues or less, or may be a range defined as a combination thereof.
- the term "expression unit for objective RNA” refers to a genetic construct configured so that the objective RNA can be expressed therefrom.
- the expression unit for the objective RNA contains a promoter sequence that functions in a coryneform bacterium and a nucleotide sequence encoding the objective RNA in the direction from 5' to 3'.
- the promoter sequence is also simply referred to as "promoter”.
- the nucleotide sequence encoding the objective RNA is also referred to as "gene encoding objective RNA” or "objective RNA gene”. It is sufficient that the objective RNA gene is ligated downstream of a promoter so that the objective RNA is expressed under control of the promoter.
- the expression unit for the objective RNA may also contain regulator sequence(s) effective for expressing the objective RNA in a coryneform bacterium, such as operator and terminator, at appropriate position(s) so that the regulator sequence(s) can function.
- regulator sequence(s) effective for expressing the objective RNA in a coryneform bacterium, such as operator and terminator, at appropriate position(s) so that the regulator sequence(s) can function.
- the terms "expression of an objective RNA gene”, “transcription of an objective RNA gene”, “expression of objective RNA”, and “transcription of objective RNA” may be used synonymously with each other.
- the expression unit for the objective RNA can be appropriately designed according to various conditions such as the type and transcription pattern of the objective RNA.
- the transcription pattern of the objective RNA is not particularly limited so long as the objective RNA is obtained.
- the objective RNA gene may be transcribed, for example, in one direction (i.e. by using either one strand of a double strand as the template), or in both directions (i.e. by using both strands of a double strand as the template). Transcription of the objective RNA gene in both directions can be performed by transcribing the gene from promoters arranged interposing the gene in mutually opposite directions (i.e. promoters arranged at 5'-side of the gene in the respective strands of a double strand). That is, the expression unit for the objective RNA may contain such two promoters.
- the two promoters may or may not be identical to each other.
- transcribing the objective RNA gene in one direction there can be typically obtained single-stranded RNA.
- transcribing the objective RNA gene in both directions there can be typically obtained double-stranded RNA.
- Double-stranded RNA can also be obtained by transcribing both strands of the double-stranded RNA from the respective expression units thereof.
- the objective RNA gene can be obtained by, for example, cloning.
- nucleotides containing the objective RNA gene such as genomic DNA and cDNA
- the objective RNA gene can also be obtained by, for example, total synthesis on the basis of the nucleotide sequence thereof (Gene, 60(1), 115-127 (1987)).
- the obtained objective RNA gene can be used as it is, or after being modified as required. That is, a variant of the objective RNA gene may be obtained by modifying the gene.
- a gene can be modified by a known technique. For example, an objective mutation can be introduced into an objective site of DNA by the site-specific mutation method.
- Examples of the site-specific mutation method include the method of utilizing PCR (Higuchi, R., 61, in PCR Technology, Erlich, H.A. Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)), and the method of utilizing phage (Kramer, W. and Frits, H.J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. in Enzymol., 154, 367 (1987)).
- a variant of the objective RNA gene may be totally synthesized.
- the expression unit for the objective RNA can be obtained by appropriately applying modification, such as introduction of a promoter sequence, to the obtained objective RNA gene.
- modification such as introduction of a promoter sequence
- other elements constituting the expression unit for the objective RNA such as a promoter sequence, or the whole of the expression unit for the objective RNA can be obtained in the same manner as the objective RNA gene.
- the promoter for expressing the objective RNA gene is not particularly limited so long as it functions in the host.
- the term "promoter that functions in a host” refers to a promoter that shows a promoter activity, i.e. gene transcription activity, in the host.
- the promoter may be a promoter derived from the host, or a heterogenous promoter.
- the promoter may be the native promoter of the objective RNA gene, or a promoter of another gene.
- the promoter may be an inducible promoter or a constitutive promoter for gene expression.
- the promoter examples include, for example, promoters of genes of the glycolytic pathway, pentose phosphate pathway, TCA cycle, amino acid biosynthesis systems, and cell surface layer proteins.
- a stronger promoter such as the following ones may also be used.
- the stronger promoter include, for example, the artificially modified P54-6 promoter (Appl. Microbiol.
- promoters include promoters derived from phages, such as F1 promoter, T7 promoter, T5 promoter, T3 promoter, and SP6 promoter. More particular examples of the promoter include F1 promoter and T7 promoter.
- the nucleotide sequence of the F1 promoter is shown as SEQ ID NO: 13.
- the nucleotide sequence of the T7 promoter is shown as SEQ ID NO: 78.
- a highly-active type of an existing promoter may also be obtained and used by using various reporter genes. For example, by making the -35 and -10 regions in a promoter region closer to the consensus sequence, the activity of the promoter can be enhanced (WO00/18935).
- highly active-type promoter include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application No. 2006134574) and pnlp8 promoter (WO2010/027045). Methods for evaluating the strength of promoters and examples of strong promoters are described in the paper of Goldstein et al. (Prokaryotic Promoters in Biotechnology, Biotechnol. Annu. Rev., 1, 105-128 (1995)), and so forth.
- the promoter may be a promoter having any of the nucleotide sequences of the promoters exemplified above (e.g. the nucleotide sequences shown as SEQ ID NO: 13 and 78).
- the promoter may also be a conservative variant of any of the nucleotide sequences of the promoters exemplified above (e.g. the nucleotide sequences shown as SEQ ID NO: 13 and 78). That is, the promoters exemplified above each can be used as they are, or after being modified as required. Promoters defined with the above-mentioned promoter names include not only the promoters exemplified above, respectively, but also include conservative variants thereof.
- the term “F1 promoter” includes not only a promoter having the nucleotide sequence shown as SEQ ID NO: 13, but also includes conservative variants thereof.
- the term “T7 promoter” includes not only a promoter having the nucleotide sequence shown as SEQ ID NO: 78, but also includes conservative variants thereof.
- the descriptions concerning conservative variants of the ribonuclease III gene mentioned later can be applied mutatis mutandis to conservative variants of promoters.
- the promoter may be a promoter having a nucleotide sequence showing a homology of, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, to the nucleotide sequence shown as SEQ ID NO: 13 or 78, so long as the original function is maintained.
- the term "original function” used for promoters refers to a function of providing the expression of a gene ligated immediately downstream thereof under certain conditions.
- the term “certain conditions” refers to conditions under which the original promoter provides the expression of a gene ligated immediately downstream thereof.
- a gene can be constitutively expressed from the F1 promoter.
- a gene can be inducibly expressed from the T7 promoter, T5 promoter, T3 promoter, or SP6 promoter in the presence of T7 RNA polymerase, T5 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase.
- a conservative variant of a promoter may have a transcription activity of, for example, 80% or more, 90% or more, or 100% or more, of that of the original promoter. The presence or absence of gene expression and intensity of gene expression (transcription activity) can be confirmed by, for example, using a reporter gene.
- a terminator for termination of gene transcription may be located downstream of the objective RNA gene.
- the terminator is not particularly limited so long as it functions in the host.
- the terminator may be a terminator derived from the host, or a heterogenous terminator.
- the terminator may be the native terminator of the objective RNA gene, or a terminator of another gene. Specific examples of the terminator include, for example, the terminator of bacteriophage BFK20.
- Methods for introducing the expression unit for the objective RNA into a coryneform bacterium are not particularly limited.
- the term "introduction of an expression unit for objective RNA” refers to making a host to harbor the expression unit for the objective RNA, and may specifically refer to expressively introducing the objective RNA gene into a host.
- the term "introduction of an expression unit for objective RNA” includes not only cases of collectively introducing the expression unit for the objective RNA that has been preliminarily constructed into a host, but also includes cases of introducing at least the objective RNA gene into a host so as to construct the expression unit for the objective RNA in the host, unless otherwise stated.
- the expression unit for the objective RNA may be present in a vector autonomously replicable separately from the chromosome, such as plasmid, or may be integrated into the chromosome. That is, the bacterium of the present invention, for example, may have the expression unit for the objective RNA on a vector, and in other words, may have a vector containing the expression unit for the objective RNA. The bacterium of the present invention, for example, may also have the expression unit for the objective RNA on the chromosome. The bacterium of the present invention may have only one copy of the expression unit for the objective RNA, or two or more copies of the expression unit for the objective RNA.
- the copy number of the expression unit for the objective RNA possessed by the bacterium of the present invention may be 5 copies/cell or more, 10 copies/cell or more, 20 copies/cell or more, 30 copies/cell or more, 50 copies/cell or more, 70 copies/cell or more, 100 copies/cell or more, 150 copies/cell or more, 200 copies/cell or more, 300 copies/cell or more, 500 copies/cell or more, 1000 copies/cell or more, or may be 2000 copies/cell or less, 1500 copies/cell or less, 1000 copies/cell or less, 500 copies/cell or less, or 300 copies/cell or less, or may be a range defined as a non-contradictory combination thereof.
- the bacterium of the present invention may have only one kind of expression unit for the objective RNA, or two or more kinds of expression units for the objective RNA.
- the copy number and kind of the expression unit for the objective RNA may also be read as the copy number and kind of the objective RNA gene, respectively.
- those expression units are harbored by the bacterium of the present invention so that the objective RNA is produced.
- all of those expression units may be harbored on a single expression vector or on the chromosome.
- those expression units may be harbored separately on a plurality of expression vectors, or separately on a single or plurality of expression vectors and the chromosome.
- the expression unit for the objective RNA can be introduced into a host by, for example, using a vector containing the expression unit for the objective RNA.
- the vector containing the expression unit for the objective RNA is also referred to as "expression vector for objective RNA".
- the expression vector for the objective RNA can be constructed by, for example, ligating the expression unit for the objective RNA with a vector.
- the expression vector for the objective RNA can also be constructed by ligating the objective RNA gene downstream of the promoter.
- a transformant into which the vector has been introduced can be obtained, namely, the expression unit for the objective RNA can be introduced into the host.
- a vector autonomously replicable in cells of the host can be used.
- the vector is preferably a multi-copy vector.
- the copy number of the vector may be 5 copies/cell or more, 10 copies/cell or more, 20 copies/cell or more, 30 copies/cell or more, 50 copies/cell or more, 70 copies/cell or more, 100 copies/cell or more, 150 copies/cell or more, 200 copies/cell or more, 300 copies/cell or more, 500 copies/cell or more, 1000 copies/cell or more, or may be 2000 copies/cell or less, 1500 copies/cell or less, 1000 copies/cell or less, 500 copies/cell or less, or 300 copies/cell or less, or may be a range defined as a non-contradictory combination thereof.
- the vector preferably contains a marker such as an antibiotic resistance gene or auxotrophy-complementing gene for selection of transformants.
- the vector may contain a promoter and/or terminator for expressing the introduced gene.
- the vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, cosmid, phagemid, or the like.
- vectors autonomously replicable in coryneform bacteria include, for example, pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol.
- plasmids obtained by improving these and having a drug resistance gene
- pCRY30 Japanese Patent Laid-open (Kokai) No. 3-210184
- pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX Japanese Patent Laid-open (Kokai) No. 2-72876 and U.S. Patent No. 5,185,262
- pCRY2 and pCRY3 Japanese Patent Laid-open (Kokai) No.
- vectors autonomously replicable in coryneform bacteria also include, for example, pVC7H1, pVC7H2, pVC7H3, pVC7H4, pVC7H5, pVC7H6, and pVC7H7 (present Examples), which are variants of pVC7.
- vectors autonomously replicable in coryneform bacteria also include, for example, pPK4H1, pPK4H2, pPK4H3, pPK4H4, pPK4H5, and pPK4H6 (present Examples), which are variants of pPK4.
- the expression unit for the objective RNA can be introduced into the chromosome of a host by, for example, using a transposon such as artificial transposon.
- a transposon such as artificial transposon
- the expression unit for the objective RNA can be introduced into the chromosome via homologous recombination or due to the transposition activity thereof.
- the expression unit for the objective RNA can also be introduced into the chromosome of a host by introduction methods utilizing homologous recombination.
- Examples of the introduction methods utilizing homologous recombination include, for example, methods of using a linear DNA, a plasmid containing a temperature sensitive replication origin, a plasmid capable of conjugative transfer, a suicide vector not having a replication origin that functions in a host, or the like. Only one copy or two or more copies of the expression unit for the objective RNA may be introduced. For example, by performing homologous recombination using a sequence present in multiple copies on a chromosome as a target, multiple copies of the expression unit for the objective RNA can be introduced into the chromosome. Examples of the sequence present in multiple copies on a chromosome include repetitive DNAs, and inverted repeats located at the both ends of a transposon.
- the objective RNA gene may be introduced into the chromosome so as to construct the expression unit for the objective RNA on the chromosome.
- the expression unit for the objective RNA can be constructed on the chromosome.
- introduction of a part of the expression unit for the objective RNA, such as the objective RNA gene, into the chromosome can be performed in the same manner as introduction of the whole of the expression unit for the objective RNA into the chromosome.
- Methods for transformation are not particularly limited, and generally used methods, such as the protoplast method (Gene, 39, 281-286(1985)), the electroporation method (Bio/Technology, 7, 1067-1070(1989)), and the electric pulse method (JP H2-207791 A), can be used.
- bacterium of the present invention has been modified so that the activity of ribonuclease III (RNaseIII) is reduced. Specifically, the bacterium of the present invention has been modified so that the activity of ribonuclease III is reduced as compared with a non-modified strain. The activity of ribonuclease III may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain. That is, the bacterium of the present invention may have been modified so that, for example, the activity of ribonuclease III is deleted (eliminated).
- RNaseIII ribonuclease III
- the objective RNA-producing ability of the bacterium can be improved, and that is, production of the objective RNA by using the bacterium can be increased.
- ribonuclease III refers to a protein that has an activity of catalyzing the reaction of cleaving specific RNA such as double-stranded RNA.
- a gene encoding ribonuclease III is also referred to as “ribonuclease III gene”.
- ribonuclease III gene examples include rnc gene.
- a protein (ribonuclease III) encoded by rnc gene is also referred to as "Rnc protein”.
- nucleotide sequences of ribonuclease III genes such as rnc genes, possessed by coryneform bacteria and the amino acid sequences of ribonucleases III encoded by these genes, such as Rnc proteins, can be obtained from, for example, public databases such as NCBI (National Center for Biotechnology Information).
- NCBI National Center for Biotechnology Information
- the nucleotide sequence of the rnc gene of the C. glutamicum ATCC 13869 strain and the amino acid sequence of the Rnc protein encoded by the gene are shown in SEQ ID NOS: 51 and 52, respectively.
- the ribonuclease III gene may be, for example, a gene having the nucleotide sequence of any of the ribonuclease III genes exemplified above (e.g. the nucleotide sequence shown as SEQ ID NO: 51).
- ribonuclease III may be, for example, a protein having the amino acid sequence of any of the ribonucleases III exemplified above (e.g. the amino acid sequence shown as SEQ ID NO: 52).
- the ribonuclease III gene may be a variant of any of the ribonuclease III genes exemplified above (e.g. a gene having the nucleotide sequence shown as SEQ ID NO: 51), so long as the original function is maintained.
- ribonuclease III may be a variant of any of the ribonucleases III exemplified above (e.g. a protein having the amino acid sequence shown as SEQ ID NO: 52), so long as the original function is maintained.
- Such a variant that maintains the original function is also referred to as "conservative variant".
- the term "rnc gene” includes not only the rnc genes exemplified above, but also includes conservative variants thereof.
- the term “Rnc protein” includes not only the Rnc proteins exemplified above, but also includes conservative variants thereof. Examples of the conservative variants include, for example, homologues and artificially modified versions of the ribonuclease III genes and ribonucleases III exemplified above.
- the expression "the original function is maintained” means that a variant of gene or protein has a function (such as activity or property) corresponding to the function (such as activity or property) of the original gene or protein. That is, the expression “the original function is maintained” used for the ribonuclease III gene means that a variant of the gene encodes a protein that maintains the original function (i.e. a protein having ribonuclease III activity). Furthermore, the expression “the original function is maintained” used for ribonuclease III means that a variant of the protein has ribonuclease III activity.
- Ribonuclease III activity can be measured by, for example, incubating the enzyme with RNA that serves as a substrate thereof (e.g. double-stranded RNA), and measuring the enzyme-dependent cleavage of the RNA.
- RNA that serves as a substrate thereof
- ribonuclease III activity is generally measured in the following manner (Methods Enzymol. 2001;342:143-58.).
- One example is a method of adding an enzyme (e.g.
- ribonuclease III activity can be calculated on the basis of the degree of the decrease in radioactivity as an indicator of cleavage of the substrate.
- another example is a method of adding 32 P-radiolabeled double-stranded RNA as a substrate to a reaction mixture containing an enzyme (30 mM Tris-HCl (pH8.0), 250 mM potassium glutamate or 160 mM NaCl, 5 mM spermidine, 0.1 mM EDTA, and 0.1 mM DTT), incubating at 37°C for 5 min, adding thereto MgCl 2 at a final concentration of 10 mM to initiate the RNA cleavage reaction, and adding thereto, after appropriate proceeding of the reaction, an equal volume of a mixture of EDTA and electrophoresis marker dye, of which the EDTA concentration is one providing a final concentration of 20 mM or more, to stop the reaction.
- an enzyme 30 mM Tris-HCl (pH8.0), 250 mM potassium glutamate or 160 mM NaCl, 5 mM spermidine, 0.1 mM EDTA, and 0.1 mM DTT
- ribonuclease III activity can be detected by applying samples after the reaction to electrophoresis using a denaturing 15%(w/v) polyacrylamide gel with TBE buffer (89 mM Tris/Tris-borate, and 2 mM EDTA) containing 7 M urea, and applying the gel to a radiation imaging analyzer to analyze cleaved RNA fragments.
- TBE buffer 89 mM Tris/Tris-borate, and 2 mM EDTA
- Homologues of the ribonuclease III gene or homologues of ribonuclease III can be easily obtained from public databases by, for example, BLAST search or FASTA search using the nucleotide sequence of any of the ribonuclease III genes exemplified above or the amino acid sequence of any of the ribonucleases III exemplified above as a query sequence.
- homologues of the ribonuclease III gene can be obtained by, for example, PCR using the chromosome of a coryneform bacterium as the template, and oligonucleotides prepared on the basis of the nucleotide sequence of any of these known ribonuclease III genes and adjacent regions thereof as primers.
- the ribonuclease III gene may be a gene encoding a protein having the amino acid sequence of any of the ribonucleases III exemplified above (e.g. the amino acid sequence shown as SEQ ID NO: 52), but which includes substitution, deletion, insertion, and/or addition of one or several amino acid residues at one or several positions, so long as the original function is maintained.
- the number meant by the term "one or several" mentioned above may differ depending on the positions of amino acid residues in the three-dimensional structure of the protein or the types of amino acid residues, specifically, it is, for example, 1 to 50, 1 to 40, or 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, particularly preferably 1 to 3.
- the aforementioned substitution, deletion, insertion, and/or addition of one or several amino acid residues are/is a conservative mutation that maintains the normal function of the protein.
- Typical examples of the conservative mutation are conservative substitutions.
- the conservative substitution is a mutation wherein substitution takes place mutually among Phe, Trp, and Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, and Val, if it is a hydrophobic amino acid; between Gln and Asn, if it is a polar amino acid; among Lys, Arg, and His, if it is a basic amino acid; between Asp and Glu, if it is an acidic amino acid; and between Ser and Thr, if it is an amino acid having a hydroxyl group.
- substitutions considered as conservative substitutions include, specifically, substitution of Ser or Thr for Ala, substitution of Gln, His, or Lys for Arg, substitution of Glu, Gln, Lys, His, or Asp for Asn, substitution of Asn, Glu, or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp, or Arg for Gln, substitution of Gly, Asn, Gln, Lys, or Asp for Glu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg, or Tyr for His, substitution of Leu, Met, Val, or Phe for Ile, substitution of Ile, Met, Val, or Phe for Leu, substitution of Asn, Glu, Gln, His, or Arg for Lys, substitution of Ile, Leu, Val, or Phe for Met, substitution of Trp, Tyr, Met, Ile, or Leu for Phe, substitution of Thr or Ala for Ser
- the ribonuclease III gene may be a gene encoding a protein having an amino acid sequence showing a homology of, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably 99% or more, to the total amino acid sequence of any of the ribonucleases III exemplified above, so long as the original function is maintained.
- “homology” means "identity”.
- the ribonucleases III gene may also be a DNA that is able to hybridize under stringent conditions with a complementary sequence of the nucleotide sequence of any of the ribonucleases III genes exemplified above (e.g. the nucleotide sequence shown as SEQ ID NO: 51) or a probe that can be prepared from the complementary sequence, so long as the original function is maintained.
- stringent conditions refers to conditions under which a so-called specific hybrid is formed, and a non-specific hybrid is not formed.
- Examples of the stringent conditions include those under which highly homologous DNAs hybridize to each other, for example, DNAs not less than 80% homologous, preferably not less than 90% homologous, more preferably not less than 95% homologous, still more preferably not less than 97% homologous, particularly preferably not less than 99% homologous, hybridize to each other, and DNAs less homologous than the above do not hybridize to each other, or conditions of washing of typical Southern hybridization, i.e., conditions of washing once, preferably 2 or 3 times, at a salt concentration and temperature corresponding to 1 x SSC, 0.1% SDS at 60°C, preferably 0.1 x SSC, 0.1% SDS at 60°C, more preferably 0.1 x SSC, 0.1% SDS at 68°C.
- the probe may be, for example, a part of a complementary sequence of the gene.
- a probe can be prepared by PCR using oligonucleotides prepared on the basis of a known gene sequence as primers and a DNA fragment containing any of these nucleotide sequences as a template.
- a DNA fragment having a length of about 300 bp can be used as the probe.
- the washing conditions of the hybridization may be, for example, 50°C, 2 x SSC and 0.1% SDS.
- ribonucleases III gene may be replaced with respective equivalent codons. That is, the ribonucleases III gene may be a variant of any of the ribonucleases III genes exemplified above due to the degeneracy of the genetic code.
- the percentage of the sequence identity between two sequences can be determined by, for example, using a mathematical algorithm.
- a mathematical algorithm include the algorithm of Myers and Miller (1988) CABIOS 4:11-17, the local homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the method for searching homology of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and an modified version of the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, such as one described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
- sequence comparison i.e. alignment
- the program can be appropriately executed by a computer.
- Examples of such a program include, but not limited to, CLUSTAL of PC/Gene program (available from Intelligenetics, Mountain View, Calif.), ALIGN program (Version 2.0), and GAP, BESTFIT, BLAST, FASTA, and TFASTA of Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignment using these programs can be performed by using, for example, initial parameters.
- the CLUSTAL program is well described in Higgins et al.
- BLAST nucleotide search can be performed by using BLASTN program with score of 100 and word length of 12.
- BLAST protein search can be performed by using BLASTX program with score of 50 and word length of 3. See http://www.ncbi.nlm.nih.gov for BLAST nucleotide search and BLAST protein search.
- Gapped BLAST (BLAST 2.0) can be used in order to obtain an alignment including gap(s) for the purpose of comparison.
- PSI-BLAST can be used in order to perform repetitive search for detecting distant relationships between sequences. See Altschul et al. (1997) Nucleic Acids Res. 25:3389 for Gapped BLAST and PSI-BLAST.
- initial parameters of each program e.g. BLASTN for nucleotide sequences, and BLASTX for amino acid sequences
- Alignment can also be manually performed.
- sequence identity between two sequences is calculated as the ratio of residues matching in the two sequences when aligning the two sequences so as to fit maximally with each other.
- the expression "the activity of a protein is reduced” means that the activity of the protein is reduced as compared with a non-modified strain. Specifically, the expression “the activity of a protein is reduced” means that the activity of the protein per cell is reduced as compared with that of a non-modified strain.
- the term "non-modified strain” used herein refers to a control strain that has not been modified so that the activity of an objective protein is reduced. Examples of the non-modified strain include a wild-type strain and parent strain. Specific examples of the non-modified strain include the respective type strains of the species of bacteria. Specific examples of the non-modified strain also include strains exemplified above in relation to the description of coryneform bacteria.
- the activity of a protein may be reduced as compared with a type strain, i.e. the type strain of the species to which the bacterium of the present invention belongs.
- the activity of a protein may also be reduced as compared with the C. glutamicum ATCC 13032 strain.
- the activity of a protein may also be reduced as compared with the C. glutamicum 2256 strain (ATCC 13869).
- the state that "the activity of a protein is reduced" also includes a state that the activity of the protein has completely disappeared.
- the expression "the activity of a protein is reduced” may mean that the number of molecules of the protein per cell is reduced, and/or the function of each molecule of the protein is reduced as compared with those of a non-modified strain. That is, the term “activity” in the expression “the activity of a protein is reduced” is not limited to the catalytic activity of the protein, but may also mean the transcription amount of a gene (i.e. the amount of mRNA) encoding the protein or the translation amount of the protein (i.e. the amount of the protein).
- the state that "the number of molecules of the protein per cell is reduced” also includes a state that the protein does not exist at all.
- the state that "the function of each molecule of the protein is reduced” also includes a state that the function of each protein molecule has completely disappeared.
- the degree of the reduction in the activity of a protein is not particularly limited, so long as the activity is reduced as compared with that of a non-modified strain.
- the activity of a protein may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.
- the modification for reducing the activity of a protein can be attained by, for example, reducing the expression of a gene encoding the protein.
- the expression "the expression of a gene is reduced” means that the expression of the gene is reduced as compared with a non-modified strain such as a wild-type strain and parent strain.
- the expression “the expression of a gene is reduced” means that the expression of the gene per cell is reduced as compared with that of a non-modified strain. More specifically, the expression “the expression of a gene is reduced” may mean that the transcription amount of the gene (i.e. the amount of mRNA) is reduced, and/or the translation amount of the gene (i.e. the amount of the protein expressed from the gene) is reduced.
- the state that "the expression of a gene is reduced” also includes a state that the gene is not expressed at all.
- the state that "the expression of a gene is reduced” is also referred to as "the expression of a gene is attenuated”.
- the expression of a gene may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.
- the reduction in gene expression may be due to, for example, a reduction in the transcription efficiency, a reduction in the translation efficiency, or a combination of them.
- the expression of a gene can be reduced by modifying an expression control sequence of the gene such as a promoter, a Shine-Dalgarno (SD) sequence (also referred to as ribosome-binding site (RBS)), and a spacer region between RBS and the start codon of the gene.
- SD Shine-Dalgarno
- RBS ribosome-binding site
- an expression control sequence preferably one or more nucleotides, more preferably two or more nucleotides, particularly preferably three or more nucleotides, of the expression control sequence are modified.
- the transcription efficiency of a gene can be reduced by, for example, replacing the promoter of the gene on a chromosome with a weaker promoter.
- weaker promoter means a promoter providing an attenuated transcription of a gene compared with an inherently existing wild-type promoter of the gene.
- weaker promoters include, for example, inducible promoters. That is, an inducible promoter may function as a weaker promoter under a non-induced condition, such as in the absence of the corresponding inducer.
- a part or the whole of an expression control sequence may be deleted.
- the expression of a gene can also be reduced by, for example, manipulating a factor responsible for expression control.
- the factor responsible for expression control examples include low molecules responsible for transcription or translation control (inducers, inhibitors, etc.), proteins responsible for transcription or translation control (transcription factors etc.), nucleic acids responsible for transcription or translation control (siRNA etc.), and so forth.
- the expression of a gene can also be reduced by, for example, introducing a mutation that reduces the expression of the gene into the coding region of the gene.
- the expression of a gene can be reduced by replacing a codon in the coding region of the gene with a synonymous codon used less frequently in a host.
- the gene expression may be reduced due to disruption of a gene as described later.
- the modification for reducing the activity of a protein can also be attained by, for example, disrupting a gene encoding the protein.
- the expression "a gene is disrupted” means that a gene is modified so that a protein that can normally function is not produced.
- the state that "a protein that normally functions is not produced” includes a state that the protein is not produced at all from the gene, and a state that the protein of which the function (such as activity or property) per molecule is reduced or eliminated is produced from the gene.
- Disruption of a gene can be attained by, for example, deleting the gene on a chromosome.
- the term "deletion of a gene” refers to deletion of a partial or entire region of the coding region of the gene. Furthermore, the whole of a gene including sequences upstream and downstream from the coding region of the gene on a chromosome may be deleted.
- the region to be deleted may be any region such as an N-terminal region (region encoding an N-terminal region of a protein), an internal region, or a C-terminal region (region encoding a C-terminal region of a protein), so long as the activity of the protein can be reduced. Deletion of a longer region can usually more surely inactivate the gene.
- the region to be deleted may be, for example, a region having a length of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the total length of the coding region of the gene. Furthermore, it is preferred that reading frames of the sequences upstream and downstream from the region to be deleted are not the same. Inconsistency of reading frames may cause a frameshift downstream of the region to be deleted.
- Disruption of a gene can also be attained by, for example, introducing a mutation for an amino acid substitution (missense mutation), a stop codon (nonsense mutation), addition or deletion of one or two nucleotide residues (frame shift mutation), or the like into the coding region of the gene on a chromosome (Journal of Biological Chemistry, 272:8611-8617 (1997); Proceedings of the National Academy of Sciences, USA, 95 5511-5515 (1998); Journal of Biological Chemistry, 26 116, 20833-20839 (1991)).
- Disruption of a gene can also be attained by, for example, inserting another nucleotide sequence into a coding region of the gene on a chromosome.
- Site of the insertion may be in any region of the gene, and insertion of a longer nucleotide sequence can usually more surely inactivate the gene.
- reading frames of the sequences upstream and downstream from the insertion site are not the same. Inconsistency of reading frames may cause a frameshift downstream of the region to be deleted.
- the other nucleotide sequence is not particularly limited so long as a sequence that reduces or eliminates the activity of the encoded protein is chosen, and examples thereof include, for example, a marker gene such as antibiotic resistance genes, and a gene useful for production of an objective substance.
- disruption of a gene may be carried out so that the amino acid sequence of the encoded protein is deleted.
- the modification for reducing the activity of a protein can be attained by, for example, deleting the amino acid sequence of the protein, specifically, modifying a gene so as to encode a protein of which the amino acid sequence is deleted.
- the term "deletion of the amino acid sequence of a protein” refers to deletion of a partial or entire region of the amino acid sequence of the protein.
- the term “deletion of the amino acid sequence of a protein” means that the original amino acid sequence disappears in the protein, and also includes cases where the original amino acid sequence is changed to another amino acid sequence.
- a region that was changed to another amino acid sequence by frameshift may be regarded as a deleted region.
- the amino acid sequence of a protein is deleted, the total length of the protein is typically shortened, but there can also be cases where the total length of the protein is not changed or is extended.
- deletion of a partial or entire region of the coding region of a gene a region encoded by the deleted region can be deleted in the encoded protein.
- a region encoded by the downstream region of the introduction site can be deleted in the encoded protein.
- a region encoded by the frameshift region can be deleted in the encoded protein.
- the aforementioned descriptions concerning the position and length of the region to be deleted in deletion of a gene can be applied mutatis mutandis to the position and length of the region to be deleted in deletion of the amino acid sequence of a protein.
- Such modification of a gene on a chromosome as described above can be attained by, for example, preparing a disruption-type gene modified so that it is unable to produce a protein that normally functions, and transforming a host with a recombinant DNA containing the disruption-type gene to cause homologous recombination between the disruption-type gene and the wild-type gene on a chromosome and thereby substitute the disruption-type gene for the wild-type gene on the chromosome.
- a marker gene selected according to the characteristics of the host such as auxotrophy
- the disruption-type gene examples include a gene of which a partial or entire region of the coding region is deleted, gene including a missense mutation, gene including a nonsense mutation, gene including a frame shift mutation, and gene including insertion of a transposon or marker gene.
- the protein encoded by the disruption-type gene has a conformation different from that of the wild-type protein, even if it is produced, and thus the function thereof is reduced or eliminated.
- Such gene disruption based on gene substitution utilizing homologous recombination has already been established, and there are methods of using a linear DNA such as a method called "Red driven integration" (Datsenko, K.A, and Wanner, B.L., Proc. Natl. Acad. Sci.
- Modification for reducing activity of a protein can also be attained by, for example, a mutagenesis treatment.
- the mutagenesis treatment include irradiation of X-ray or ultraviolet and treatment with a mutation agent such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).
- MNNG N-methyl-N'-nitro-N-nitrosoguanidine
- EMS ethyl methanesulfonate
- MMS methyl methanesulfonate
- Such methods for reducing the activity of a protein as mentioned above may be used independently or in an arbitrary combination.
- a part or all of the plurality of subunits may be modified, so long as the activity of the protein is eventually reduced. That is, for example, a part or all of a plurality of genes that encode the respective subunits may be disrupted or the like.
- a part or all of the activities of the plurality of isozymes may be reduced, so long as the activity of the protein is eventually reduced. That is, for example, a part or all of a plurality of genes that encode the respective isozymes may be disrupted or the like.
- a reduction in the activity of a protein can be confirmed by measuring the activity of the protein.
- a reduction in the activity of a protein can also be confirmed by confirming a reduction in the expression of a gene encoding the protein.
- a reduction in the expression of a gene can be confirmed by confirming a reduction in the transcription amount of the gene or a reduction in the amount of the protein expressed from the gene.
- a reduction in the transcription amount of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain.
- Examples of the method for evaluating the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-seq, and so forth (Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001).
- the amount of mRNA (such as the number of molecules of the mRNA per cell) is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.
- the amount of the protein (such as the number of molecules of the protein per cell) is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.
- Disruption of a gene can be confirmed by determining nucleotide sequence of a part or the whole of the gene, restriction enzyme map, full length, or the like of the gene depending on the means used for the disruption.
- the aforementioned methods for reducing the activity of a protein can be applied to reduction in the activities of arbitrary proteins and reduction in the expression of arbitrary genes, as well as reduction in ribonucleases III activity.
- the objective RNA can be produced by using the thus-obtained bacterium of the present invention.
- the method of the present invention is a method for producing the objective RNA, the method comprising culturing the bacterium of the present invention, and collecting the transcribed objective RNA.
- the method of the present invention is a method for producing the objective RNA, the method comprising culturing the bacterium of the present invention in a medium, to transcribe the objective RNA and accumulate the objective RNA in cells of the bacterium, and collecting the objective RNA from the cells.
- the bacterium of the present invention can be cultured according to, for example, culture conditions usually used for culturing bacteria such as coryneform bacteria.
- the bacterium of the present invention can be cultured in, for example, a usual medium containing a carbon source, a nitrogen source, and inorganic ions.
- organic micronutrients such as vitamins and amino acids can also be added as required.
- the carbon source for example, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and others can be used.
- the nitrogen source for example, ammonia gas, aqueous ammonia, ammonium salts, and others can be used.
- the inorganic ions for example, calcium ions, magnesium ions, phosphate ions, potassium ions, iron ions, and so forth can be appropriately used as required.
- the culture can be performed within appropriate ranges of pH 5.0 to 8.5 and 15 to 37°C under aerobic conditions for 10 to 120 hours.
- culture conditions for L-amino acid production using bacteria such as coryneform bacteria and culture conditions for methods of secretory production of a protein using bacteria such as coryneform bacteria can be referred to (WO01/23591, WO2005/103278, WO2013/065869, WO2013/065772, WO2013/118544, WO2013/062029, etc.).
- an inducible promoter is used for expression of the objective RNA
- the expression of the objective RNA can be appropriately induced.
- the objective RNA is transcribed and accumulated in cells of the bacterium.
- the expression and accumulation of the objective RNA can be confirmed by, for example, applying a fraction containing a cell extract as a sample to electrophoresis, and detecting a band corresponding to the molecular weight of the objective RNA.
- the objective RNA can be collected from the cells by appropriate methods used for separation and purification of compounds. Examples of such methods include, for example, centrifugation, salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography, affinity chromatography, and electrophoresis. These methods can be independently used, or can be used in an appropriate combination. Specifically, for example, the cells can be disrupted with ultrasonic waves or the like, a supernatant can be obtained by removing the cells from the cell-disrupted suspension by centrifugation or the like, and the objective RNA can be collected from the supernatant by the ion exchange resin method or the like.
- the collected objective RNA may be a free compound, a salt thereof, or a mixture thereof.
- the collected objective RNA may also be a complex with a high-molecular-weight compound such as a protein. That is, in the present invention, the term "objective RNA" may refer to the objective RNA in a free form, a salt thereof, a complex thereof with a high-molecular-weight compound such as a protein, or a mixture thereof, unless otherwise stated.
- the salt include, for example, ammonium salt and sodium salt.
- the collected objective RNA may contain, for example, such components as bacterial cells, medium components, moisture, and by-product metabolites of the bacterium, in addition to the objective RNA.
- the objective RNA may also be purified at a desired extent. Purity of the collected objective RNA may be, for example, 30% (w/w) or higher, 50% (w/w) or higher, 70% (w/w) or higher, 80% (w/w) or higher, 90% (w/w) or higher, or 95% (w/w) or higher.
- rnc gene a disruption strain of a ribonuclease III (RNaseIII) homologue gene (hereinafter also referred to as "rnc gene") of the C. glutamicum 2256 strain (ATCC 13869 strain, hereinafter also referred to simply as "2256 strain") was constructed in the following manner.
- genome DNA was obtained from cells of the 2256 strain with DNeasy Blood & Tissue Kit (QIAGEN).
- PCR amplification was performed by using this genome DNA as the template and PrimeSTAR GXL DNA Polymerase (TAKARA BIO), as well as primers of SEQ ID NOS: 1 and 2 to obtain a DNA fragment of about 1 kb containing an upstream region of the rnc gene, and primers of SEQ ID NOS: 3 and 4 to obtain a DNA fragment of about 1 kb containing a downstream region of the rnc gene.
- the PCR conditions were set according to a protocol recommended by the manufacturer.
- Competent cells of the Escherichia coli JM109 strain were transformed with the reaction mixture, applied to LB agar medium containing 25 ⁇ g/mL of kanamycin, and cultured at 37°C overnight. Then, single colonies were isolated from colonies that appeared on the agar medium, to obtain transformants resistant to kanamycin. Plasmids were extracted from the obtained transformants in the usual manner. A plasmid containing the DNA fragments of the upstream and downstream regions of the rnc gene was identified by a structural analysis, and designated as pBS4S ⁇ rnc (Fig. 1).
- This plasmid is not able to autonomously replicate in coryneform bacteria. Therefore, if coryneform bacteria are transformed with this plasmid, transformants in which this plasmid is incorporated into the chromosome by homologous recombination and which thereby have kanamycin resistance appear, although it occurs at an extremely low frequency.
- the 2256 strain was transformed with a high concentration of the plasmid pBS4 ⁇ rnc by the electric pulse method, applied to CM-Dex agar medium (5 g/L of glucose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1 g/L of KH 2 PO 4 , 0.4 g/L of MgSO 4 -7H 2 O, 0.01 g/L of FeSO 4 -7H 2 O, 0.01 g/L of MnSO 4 -7H 2 O, 3 g/L of urea, 1.2 g/L of soybean hydrolysate, adjusted to pH 7.5 with KOH, 20 g/L of agar) containing 25 ⁇ g/mL of kanamycin, and cultured at 30°C overnight.
- CM-Dex agar medium 5 g/L of glucose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1 g/L of KH 2 PO 4 , 0.4
- CM-Dex medium having the same composition as that of CM-Dex agar medium except that it does not contain agar
- kanamycin not containing kanamycin at 30°C overnight.
- the culture broth was appropriately diluted, applied to 10%(w/v)-sucrose-containing Dex-S10 agar medium (100 g/L of sucrose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1 g/L of KH 2 PO 4 , 0.4 g/L of MgSO 4 -7H 2 O, 0.01 g/L of FeSO 4 -7H 2 O, 0.01 g/L of MnSO 4 -4-5H 2 O, 3 g/L of urea, 1.2 g/L of soybean protein hydrolysate solution, 10 ⁇ g/L of biotin, adjusted to pH 7.5 with KOH, 20 g/L of agar) not containing kanamycin, and cultured at 30°C overnight.
- 10%(w/v)-sucrose-containing Dex-S10 agar medium 100 g/L of sucrose, 10 g/L of polypeptone, 10 g/L of yeast extract, 1 g
- strains were each considered to be a strain in which the sacB gene was removed as a result of the 2 nd homologous recombination and which thereby became insensitive to sucrose.
- the thus-obtained strains would include strain(s) of which the rnc gene was replaced with the deficient-type, and strain(s) of which the rnc gene reverted to the wild-type.
- the colonies that appeared were applied to colony PCR using KOD FX NEO (TOYOBO), to select rnc gene-deficient strains.
- some strains provided a DNA fragment having a shorter length in PCR amplification than that observed for the case of using the genome DNA of the 2256 strain (wild-type) as the template.
- one strain thereof was selected as an rnc gene-deficient strain, and designated as 2256 ⁇ rnc.
- the length of the PCR-amplified DNA fragment observed for the case of the wild-type rnc gene is about 3 kbp
- the length of the PCR-amplified DNA fragment observed for the case of deficient-type rnc gene is about 2 kbp (Fig. 2).
- the 2256 strain has an endogenous plasmid pAM330 (Yamaguchi, Ryuji, et al. "Determination of the complete nucleotide sequence of Brevibacterium lactofermentum plasmid pAM330 and analysis of its genetic information.” Agricultural and biological chemistry 50.11 (1986): 2771-2778.). There was constructed a plasmid pVC7-sacB, which corresponds to a plasmid pVC7 (JP1997-070291A) incorporated with a sacB gene.
- pVC7 is a composite plasmid of pAM330 and an Escherichia coli-universal vector pHSG399 (TAKARA BIO). Specifically, PCR amplification was performed by using pBS4S as the template, primers of SEQ ID NOS: 9 and 10, and PrimeSTAR GXL DNA Polymerase to obtain an amplified fragment of the sacB gene. Separately, PCR amplification was performed by using pVC7 as the template, primers of SEQ ID NOS: 11 and 12, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of pVC7. Both the amplified fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit (Clontech).
- strains 2256 and 2256 ⁇ rnc were each introduced with pVC7-sacB by the electric pulse method, applied to CM-Dex agar medium containing 5 ⁇ g/mL of chloramphenicol, and cultured at 30°C overnight, to obtain a plurality of transformants for each of strains 2256/pVC7-sacB and 2256 ⁇ rnc/pVC7-sacB. Then, from them, there were obtained strains 2256 ⁇ pAM330/pVC7-sacB and 2256 ⁇ rnc ⁇ pAM330/pVC7-sacB, of which the endogenous plasmid pAM330 was removed.
- strains were each applied to Dex-S10 agar medium, and cultured at 30°C overnight, to obtain strains 2256 ⁇ pAM330 and 2256 ⁇ rnc ⁇ pAM330, which were cured of pVC7-sacB to thereby become insensitive to sucrose.
- a promoter functional region (SEQ ID NO: 13; also referred to as "F1 promoter”; Koptides, M., et al., (1992). Characterization of bacteriophage BFK20 from Brevibacterium flavum. Microbiology, 138(7), 1387-1391.) from the promoter sequence (Accession No. L13772) of a bacteriophage BFK20, which is infectious to coryneform bacteria, a terminator (ter) region of the same (SEQ ID NO: 14; Bukovska, G., et al., (2006). Complete nucleotide sequence and genome analysis of bacteriophage BFK20-a lytic phage of the industrial producer Brevibacterium flavum.
- a DNA fragment of the U1Ainsert RNA transcription unit was prepared by chemical synthesis (Eurofins Genomics). Then, PCR amplification was performed by using the DNA fragment as the template, primers of SEQ ID NOS: 17 and 18, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of the U1Ainsert RNA transcription unit. Separately, PCR amplification was performed by using pVC7 as the template, primers of SEQ ID NOS: 19 and 20, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of pVC7. These amplified fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit (Clontech).
- a DNA fragment of Hv-iap (SEQ ID NO: 21), which is a partial sequence of cDNA of an iap gene encoding an inhibitor of apoptosis protein IAP of Henosepilachna vigintioctopunctata, was prepared by chemical synthesis on the basis of information described in WO2010/140675. There was constructed a plasmid containing a DNA sequence containing the F1 promoter and the Hv-iap sequence ligated immediately downstream thereof in the following manner (Fig. 5A).
- PCR amplification was performed by using pVC7 as the template, primers of SEQ ID NOS: 20 and 22, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of pVC7.
- PCR amplification was performed by using the DNA fragment of SEQ ID NO: 16 as the template, primers of SEQ ID NOS: 23 and 24, and PrimeSTAR HS (TAKARA BIO) to obtain a DNA fragment containing the F1 promoter sequence.
- PCR amplification was performed by using the DNA fragment of SEQ ID NO: 21 as the template, primers of SEQ ID NOS: 25 and 26, and PrimeSTAR HS (TAKARA BIO) to obtain a DNA fragment of the Hv-iap sequence.
- PCR amplification was performed by using pVC7 as the template, primers of SEQ ID NOS: 20 and 27, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of pVC7.
- PCR amplification was performed by using the DNA fragment of SEQ ID NO: 16 as the template, primers of SEQ ID NOS: 28 and 29, and PrimeSTAR HS (TAKARA BIO) to obtain an amplified fragment of the F1 promoter sequence. Both the amplified fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit (Clontech).
- inverse PCR was performed by using pVC7-Pf1rev as the template, primers of SEQ ID NOS: 20 and 30, and KOD -Plus- Mutagenesis Kit (TOYOBO). Then, the amplified DNA fragment was subject to DpnI treatment, phosphorylation reaction, and self-ligation reaction to thereby be cyclized, and introduced into competent cells of the Escherichia coli JM109 strain (TAKARA BIO). The cells were applied to LB agar medium containing 25 ⁇ g/mL of chloramphenicol, and cultured at 37°C overnight.
- Plasmids were extracted from the obtained transformants in the usual manner. An objective plasmid was identified by DNA sequencing analysis, and designated as pVC7-KpnI-XhoI-Pf1rev (Fig. 5B).
- PCR was performed by using pVC7-Pf1-Hv-iap as the template, primers of SEQ ID NOS: 31 and 32, and PrimeSTAR HS (TAKARA BIO) to obtain a DNA fragment containing the KpnI restriction enzyme site, F1 promoter region, Hv-iap region, and XhoI restriction enzyme site in this order.
- This DNA fragment and pVC7-KpnI-XhoI-Pf1rev were each digested with restriction enzymes KpnI and XhoI, and purified with MinElute PCR Purification Kit (QIAGEN). Both the purified products were mixed, and mutually ligated by a ligation reaction using Ligation high Ver.2 (TOYOBO).
- Plasmids were extracted from the obtained transformants in the usual manner. An objective plasmid was identified by DNA sequencing analysis, and designated as pVC7-Pf1-Hv-iap-Pf1rev (Fig. 5B).
- Colonies of the transformants obtained above were each spread on CM-Dex agar medium containing 5 ⁇ g/mL of chloramphenicol, and cultured at 30°C for about 16 hr. Then, a part of the cultured cells was used for test-tube culture. Culture was carried out in 2 mL of CM-Dex medium containing 5 ⁇ g/mL of chloramphenicol at 30°C with shaking for 24 hr. Then, 200 ⁇ L of the culture broth was treated with RNAprotect Bacteria Reagent, and the supernatant was removed.
- SIGMA 15 mg/mL of lysozyme
- TAKARA BIO 20 mg/mL proK
- plasmid was extracted from cells of each of the clones with QIAprep Spin Miniprep Kit (QIAGEN), and subject to agarose gel electrophoresis to compare the plasmid amounts.
- QIAprep Spin Miniprep Kit QIAGEN
- agarose gel electrophoresis As a result, it was observed that a plasmid extracted from one of the clones provides a DNA band clearly bolder than those extracted from the other clones.
- this plasmid was anew introduced into the 2256 strain, the plasmid was similarly extracted from the transformant, and the yield amount thereof was analyzed by agarose gel electrophoresis.
- this plasmid provides a DNA band clearly bolder than that of pVC7 as a control, and that is, it was revealed that this plasmid is a plasmid maintained at a high copy number in cells of coryneform bacteria as compared with the original plasmid pVC7.
- This plasmid was designated as pVC7H1.
- the mutation site of the obtained pVC7H1 was analyzed with a DNA sequencer, Genetic Analyzer 3500xl (Applied Biosystems). As a result, it was revealed that, in pVC7H1, the 1172 nd nucleotide of the total 6679 bp nucleotide sequence of pVC7 has been mutated from cytosine (C) to adenine (A), in which the nucleotide A at 2 nd position counted from the 5' terminus of the digestion recognition site of the restriction enzyme HindIII is regarded as "+1".
- this mutation is referred to as "C1172A”.
- this mutation is a cause of high copy number variation of pVC7H1.
- the mutant plasmids were each constructed with KOD -Plus- Mutagenesis Kit (TOYOBO). There were constructed pVC7H2 by using primers of SEQ ID NOS: 33 and 34, pVC7H3 by using primers of SEQ ID NOS: 33 and 35, pVC7H4 by using primers of SEQ ID NOS: 36 and 37, pVC7H5 by using primers of SEQ ID NOS: 36 and 38, pVC7H6 by using primers of SEQ ID NOS: 33 and 39, and pVC7H7 by using primers of SEQ ID NOS: 33 and 40, in combination with the plasmid pVC7 as the template, according to the construction protocol attached to the kit (Table 1). The total nucleotide sequences thereof were confirmed to be correct with a DNA sequencer.
- the constructed plasmids were each transformed into the 2256 ⁇ pAM330 strain in the usual manner.
- CM-Dex agar medium containing 5 ⁇ g/mL of chloramphenicol was used as the selection medium. Colonies were well formed in cases of transformation of any of the plasmids. Hence, colonies harboring the respective plasmids were each inoculated into the same agar medium with an inoculation loop, and cultured at 30°C for about 1 day. Then, grown cells of each strain in an amount of about 2-cm-square region were inoculated into CM-Dex medium containing 5 ⁇ g/mL of chloramphenicol, and cultured at 30°C with shaking overnight.
- the harbored plasmid was extracted from each culture broth in the usual manner. A part of the prepared plasmid solution was subject to agarose gel electrophoresis to analyze the DNA band of each plasmid. The results are shown in Table 2.
- pVC7H2 was used for the following experiments. It is estimated that the copy number of pVC7 is about 10 copies/cell, the copy number of pVC7H1 is about 100 copies/cell, and the copy number of pVC7H2 is about 250 copies/cell.
- RNA production using high copy number variation plasmids High copy number variations of pVC7-Pf1-U1Ainsert and pVC7-Pf1-Hv-iap-Pf1rev were constructed in the following manner. Specifically, inverse PCR was performed by using pVC7-Pf1-U1Ainsert or pVC7-Pf1-Hv-iap-Pf1rev as the template, primers of SEQ ID NOS: 33 and 41 for introduction of pVC7H1 mutation or primers of SEQ ID NOS: 33 and 34 for introduction of pVC7H2 mutation, and KOD -Plus- Mutagenesis Kit (TOYOBO).
- the obtained DNA fragments were subject to DpnI treatment, phosphorylation reaction, and self-ligation reaction to thereby be cyclized, and transformed into competent cells of the Escherichia coli JM109 strain (TAKARA BIO).
- the cells were applied to LB agar medium containing 25 ⁇ g/mL of chloramphenicol, and cultured at 37°C overnight. Then, single colonies were isolated from colonies that appeared to obtain transformants. Plasmids were extracted from the obtained transformants in the usual manner.
- the 2256 ⁇ rnc ⁇ pAM330 strain was introduced with each of the plasmids pVC7, pVC7-Pf1-U1Ainsert, pVC7H1-Pf1-U1Ainsert, pVC7H2-Pf1-U1Ainsert, pVC7-Pf1-Hv-iap-Pf1rev, pVC7H1-Pf1-Hv-iap-Pf1rev, and pVC7H2-Pf1-Hv-iap-Pf1rev by the electric pulse method, applied to CM-Dex agar medium containing 5 ⁇ g/mL of chloramphenicol, and cultured at 30°C overnight.
- RNA produced by the strains was evaluated.
- a part of cells cultured on the agar medium from a colony of each of the transformants was inoculated into 2 mL of CM-Dex medium containing 5 ⁇ g/mL of chloramphenicol, and cultured at 30°C with shaking for 24 hr.
- RNA was extracted from 200 ⁇ L of the culture broth in the same manner including RNAprotect Bacteria Reagent treatment as the above-mentioned Example, and finally the RNA sample was dissolved with 50 ⁇ L of RNase-free water to prepare a total RNA solution.
- RNA solutions were subject to total RNA analysis by PAGE under non-denaturing conditions using Novex TBE Gels (6%).
- the accumulation amount of U1Ainsert-RNA was increased in the order of pVC7-Pf1-U1Ainsert ⁇ pVC7H1-Pf1-U1Ainsert approximately equal to pVC7H2-Pf1-U1Ainsert (Fig. 7).
- the accumulation amount of Hv-iap-dsRNA was increased in the order of pVC7-Pf1-Hv-iap-Pf1rev ⁇ pVC7H1-Pf1-Hv-iap-Pf1rev ⁇ pVC7H2-Pf1-Hv-iap-Pf1rev (Fig. 7).
- the intracellular accumulation amount of objective RNA is increased by increasing the copy number of a plasmid for transcription of the RNA.
- RNA production by T7-promoter-induced-expression system in E. coli As described previously (Timmons, L., Court, D. L., & Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene, 263(1), 103-112.), there has been reported an RNA production system using T7 RNA polymerase in the Escherichia coli HT115(DE3) strain, which is a rnc gene deficient strain. Hence, a comparison was performed between RNA production by F1-promoter-expression system in the C. glutamicum 2256 ⁇ rnc ⁇ pAM330 strain and RNA production by T7-promoter-induced-expression system in E. coli.
- a plasmid, pL4440-Pt7-U1Ainsert, for transcription of the U1A-binding sequence under control of T7 promoter in a single direction and a plasmid, pL4440-Pt7-Hv-iap-Pt7rev, for transcription of Hv-iap RNA under control of T7 promoter in dual directions were constructed in the following manner.
- PCR was performed by using a plasmid pL4440 (GE Healthcare) as the template, primers of SEQ ID NOS: 42 and 43, and KOD FX NEO to obtain a DNA fragment of pL4440.
- PCR was performed by using pVC7-Pf1-U1Ainsert as the template, primers of SEQ ID NOS: 44 and 45, and PrimeSTAR HS (TAKARA BIO) to obtain a DNA fragment containing the U1Ainsert sequence.
- a DNA chain of SEQ ID NO: 46 and a DNA chain of SEQ ID NO: 47, wherein the DNA chain of SEQ ID NO: 47 has a sequence corresponding to the complementary sequence of the DNA chain of SEQ ID NO: 46, were mixed and mutually annealed, and purified with MinElute PCR Purification Kit to obtain a DNA fragment of the T7 terminator sequence. These three DNA fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit. Then, competent cells of the Escherichia coli JM109 strain were transformed with the reaction mixture, applied to LB agar medium containing 100 ⁇ g/mL of ampicillin, and cultured at 37°C overnight. Then, single colonies were isolated from colonies that appeared. Plasmids were extracted from the obtained transformants in the usual manner. Objective plasmids were identified by DNA sequencing analysis, and one of them was designated as pL4440-Pt7-U1Ainsert (Fig. 8).
- PCR was performed by using the plasmid pL4440 as the template, primers of SEQ ID NOS: 42 and 48, and KOD FX NEO to obtain a DNA fragment of pL4440.
- PCR was performed by using a plasmid pVC7-Pt7-Hv-iap-Pt7rev (described below in Example ⁇ 9> (2-2)) as the template, primers of SEQ ID NOS: 49 and 50, and KOD FX NEO to obtain a DNA fragment of the "Pt7-Hv-iap-Pt7rev" region.
- These DNA fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit.
- the E. coli HT115(DE3) strain was introduced with each of pL4440-Pt7-U1Ainsert and pL4440-Pt7-Hv-iap-Pt7rev by the electric pulse method to obtain transformant strains HT115(DE3)/pL4440-Pt7-U1Ainsert and HT115(DE3)/pL4440-Pt7-Hv-iap-Pt7rev.
- the strains were each cultured in LB liquid medium containing 100 ⁇ g/mL of ampicillin in a test tube at 37°C with shaking overnight to prepare a seed culture broth.
- the seed culture broth was added to LB liquid medium containing 100 ⁇ g/mL of ampicillin in a volume of 1/50 of the medium to start main culture at 37°C.
- IPTG was added thereto at a final concentration of 1 mM, culture was further continued, and sampling was carried out at 4 hr, 8hr, and 24 hr after addition of IPTG.
- a 200- ⁇ L aliquot of the culture broth was centrifuged (13,800 ⁇ g, 2 min) to collect cells.
- RNA was extracted with TRIzol (Thermo Fisher Scientific) according to the protocol thereof, and dissolved with 50 ⁇ L of RNase-free water to prepare a total RNA solution. For comparison, the total RNA solution of C.
- Example ⁇ 7> was used. PAGE was carried out under non-denaturing conditions by using Novex TBE Gels (6%) and 1 ⁇ L each of the RNA samples.
- the amount of U1Ainsert-ssRNA produced by F1-promoter-expression system in the C. glutamicum was larger than the amount of U1Ainsert-ssRNA produced by T7-promoter-induced-expression system in E. coli (Fig. 10).
- the amount of Hv-iap-dsRNA produced by F1-promoter-expression system in the C. glutamicum was larger than the amount of Hv-iap-dsRNA produced by T7-promoter-induced-expression system in E. coli (Fig. 10).
- coryneform bacteria such as C. glutamicum are useful for production of objective RNA.
- RNA production by T7-promoter-induced-expression system in C. glutamicum (1) Production of hairpin like structured RNA (1-1) Construction of plasmid pPK-T7lac-vd-antiOlac for transcription of hairpin like structured RNA A plasmid, pPK-T7lac-vd-antiOlac, for transcription of a hairpin like structured RNA as objective RNA under control of T7 promoter in a single direction was constructed in the following manner.
- pPK-T7lac (1-1-2) Construction of pPK-T7lac
- a fragment (131 bp) containing terminator T7 (T T7 ) was amplified from pET22b(+) (Novagen) with primers of SEQ ID NOS: 54 and 55 by PCR.
- the fragment was digested with KpnI and SalI, and cloned into pPK4 (U.S. patent No. 6,090,597) linearized by the same restriction enzymes.
- the constructed plasmid was named pPK4 XB - T7 ter (Fig. 11).
- kanamycin-resistance transformants Plasmids were extracted from the transformants, and the objective plasmid was selected and named pPK-T7lac-vd-antiOlac (Fig. 13).
- a DNA fragment-A of SEQ ID NO: 58 (161 bp) containing the strong promoter was prepared by chemical synthesis. Separately, a DNA fragment-B was amplified by PCR using primers of SEQ ID NOS: 60 (L54-Cm) and 61 (R55-Cm) with pVC7 plasmid as the template. Overlapping-PCR was carried out by using the fragment-A and fragment-B, and the resulting PCR fragment was amplified again by PCR using flanking primers of SEQ ID NOS: 62 (L54-hpa) and 61 (R55-Cm).
- the amplified fragment was digested with HpaI and NcoI, and ligated into HpaI-NcoI sites on pVC7.
- the ligation mixture was transformed into competent cells of the E. coli TG1 strain (Zymo Research) to obtain Cm r transformants. Plasmids were extracted from the transformants, and the objective plasmid was selected and named pVC54 (Fig. 14).
- a DNA fragment (4452 bp) containing lacI-PlacUV5-gene1 was cut out from pAR1219 (Proc Natl Acad Sci U S A. 1984 Apr; 81(7): 2035-2039) with BamHI, and cloned into the unique BamHI site of pBS5t-ptrB*-2Ter.
- the lacI-PlacUV5-gene1 fragment contains gene1 encoding T7 RNA polymerase and expressed under control of lacUV5 promoter (PlacUV5).
- a plasmid with genetic orientation of gene1 to tL3 was selected and named pBS5t-ptrB*-T7pol (Fig. 17).
- Plasmid pVC54-T7pol for expression of T7RNA polymerase was constructed from pVC54 and pBS5t-ptrB*-T7pol in the following manner.
- a DNA fragment containing lacI-PlacUV5-gene1 was cut out from pBS5t-ptrB*-T7pol with BglII. Then, the DNA fragment was ligated into pVC54 at BamHI site to construct pVC54-T7pol (Fig. 18).
- RNA-producing C. glutamicum The C. glutamicum 2256 ⁇ rnc ⁇ pAM330 strain was initially transformed with pVC54-T7Pol by electro-transformation method. Transformants were selected on a plate with CM2G medium (5 g/L of glucose, 10 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of NaCl, adjusted to pH 7.0) supplemented with 10 ⁇ g/mL of chloramphenicol (Cm) in 24 hr growth at 30°C. Then, a single colony isolation method was applied for obtaining colonies of two independent clones of C. glutamicum 2256 ⁇ rnc ⁇ pAM330 having pVC54-T7Pol.
- CM2G medium 5 g/L of glucose, 10 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of NaCl, adjusted to pH 7.0
- Cm chloramphenicol
- RNA production by T7-promoter-induced-expression system in C. glutamicum The C. glutamicum 2256 ⁇ rnc ⁇ pAM330/pVC54-T7Pol/pPK-T7lac-vd-antiOlac strain (each of clones A and B) was inoculated into 5 ml of CM2G medium supplemented with 10 ⁇ g/mL of Cm and 25 ⁇ g/ml of Km, and cultured overnight at 32 °C. On the next morning, the strain was inoculated into the same fresh medium (5 ml) at the OD 600 value of 0.2-0.3, and cultured at 32 °C for 4-5 hr.
- the induction was made by addition of 2 mM IPTG into the culture broth. After induction, the cultivation was continued for additional 3 hr and 19 hr, and also the OD 600 values of the culture broths were measured. Then, an aliquot (1-2 ml) of each of the culture broths was immediately mixed with RNAprotect Bacteria Reagent (QIAGEN 76506) according to manufacture recommendation, and pellet of cells was immediately frozen at -70°C.
- RNA isolation procedure using Trizol LS was applied.
- the isolated total RNA was diluted in 30 ⁇ l of DEPC-treated water to prepare a total RNA solution, the concentration of RNA in the solution was measured by Nanodrop spectrophotometer, and then an aliquot of the solution was applied for denaturing urea-PAGE (5%).
- Typically 3 ⁇ g each of total RNA solutions was applied to a well of the gel.
- staining of RNA in the gel was done with ethidium bromide (EthBr).
- EthBr ethidium bromide
- a DNA fragment containing gene 1 (T7pol) encoding T7 RNA polymerase was amplified by PCR using KOD FX NEO polymerase, primers of SEQ ID NOS: 67 and 68, and pVC54-T7pol as the template.
- Another DNA fragment was obtained by PCR using primers of SEQ ID NOS: 69 and 70, and a plasmid pPK4 (U.S. patent NO. 6,090,597) as the template. Both the DNA fragments were mixed, and ligated to each other by using In-Fusion HD Cloning Kit (TAKARA BIO). Then, E.
- coli JM109 competent cells were transformed with the reaction mixture, applied to LB agar medium containing Km (50 ⁇ g/mL), and cultured at 37°C for 16 hr to obtain Km R transformants. Among them, several colonies were isolated, and plasmids were extracted from the transformants. After the confirmation of the DNA sequences of the plasmids, an objective plasmid was selected and named pPK4-T7pol (Fig. 20).
- a DNA fragment-P of SEQ ID NO: 73 containing T7 promoter (forward-direction), KpnI restriction site, XhoI restriction site, and T7-promoter (reversed-direction) in this order, and another DNA fragment-Q of SEQ ID NO: 74 containing the complimentary sequence of the fragment-P were prepared by chemical synthesis, and both the single-stranded DNA fragments were mixed and annealed to produce a DNA fragment-R. Then, both the DNA fragment-N and DNA fragment-R were ligated to each other by using In-Fusion HD Cloning Kit. E.
- coli JM109 competent cells were transformed with the reaction mixture, applied to LB agar medium containing Cm (25 ⁇ g/ml), and cultured at 37°C for 16 hr to obtain Cm R transformants. Among them, several colonies were isolated, and plasmids were extracted from the transformants. After the confirmation of the DNA sequences of the plasmids, an objective plasmid was selected and named pVC7-Pt7-KpnI-XhoI-Pt7rev (Fig. 21).
- E. coli JM109 competent cells were transformed with the reaction mixture, applied to LB agar medium containing Cm (25 ⁇ g/ml), and cultured at 37°C for 16 hr to obtain Cm R transformants. Among them, several colonies were isolated, and plasmids were purified from the transformants. After the confirmation of the DNA sequences of the plasmids, an objective plasmid was selected and named pVC7-Pt7-Hv-iap-Pt7rev (Fig. 21).
- RNA production by T7-promoter-induced-expression system in C. glutamicum The C. glutamicum 2256 ⁇ rnc ⁇ pAM330/pPK4-T7pol/pVC7-Pt7-Hv-iap-Pt7rev strain was grown at 30°C in a test tube containing CM-Dex medium containing Km (25 ⁇ g/ml) and Cm (5 ⁇ g/ml) for 16 hr to prepare a seed culture broth. Then, the seed culture broth was inoculated into a fresh CM-Dex medium with a one-tenth of the volume of the medium to start main culture at 30°C.
- RNA production by using T7-promoter-induced-expression system in C. glutamicum was again confirmed.
- the plasmid pPK4 is a composite plasmid of a plasmid pHSG399 usable for E. coli and a plasmid pHM1519 possessed by C. glutamicum ATCC 13058, and hence, pPK4 serves as a shuttle vector replicable in both bacteria (U.S. patent No. 6,090,597).
- the mutant plasmids were each constructed with KOD -Plus- Mutagenesis Kit (TOYOBO). There were constructed pPK4H1 by using primers of SEQ ID NOS: 79 and 80, pPK4H2 by using primers of SEQ ID NOS: 81 and 82, pPK4H3 by using primers of SEQ ID NOS: 83 and 82, pPK4H4 by using primers of SEQ ID NOS: 84 and 82, pPK4H5 by using primers of SEQ ID NOS: 85 and 82, and pPK4H6 by using primers of SEQ ID NOS: 86 and 82, in combination with the plasmid pPK4 as the template, according to the construction protocol attached to the kit (Table 3). The total nucleotide sequences thereof were confirmed to be correct with a DNA sequencer.
- RNA production using pPK4H1 The expression system of U1A-RNA was integrated into the plasmid pPK4H1 in the following manner.
- PCR amplification was performed by using pVC7-Pf1-U1Ainsert as the template, primers of SEQ ID NOS: 87 and 88, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of the U1Ainsert RNA transcription unit.
- PCR amplification was performed by using pPK4H1 as the template, primers of SEQ ID NOS: 89 and 90, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of pPK4H1. These amplified fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit (Clontech).
- the C. glutamicum strain 2256 ⁇ rnc ⁇ pAM330 was introduced with each of the plasmids pPK4H1 and pPK4H1-Pf1-U1Ainsert by the electric pulse method, applied to CM-Dex agar medium containing 25 ⁇ g/mL of kanamycin, and cultured at 30°C overnight. Thereby, transformant strains 2256 ⁇ rnc ⁇ pAM330/pPK4H1 and 2256 ⁇ rnc ⁇ pAM330/pPK4H1-Pf1-U1Ainsert were obtained.
- CM-Dex agar medium containing 25 ⁇ g/mL of kanamycin, and cultured at 30°C for about 16 hr. Then, a part of the cultured cells was used for test-tube culture. Culture was carried out in 2 mL of CM-Dex medium containing 25 ⁇ g/mL of kanamycin at 30°C with shaking for 24 hr. Then, 200 ⁇ L of the culture broth was treated with RNAprotect Bacteria Reagent, and the supernatant was removed.
- SIGMA 15 mg/mL of lysozyme
- TAKARA BIO 20 mg/mL proK
- RNA band derived from U1Ainsert was confirmed at a predicted position only for the strain 22256 ⁇ rnc ⁇ pAM330/pPK4H1-Pf1-U1Ainsert (Fig. 23).
- a large amount of objective RNA was successfully produced in a Corynebacterium bacterium as a host even when using a pHM1519-derived vector, which is different from pVC7-derived vectors used in former Examples.
- RNA can be efficiently produced.
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Abstract
Description
[1] A method for producing objective RNA, the method comprising:
culturing a coryneform bacterium having an expression unit for the objective RNA in a medium, to express the objective RNA and accumulate the objective RNA in cells of the bacterium; and
collecting the objective RNA from the cells,
wherein the bacterium has been modified so that the activity of ribonuclease III is reduced as compared with a non-modified strain.
[2] The method mentioned above, wherein the ribonuclease III is a protein defined in (a), (b), or (c) mentioned below:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 52;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 52, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having ribonuclease III activity;
(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 52, and having ribonuclease III activity.
[3] The method mentioned above, wherein the activity of ribonuclease III is reduced by attenuating the expression of a gene encoding ribonuclease III, or by disrupting the gene.
[4] The method mentioned above, wherein the activity of ribonuclease III is reduced by deletion of a gene encoding ribonuclease III.
[5] The method mentioned above, wherein the bacterium has the expression unit at a copy number of 5 copies/cell or more.
[6] The method mentioned above, wherein the bacterium has the expression unit at a copy number of 70 copies/cell or more.
[7] The method mentioned above, wherein the bacterium has a vector containing the expression unit.
[8] The method mentioned above, wherein the expression unit contains a promoter sequence that functions in the coryneform bacterium and a nucleotide sequence encoding the objective RNA in the direction from 5' to 3'.
[9] The method mentioned above, wherein the promoter sequence is a promoter derived from a phage.
[10] The method mentioned above, wherein the promoter sequence is F1 promoter or T7 promoter.
[11] The method mentioned above, wherein the promoter sequence is a promoter defined in (a) or (b) mentioned below:
(a) a promoter comprising the nucleotide sequence of SEQ ID NO: 13 or 78;
(b) a promoter comprising an nucleotide sequence showing an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 13 or 78.
[12] The method mentioned above, wherein the bacterium is a bacterium belonging to the genus Corynebacterium.
[13] The method mentioned above, wherein the bacterium is Corynebacterium glutamicum.
The bacterium of the present invention is a coryneform bacterium having an expression unit for objective RNA, which has been modified so that the activity of ribonuclease III is reduced.
In the present invention, the term "bacterium having an objective RNA-producing ability" refers to a bacterium having an ability to express and accumulate the objective RNA in cells of the bacterium in such a degree that the objective RNA can be collected, when the bacterium is cultured in a medium. The bacterium having the objective RNA-producing ability may be a bacterium that is able to accumulate the objective RNA in cells of the bacterium in an amount larger than that obtainable with a non-modified strain. The term "non-modified strain" refers to a control strain that has not been modified so that the activity of ribonuclease III is reduced. That is, examples of the non-modified strain include a wild-type strain and parental strain. The bacterium having the objective RNA-producing ability may also be a bacterium that is able to accumulate the objective RNA in cells of the bacterium in an amount of 1 mg/L-culture or more, 2 mg/L-culture or more, 5 mg/L-culture or more, 10 mg/L-culture or more, 20 mg/L-culture or more, 50 mg/L-culture or more, or 100 mg/L-culture or more.
Corynebacterium acetoacidophilum
Corynebacterium acetoglutamicum
Corynebacterium alkanolyticum
Corynebacterium callunae
Corynebacterium crenatum
Corynebacterium glutamicum
Corynebacterium lilium
Corynebacterium melassecola
Corynebacterium thermoaminogenes (Corynebacterium efficiens)
Corynebacterium herculis
Brevibacterium divaricatum (Corynebacterium glutamicum)
Brevibacterium flavum (Corynebacterium glutamicum)
Brevibacterium immariophilum
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum
Brevibacterium saccharolyticum
Brevibacterium thiogenitalis
Corynebacterium ammoniagenes (Corynebacterium stationis)
Brevibacterium album
Brevibacterium cerinum
Microbacterium ammoniaphilum
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium alkanolyticum ATCC 21511
Corynebacterium callunae ATCC 15991
Corynebacterium crenatum AS1.542
Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734
Corynebacterium lilium ATCC 15990
Corynebacterium melassecola ATCC 17965
Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC 13868
Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020
Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC 14068
Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869
Brevibacterium roseum ATCC 13825
Brevibacterium saccharolyticum ATCC 14066
Brevibacterium thiogenitalis ATCC 19240
Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872
Brevibacterium album ATCC 15111
Brevibacterium cerinum ATCC 15112
Microbacterium ammoniaphilum ATCC 15354
The bacterium of the present invention has the expression unit for the objective RNA. A coryneform bacterium having the expression unit for the objective RNA can be obtained by introducing the expression unit for the objective RNA to a coryneform bacterium.
The bacterium of the present invention has been modified so that the activity of ribonuclease III (RNaseIII) is reduced. Specifically, the bacterium of the present invention has been modified so that the activity of ribonuclease III is reduced as compared with a non-modified strain. The activity of ribonuclease III may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain. That is, the bacterium of the present invention may have been modified so that, for example, the activity of ribonuclease III is deleted (eliminated). By modifying a coryneform bacterium so that the activity of ribonuclease III is reduced, the objective RNA-producing ability of the bacterium can be improved, and that is, production of the objective RNA by using the bacterium can be increased.
The objective RNA can be produced by using the thus-obtained bacterium of the present invention. The method of the present invention is a method for producing the objective RNA, the method comprising culturing the bacterium of the present invention, and collecting the transcribed objective RNA. By culturing the bacterium of the present invention in a medium, the objective RNA can be transcribed and accumulated in cells of the bacterium. That is, specifically, the method of the present invention is a method for producing the objective RNA, the method comprising culturing the bacterium of the present invention in a medium, to transcribe the objective RNA and accumulate the objective RNA in cells of the bacterium, and collecting the objective RNA from the cells.
A disruption strain of a ribonuclease III (RNaseIII) homologue gene (hereinafter also referred to as "rnc gene") of the C. glutamicum 2256 strain (ATCC 13869 strain, hereinafter also referred to simply as "2256 strain") was constructed in the following manner.
The 2256 strain has an endogenous plasmid pAM330 (Yamaguchi, Ryuji, et al. "Determination of the complete nucleotide sequence of Brevibacterium lactofermentum plasmid pAM330 and analysis of its genetic information." Agricultural and biological chemistry 50.11 (1986): 2771-2778.). There was constructed a plasmid pVC7-sacB, which corresponds to a plasmid pVC7 (JP1997-070291A) incorporated with a sacB gene. pVC7 is a composite plasmid of pAM330 and an Escherichia coli-universal vector pHSG399 (TAKARA BIO). Specifically, PCR amplification was performed by using pBS4S as the template, primers of SEQ ID NOS: 9 and 10, and PrimeSTAR GXL DNA Polymerase to obtain an amplified fragment of the sacB gene. Separately, PCR amplification was performed by using pVC7 as the template, primers of SEQ ID NOS: 11 and 12, and KOD FX NEO (TOYOBO) to obtain an amplified fragment of pVC7. Both the amplified fragments were mixed, and mutually ligated by using In-Fusion HD Cloning Kit (Clontech). Then, competent cells of the Escherichia coli JM109 strain (TAKARA BIO) were transformed with the reaction mixture, applied to LB agar medium containing 25 μg/mL of chloramphenicol, and cultured at 37°C overnight. Then, single colonies were isolated from colonies that appeared. Plasmids were extracted from the obtained transformants in the usual manner. An objective plasmid was identified by DNA sequencing analysis, and designated as pVC7-sacB (Fig. 3). The
A plasmid, pVC7-Pf1-U1Ainsert, for transcription of a U1A-binding sequence as objective RNA under control of F1 promoter in a single direction was constructed in the following manner.
A plasmid, pVC7-Pf1-Hv-iap-Pf1rev, for transcription of Hv-iap RNA as objective RNA under control of F1 promoter in dual directions was constructed in the following manner.
The C. glutamicum strains 2256ΔpAM330 and 2256ΔrncΔpAM330 were introduced with each of the plasmids pVC7, pVC7-Pf1-U1Ainsert, and pVC7-Pf1-Hv-iap-Pf1rev by the electric pulse method, applied to CM-Dex agar medium containing 5 μg/mL of chloramphenicol, and cultured at 30°C overnight. Thereby, transformant strains 2256ΔpAM330/pVC7 and 2256ΔrncΔpAM330/pVC7, 2256ΔpAM330/pVC7-Pf1-U1Ainsert and 2256ΔrncΔpAM330/pVC7-Pf1-U1Ainsert, and 2256ΔpAM330/pVC7-Pf1-Hv-iap-Pf1rev and 2256ΔrncΔpAM330/pVC7-Pf1-Hv-iap-Pf1rev were obtained.
Sixty clones of a
High copy number variations of pVC7-Pf1-U1Ainsert and pVC7-Pf1-Hv-iap-Pf1rev were constructed in the following manner. Specifically, inverse PCR was performed by using pVC7-Pf1-U1Ainsert or pVC7-Pf1-Hv-iap-Pf1rev as the template, primers of SEQ ID NOS: 33 and 41 for introduction of pVC7H1 mutation or primers of SEQ ID NOS: 33 and 34 for introduction of pVC7H2 mutation, and KOD -Plus- Mutagenesis Kit (TOYOBO). Then, the obtained DNA fragments were subject to DpnI treatment, phosphorylation reaction, and self-ligation reaction to thereby be cyclized, and transformed into competent cells of the Escherichia coli JM109 strain (TAKARA BIO). The cells were applied to LB agar medium containing 25 μg/mL of chloramphenicol, and cultured at 37°C overnight. Then, single colonies were isolated from colonies that appeared to obtain transformants. Plasmids were extracted from the obtained transformants in the usual manner. Objective plasmids were identified by DNA sequencing analysis, and designated as pVC7H1-Pf1-U1Ainsert, pVC7H2-Pf1-U1Ainsert, pVC7H1-Pf1-Hv-iap-Pf1rev, and pVC7H2-Pf1-Hv-iap-Pf1rev.
As described previously (Timmons, L., Court, D. L., & Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene, 263(1), 103-112.), there has been reported an RNA production system using T7 RNA polymerase in the Escherichia coli HT115(DE3) strain, which is a rnc gene deficient strain. Hence, a comparison was performed between RNA production by F1-promoter-expression system in the C. glutamicum 2256ΔrncΔpAM330 strain and RNA production by T7-promoter-induced-expression system in E. coli.
(1) Production of hairpin like structured RNA
(1-1) Construction of plasmid pPK-T7lac-vd-antiOlac for transcription of hairpin like structured RNA
A plasmid, pPK-T7lac-vd-antiOlac, for transcription of a hairpin like structured RNA as objective RNA under control of T7 promoter in a single direction was constructed in the following manner.
A DNA fragment of SEQ ID NO: 53 encoding the hairpin like structured RNA was prepared by chemical synthesis based on the genome sequence of a potato spindle tuber viroid (PSTVd).125 (Mol Biol (Mosk). 2013 Jan-Feb;47(1):94-106.). The DNA fragment was cloned into pUC57 (ATG Servis-Gen, St. Petersburg, Russia) to construct pUC57-VD.
At first, a fragment (131 bp) containing terminator T7 (TT7) was amplified from pET22b(+) (Novagen) with primers of SEQ ID NOS: 54 and 55 by PCR. Then, the fragment was digested with KpnI and SalI, and cloned into pPK4 (U.S. patent No. 6,090,597) linearized by the same restriction enzymes. The constructed plasmid was named pPK4 XB-T7 ter (Fig. 11).
A DNA fragment VD-aOlac was cut out from pUC57-VD with XbaI and XhoI, and cloned into pPK-T7lac with T4DNA ligase. The ligation mixture was transformed into competent cells of the E. coli LE392 strain (Promega) by electroporation. Standard electroporation procedure for E. coli cells was applied. After growth of cells for 1.5 hr at 37°C, the cells were seeded on LB agar-medium plate supplemented with 50 μg/ml of kanamycin (Km) to obtain kanamycin-resistance (Kmr) transformants. Plasmids were extracted from the transformants, and the objective plasmid was selected and named pPK-T7lac-vd-antiOlac (Fig. 13).
A plasmid, pVC54-T7Pol, for expression of T7 RNA polymerase was constructed in the following manner.
For the construction of pVC54, the region of SEQ ID NO: 58 containing the promoter region for chloramphenicol-resistance (Cmr) gene and a portion derived from pAM330 of pVC7 (U.S. patent No. 5,804,414) was displaced by a region containing a strong promoter in the following manner.
A DNA fragment (2021 bp) containing a part of ptrB and hemH genes was amplified by PCR using primers of SEQ ID NOS: 63 (x550) and 64 (x551) with genomic DNA of the 2256 strain as the template. The amplified fragment was digested with BglII, and cloned into BamHI site of pBS5t (WO2006/057450). The resulting plasmid was named pBS5t-ptrB* (Fig. 15). It contains unique NheI site in the part of ptrB gene.
The Plasmid pVC54-T7pol for expression of T7RNA polymerase was constructed from pVC54 and pBS5t-ptrB*-T7pol in the following manner.
The C. glutamicum 2256ΔrncΔpAM330 strain was initially transformed with pVC54-T7Pol by electro-transformation method. Transformants were selected on a plate with CM2G medium (5 g/L of glucose, 10 g/L of tryptone, 10 g/L of yeast extract, 5 g/L of NaCl, adjusted to pH 7.0) supplemented with 10 μg/mL of chloramphenicol (Cm) in 24 hr growth at 30°C. Then, a single colony isolation method was applied for obtaining colonies of two independent clones of C. glutamicum 2256ΔrncΔpAM330 having pVC54-T7Pol. Those two clones were subject to another transformation with pPK-T7lac-vd-antiOlac. Transformants were selected on a plate with CM2G medium supplemented with 10 μg/mL of Cm and 25 μg/ml of Km in 24 hr growth at 30°C. Again, single colony isolation method was applied for obtaining colonies of two independent clones (named clones A and B) of C. glutamicum 2256ΔrncΔpAM330 having pVC54-T7Pol and pPK-T7lac-vd-antiOlac (named C. glutamicum 2256ΔrncΔpAM330/pVC54-T7Pol/pPK-T7lac-vd-antiOlac).
The C. glutamicum 2256ΔrncΔpAM330/pVC54-T7Pol/pPK-T7lac-vd-antiOlac strain (each of clones A and B) was inoculated into 5 ml of CM2G medium supplemented with 10 μg/mL of Cm and 25 μg/ml of Km, and cultured overnight at 32 °C. On the next morning, the strain was inoculated into the same fresh medium (5 ml) at the OD600 value of 0.2-0.3, and cultured at 32 °C for 4-5 hr. At this time, the induction was made by addition of 2 mM IPTG into the culture broth. After induction, the cultivation was continued for additional 3 hr and 19 hr, and also the OD600 values of the culture broths were measured. Then, an aliquot (1-2 ml) of each of the culture broths was immediately mixed with RNAprotect Bacteria Reagent (QIAGEN 76506) according to manufacture recommendation, and pellet of cells was immediately frozen at -70°C.
(2-1) Construction of plasmid pPK4-T7pol for expression of T7 RNA polymerase
A plasmid, pPK4-T7pol, for expression of T7 RNA polymerase was constructed in the following manner.
A plasmid, pVC7-Pt7-Hv-iap-Pt7rev, for transcription of Hv-iap RNA as objective RNA under control of T7 promoter in dual directions was constructed in the following manner.
A DNA fragment-N was obtained by PCR using KOD FX NEO, primers of SEQ ID NOS: 71 and 72, and pVC7 as the template. Separately, a DNA fragment-P of SEQ ID NO: 73 containing T7 promoter (forward-direction), KpnI restriction site, XhoI restriction site, and T7-promoter (reversed-direction) in this order, and another DNA fragment-Q of SEQ ID NO: 74 containing the complimentary sequence of the fragment-P were prepared by chemical synthesis, and both the single-stranded DNA fragments were mixed and annealed to produce a DNA fragment-R. Then, both the DNA fragment-N and DNA fragment-R were ligated to each other by using In-Fusion HD Cloning Kit. E. coli JM109 competent cells were transformed with the reaction mixture, applied to LB agar medium containing Cm (25 μg/ml), and cultured at 37°C for 16 hr to obtain CmR transformants. Among them, several colonies were isolated, and plasmids were extracted from the transformants. After the confirmation of the DNA sequences of the plasmids, an objective plasmid was selected and named pVC7-Pt7-KpnI-XhoI-Pt7rev (Fig. 21).
A DNA fragment-S containing KpnI restriction site, Hv-iap, and XhoI restriction site in this order was amplified by PCR using a DNA fragment of SEQ ID NO: 75 as the template, and primers of SEQ ID NOS: 76 and 77. Then, the DNA fragment-S and pVC7-Pt7-KpnI-XhoI-Pt7rev were each digested with KpnI and XhoI, and purified with MinElute PCR Purification Kit (Qiagen). Both the purified products were mixed, and ligated to each other by using Ligation high ver. 2 (TOYOBO). E. coli JM109 competent cells were transformed with the reaction mixture, applied to LB agar medium containing Cm (25 μg/ml), and cultured at 37°C for 16 hr to obtain CmR transformants. Among them, several colonies were isolated, and plasmids were purified from the transformants. After the confirmation of the DNA sequences of the plasmids, an objective plasmid was selected and named pVC7-Pt7-Hv-iap-Pt7rev (Fig. 21).
The C. glutamicum 2256ΔrncΔpAM330 strain was introduced with pPK4-T7pol by electroporation. The cell suspension was applied to CM-Dex agar medium containing Km (25 μg/ml), and cultured at 30°C for 16 hr to obtain transformants. Subsequently, one of the transformants was introduced with pVC7-Pt7-Hv-iap-Pt7rev. The cell suspension was applied to CM-Dex agar medium containing Km (25 μg/ml) and Cm (5 μg/ml), and cultured at 30°C for 24 hr to obtain transformants. Thus, finally C. glutamicum 2256ΔrncΔpAM330/pPK4-T7pol/pVC7-Pt7-Hv-iap-Pt7rev was obtained.
The C. glutamicum 2256ΔrncΔpAM330/pPK4-T7pol/pVC7-Pt7-Hv-iap-Pt7rev strain was grown at 30°C in a test tube containing CM-Dex medium containing Km (25 μg/ml) and Cm (5 μg/ml) for 16 hr to prepare a seed culture broth. Then, the seed culture broth was inoculated into a fresh CM-Dex medium with a one-tenth of the volume of the medium to start main culture at 30°C. After 6 hr incubation, an aliquot of the culture broth was sampled, and the rest of the culture broth was added with 2 mM IPTG and further cultured. At 3 hr and 27 hr after IPTG addition, aliquots of the culture broth were sampled. RNA was extracted from 200 μL of the culture broth in the same manner including RNAprotect Bacteria Reagent treatment as the above-mentioned Example, and finally the RNA sample was dissolved with 50 μL of RNase-free water to prepare a total RNA solution.
The plasmid pPK4 is a composite plasmid of a plasmid pHSG399 usable for E. coli and a plasmid pHM1519 possessed by C. glutamicum ATCC 13058, and hence, pPK4 serves as a shuttle vector replicable in both bacteria (U.S. patent No. 6,090,597).
The expression system of U1A-RNA was integrated into the plasmid pPK4H1 in the following manner.
SEQ ID NOS:
1-12: Primers
13: Nucleotide sequence of F1 promoter
14: Nucleotide sequence of terminator region of BFK20
15: Nucleotide sequence of U1A-binding sequence
16: Nucleotide sequence of transcription unit for U1Ainsert RNA
17-20: Primers
21: Nucleotide sequence of Hv-iap
22-50: Primers
51: Nucleotide sequence of rnc gene of C. glutamicum 2256 (ATCC 13869)
52: Amino acid sequence of Rnc protein of C. glutamicum 2256 (ATCC 13869)
53: Nucleotide sequence of DNA fragment
54-57: Primers
58: Nucleotide sequence of DNA fragment
59: Nucleotide sequence of DNA fragment-A
60-72: Primers
73: Nucleotide sequence of DNA fragment-P
74: Nucleotide sequence of DNA fragment-Q
75: Nucleotide sequence of DNA fragment
76-77: Primers
78: Nucleotide sequence of T7 promoter
79-90: Primers
Claims (13)
- A method for producing objective RNA, the method comprising:
culturing a coryneform bacterium having an expression unit for the objective RNA in a medium, to express the objective RNA and accumulate the objective RNA in cells of the bacterium; and
collecting the objective RNA from the cells,
wherein the bacterium has been modified so that the activity of ribonuclease III is reduced as compared with a non-modified strain.
- The method according to claim 1, wherein the ribonuclease III is a protein defined in (a), (b), or (c) mentioned below:
(a) a protein comprising the amino acid sequence of SEQ ID NO: 52;
(b) a protein comprising the amino acid sequence of SEQ ID NO: 52, but which includes substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues, and having ribonuclease III activity;
(c) a protein comprising an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of SEQ ID NO: 52, and having ribonuclease III activity.
- The method according to claim 1 or 2, wherein the activity of ribonuclease III is reduced by attenuating the expression of a gene encoding ribonuclease III, or by disrupting the gene.
- The method according to any one of claims 1 to 3, wherein the activity of ribonuclease III is reduced by deletion of a gene encoding ribonuclease III.
- The method according to any one of claims 1 to 4, wherein the bacterium has the expression unit at a copy number of 5 copies/cell or more.
- The method according to any one of claims 1 to 5, wherein the bacterium has the expression unit at a copy number of 70 copies/cell or more.
- The method according to any one of claims 1 to 6, wherein the bacterium has a vector containing the expression unit.
- The method according to any one of claims 1 to 7, wherein the expression unit contains a promoter sequence that functions in the coryneform bacterium and a nucleotide sequence encoding the objective RNA in the direction from 5' to 3'.
- The method according to claim 8, wherein the promoter sequence is a promoter derived from a phage.
- The method according to claim 8 or 9, wherein the promoter sequence is F1 promoter or T7 promoter.
- The method according to any one of claims 8 to 10, wherein the promoter sequence is a promoter defined in (a) or (b) mentioned below:
(a) a promoter comprising the nucleotide sequence of SEQ ID NO: 13 or 78;
(b) a promoter comprising an nucleotide sequence showing an identity of 90% or higher to the nucleotide sequence of SEQ ID NO: 13 or 78.
- The method according to any one of claims 1 to 11, wherein the bacterium is a bacterium belonging to the genus Corynebacterium.
- The method according to any one of claims 1 to 12, wherein the bacterium is Corynebacterium glutamicum.
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CN201880021697.7A CN110494567A (en) | 2017-03-28 | 2018-02-02 | The method for producing RNA |
JP2019554009A JP2020512007A (en) | 2017-03-28 | 2018-02-02 | Method for producing RNA |
BR112019018859A BR112019018859A2 (en) | 2017-03-28 | 2018-02-02 | method to produce objective rna |
US16/582,385 US11046986B2 (en) | 2017-03-28 | 2019-09-25 | Method for producing RNA |
US17/326,677 US11884951B2 (en) | 2017-03-28 | 2021-05-21 | Method for producing RNA |
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EP3601583A1 (en) * | 2017-03-28 | 2020-02-05 | Ajinomoto Co., Inc. | Method for producing rna |
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