WO2002097064A1 - Micro-organismes hotes - Google Patents
Micro-organismes hotes Download PDFInfo
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- WO2002097064A1 WO2002097064A1 PCT/JP2002/005151 JP0205151W WO02097064A1 WO 2002097064 A1 WO2002097064 A1 WO 2002097064A1 JP 0205151 W JP0205151 W JP 0205151W WO 02097064 A1 WO02097064 A1 WO 02097064A1
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- Prior art keywords
- gene
- microorganism
- region
- genes
- sporulation
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to host microorganisms used for producing useful proteins or polypeptides, and recombinant microorganisms.
- the industrial production of useful substances by microorganisms includes a wide variety of foods such as alcoholic beverages, miso, and soy sauce, as well as amino acids, organic acids, nucleic acids, antibiotics, sugars, lipids, and proteins. Its use has been extended to a wide range of fields, from food, medicine, daily necessities such as detergents and cosmetics, to raw materials for various chemical products.
- microorganisms originally have a wide variety of genes to respond to environmental changes in the natural world, and in industrial production of proteins, etc., which use limited production media, productivity is not always efficient. The situation was not entirely relevant.
- strains in which genes involved in the early stage of sporulation are deleted or inactivated independently have been constructed, but the effect of improving productivity is not sufficient. Not.
- the present invention relates to a host microorganism capable of improving the productivity of protein or polypeptide by deleting or inactivating genes unnecessary or harmful to the production of proteins or polypeptides from the genome.
- the purpose is to provide.
- the present invention provides a recombinant microorganism obtained by introducing a gene encoding a protein or polypeptide bound downstream of a transcription initiation control region, a translation initiation control region, or a secretion signal region into the host microorganism. It is an object of the present invention to provide a method for producing a protein or polypeptide using a recombinant microorganism. Disclosure of the invention
- the present inventors diligently searched for genes that are unnecessary or harmful to the production of useful proteins or polypeptides among various genes encoded on the microorganism genome, and found that specific genes involved in sporulation were identified. After deleting or inactivating the gene from the genome, a gene encoding the protein or polypeptide of interest is introduced by binding an appropriate transcription initiation control region, translation initiation control region or secretory signal region. It has been found that the productivity of the target protein or polypeptide is improved as compared to before deletion or inactivation.
- the present invention relates to a microorganism in which one or more genes selected from a group of genes involved in spore formation are deleted or inactivated in the middle to late stages of sporulation, a transcription initiation control region, a translation initiation control region, It is intended to provide a recombinant microorganism obtained by introducing a gene encoding a protein or polypeptide bound downstream of a secretory signal region, and a method for producing a protein or polypeptide using the recombinant microorganism. .
- Parent microorganisms for constructing the microorganism of the present invention include those involved in sporulation. Any microorganism having a spore may be used, and a microorganism that forms a spore is more preferable. These may be wild-type or mutated. Specific examples include Bacillus bacterium such as Bacillus subtilis, Clostridium bacterium, and yeast. Among them, Bacillus bacterium is preferable. Furthermore, Bacillus subtilis is particularly preferred because its whole genome information is clarified, genetic engineering and genomic engineering techniques have been established, and it has the ability to secrete and produce proteins and extracellular cells.
- Bacillus bacterium such as Bacillus subtilis, Clostridium bacterium, and yeast. Among them, Bacillus bacterium is preferable.
- Bacillus subtilis is particularly preferred because its whole genome information is clarified, genetic engineering and genomic engineering techniques have been established, and it has the ability to secrete and produce proteins and extracellular cells.
- the objective protein or polypeptide to be produced using the microorganism of the present invention is, for example, an enzyme or a physiologically useful enzyme for use in foods, pharmaceuticals, cosmetics, detergents, textile treatment, medical test agents, etc.
- examples include proteins and polypeptides such as active factors. It is known that 250 or more genes scattered on the genome are involved in sporulation, but the genes to be deleted or inactivated in the present invention encode a sporulation stage specific sigma factor. Genes that promote spore formation, such as genes that cause spore formation, and genes that are involved in the expression of the ⁇ -factor genes and the activation of the ⁇ -factor, are preferred.
- genes that are transcribed by the sigma factor and are involved in the promotion of sporulation are also included in the early stage of the sporulation stage (phase 0 to stage I) is more promising than that in the logarithmic growth phase.
- Genes that are known to increase the production of various extracellular enzymes such as oral thease and amylase are specifically expressed as genes to be deleted or inactivated during the middle to late stages of the sporulation stage.
- Those involved in sporulation are desirable.
- genes involved in sporulation stage II, stage III, stage IV, or stage V are preferred, especially sporulation stage II or stage III, and optimally sporulation.
- Genes involved in phase II are preferred. The present inventors have found that such genes are not directly involved in the production of the target protein and are not necessary for the growth of microorganisms in a usual industrial production medium.
- each gene of Bacillus subtilis shown in Table 1 and Table 2 has 70% or more, preferably 80% or more, more preferably 90% or more homology with each gene of Table 1, Genes derived from other microorganisms, preferably from Bacillus bacteria, are considered to correspond to the genes described in Table 1, and are included in the genes to be deleted or inactivated in the present invention.
- the amino acid sequence homology is calculated by the Lipman_Pearson method (Science, 227, 1435, (1985)). Deletion or inactivation of one or more genes selected from such a group of genes reduces consumption of chemical energy involved in sporulation, and prolongs the production period of protein or polypeptide. Thus, in the production of the protein or polypeptide, the productivity can be improved.
- the number of genes to be deleted or inactivated may be 1 or more, and may be 3 or more, or 5 or more, preferably 2 or 3, and particularly preferably 2.
- deletion or inactivation of a group of genes other than those described above can be combined, and a greater effect on productivity improvement is expected.
- Gene groups can be deleted or inactivated by known methods, for example, by sequentially deleting or inactivating target genes or by random deletion or inactivating mutations, followed by an appropriate method.
- a method of deleting or inactivating a gene group by performing protein productivity evaluation and gene analysis can be used.
- a method by homologous recombination may be used. That is, after cloning the DNA fragment containing the target gene into an appropriate plasmid vector, the entire region or a partial region of the gene is deleted using conventional genetic engineering techniques while leaving the DNA fragments on both sides. After making modifications such as giving a nonsense mutation to the structural gene by base substitution or frame shift, or inserting another DNA fragment into the target gene fragment isolated by cloning or PCR, the modified gene is The target gene on the genome is deleted or deleted by incorporation of the DNA fragment containing it into the parent microorganism and causing homologous recombination with the parent microorganism genome in both regions outside the target gene. Can be replaced with an inactivated gene fragment.
- Bacillus subtilis is used as a parent microorganism for constructing the microorganism of the present invention
- methods for deleting or inactivating a target gene by homologous recombination Mol. Gen. Genet., 223, 268 (1990)
- the host microorganism of the present invention can be obtained by repeating such a method.
- a method of causing homologous recombination in the same manner as described above using a randomly cloned DNA fragment, or irradiating a parent microorganism with a ray or the like may be used. It can also be carried out by such means.
- Encode the protein or polypeptide of interest in a microorganism obtained by deleting or inactivating one or more genes selected from the group of genes involved in spore formation in the middle to late stages of sporulation thus obtained.
- a microorganism host microorganism obtained by deleting or inactivating one or more genes selected from the group of genes involved in spore formation in the middle to late stages of sporulation thus obtained.
- the recombinant microorganism of the present invention can be obtained.
- the target protein or polypeptide gene is not particularly limited, and includes various industrial enzymes such as detergents, foods, fibers, feeds, chemicals, medical care, diagnostics, and bioactive peptides.
- industrial enzymes such as detergents, foods, fibers, feeds, chemicals, medical care, diagnostics, and bioactive peptides.
- the functions of industrial enzymes are classified into oxidoreductases (Oxidoreductase), transferases (Transferases), hydrolases (Hydrolases), lyases (Lyases), isomerases (Isomerase), and synthases (Ligase / Synthetase) and the like, and preferably include genes for hydrolases such as cellulase, ⁇ -amylase, and protease.
- cellulases belonging to family 15 in the classification of polysaccharide hydrolases there are cellulases belonging to family 15 in the classification of polysaccharide hydrolases (Biochem. J., 280, 309 (1991)).
- cellulases derived from microorganisms, particularly from Bacillus bacteria are mentioned. No.
- Bacillus bacterium-derived al force recellulase ⁇ ⁇ ⁇ having the sequence shown in SEQ ID NO: 1 or 2, 70%, preferably 80%, more preferably the sequence shown in SEQ ID NO: 1 or 2 Is a cellulase having a sequence having 90% or more homology.
- the amino acid sequence homology is calculated by the LipmaiHPearsoii method (Science, 227, 1435, (1985)).
- a-amylase examples include a microorganism-derived human amylase, and a liquefied amylase derived from a bacterium of the genus Bacillus is particularly preferred.
- proteases include serine proteases derived from microorganisms, particularly serine proteases derived from Bacillus bacteria, and metal proteases.
- the target protein or polypeptide gene contains, upstream thereof, a regulatory region involved in transcription, translation and secretion of the gene, that is, a promoter and a transcription initiation site. It is desirable that a transcription initiation control region, a translation initiation region including a ribosome binding site and an initiation codon, or a secretory signal peptide region be bound in an appropriate form.
- a cellulase gene derived from a bacterium belonging to the genus Bacillus described in JP-A-2000-210081 and JP-A-4-190793, etc., and 1 kb or less, preferably 0.
- the above control region derived from a region within 6 kb, more specifically, the sequence shown in SEQ ID NO: 1 or 2, or a base sequence having a certain degree of homology with these and having a similar control function, etc. Is desirable.
- the recombinant microorganism of the present invention is obtained by incorporating a recombinant plasmid, in which the above-mentioned DNA fragment containing the target protein or polypeptide gene is ligated with an appropriate plasmid vector, into host microorganism cells by a general transformation method. Obtainable.
- the recombinant microorganism of the present invention can also be obtained by using a DNA fragment obtained by linking the DNA fragment with an appropriate homologous region to the genome of the host microorganism and directly integrating the DNA fragment into the genome of the host microorganism.
- Production of the target protein or polypeptide using the recombinant microorganism of the present invention is performed by inoculating the strain into a medium containing an assimilable carbon source, a nitrogen source, and other essential components, and culturing by a usual microorganism culture method. After the cultivation, the protein or polypeptide may be collected and purified.
- a host microorganism in which the gene involved in sporulation has been deleted or inactivated, and a recombinant microorganism can be constructed using the host microorganism, and a useful protein can be constructed by using this.
- the polypeptide can be efficiently produced.
- (3 ⁇ 4-amylase or cellulase is produced using Bacillus subtilis) will be specifically described.
- genomic gene extracted from the Bacillus subtilis strain used as the host splicing by overlap extention One PCR method (Gene, 77, 61, (1989)) was used to insert a DNA fragment upstream of the start codon of the sigF gene and a DNA fragment downstream of the stop codon between chloramphene. Prepare a DNA fragment ligated by inserting a marker gene such as the Nicol resistance gene.
- a host Bacillus subtilis strain is transformed with the obtained DNA fragment by the competent method, and a transformant is isolated using chloramphenicol resistance or the like as an index, whereby the upstream and downstream of the sigF gene are homologous. It is possible to obtain a transformant in which recombination has occurred and in which the sig F gene on the genome has been replaced with a major gene such as a clonal ramphenicol resistance gene.
- a plasmid containing a gene encoding monoamylase or cellulase is introduced into the obtained transformant and the original B. subtilis strain as a control, and the obtained recombinant is shaken under appropriate conditions, for example, in a nutrient medium.
- the ⁇ -amylase activity or cellulase activity of the culture supernatant is measured and compared with the productivity of the original host Bacillus subtilis strain, whereby the desired product is deleted by deleting the sigF gene. Production can be confirmed. Further, ⁇ -amylase or cellulase can be obtained by collecting and purifying from this culture solution.
- (C) was ligated by SOE-PCR in the order of (A), (B) and (C) to obtain a 3.9 kb DNA fragment.
- Bacillus subtilis 168 strain was transformed by the combitent method, and LB agar containing chloramphenicol was used. Colonies grown on the medium were isolated as transformants. In the transformants obtained as a result, the region containing the sigF gene (2442632-2443318) on the genome was deleted, and it was confirmed by PCR and sequencing that the chloramphenicol gene was substituted.
- the region (2652156-2652723) or the region containing the region from the spoIVC B gene to the spoIIIC gene (2652262—2701023) (2652156-2701031) is deleted, and the spore-forming gene replaced with the chloramphenicol resistance gene is deleted. Each strain was obtained.
- each of the gene-deleted strains obtained in Example 1 and 168 strains of Bacillus subtilis as controls, an alkaline cellulase gene derived from Bacillus s. KSM-S237 strain Japanese Patent Laid-Open No. 2000-210081) 3.1 kb
- the strain obtained in this manner was shake-cultured overnight in 1 OmL of LB medium 3, and 0.05 mL of this culture solution was further added to 5 OmL of 2 XL-mal !!
- waste of the culture medium such as energy loss, production of by-products, and a decrease in the specific production rate can be significantly reduced when producing the target protein or polypeptide.
- the target product can be efficiently produced by prolonging the production period of the protein or polypeptide.
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- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP02728174A EP1391502B1 (en) | 2001-05-29 | 2002-05-28 | Host microorganisms |
US10/479,214 US7585674B2 (en) | 2001-05-29 | 2002-05-28 | Host microorganisms |
DK02728174.0T DK1391502T3 (da) | 2001-05-29 | 2002-05-28 | Værtsmikroorganismer |
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WO2002097064A1 true WO2002097064A1 (fr) | 2002-12-05 |
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US (1) | US7585674B2 (ja) |
EP (1) | EP1391502B1 (ja) |
CN (1) | CN100439492C (ja) |
DK (1) | DK1391502T3 (ja) |
WO (1) | WO2002097064A1 (ja) |
Cited By (1)
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WO2005045045A2 (en) * | 2003-11-07 | 2005-05-19 | Kao Corporation | Recombinant microorganism |
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WO2006007530A2 (en) * | 2004-07-01 | 2006-01-19 | Rice University | Blocking sporulation by inhibiting spoiie |
JP4485341B2 (ja) * | 2004-12-20 | 2010-06-23 | 花王株式会社 | 組換え微生物 |
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- 2002-05-28 CN CNB028111184A patent/CN100439492C/zh not_active Expired - Fee Related
- 2002-05-28 EP EP02728174A patent/EP1391502B1/en not_active Expired - Lifetime
- 2002-05-28 DK DK02728174.0T patent/DK1391502T3/da active
- 2002-05-28 WO PCT/JP2002/005151 patent/WO2002097064A1/ja active Application Filing
- 2002-05-28 US US10/479,214 patent/US7585674B2/en not_active Expired - Fee Related
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005045045A2 (en) * | 2003-11-07 | 2005-05-19 | Kao Corporation | Recombinant microorganism |
WO2005045045A3 (en) * | 2003-11-07 | 2006-01-12 | Kao Corp | Recombinant microorganism |
US7563611B2 (en) | 2003-11-07 | 2009-07-21 | Kao Corporation | Recombinant microorganism |
Also Published As
Publication number | Publication date |
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US20040248279A1 (en) | 2004-12-09 |
CN1513057A (zh) | 2004-07-14 |
DK1391502T3 (da) | 2012-05-21 |
EP1391502A1 (en) | 2004-02-25 |
US7585674B2 (en) | 2009-09-08 |
EP1391502A4 (en) | 2005-01-26 |
CN100439492C (zh) | 2008-12-03 |
EP1391502B1 (en) | 2012-04-25 |
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