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WO1989004866A1 - Cellules de bacille - Google Patents

Cellules de bacille Download PDF

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Publication number
WO1989004866A1
WO1989004866A1 PCT/GB1988/001026 GB8801026W WO8904866A1 WO 1989004866 A1 WO1989004866 A1 WO 1989004866A1 GB 8801026 W GB8801026 W GB 8801026W WO 8904866 A1 WO8904866 A1 WO 8904866A1
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Prior art keywords
npr
apr
strains
protease
strain
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Ceased
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PCT/GB1988/001026
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English (en)
Inventor
Andrew Mountain
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Celltech R&D Ltd
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Celltech Ltd
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Publication date
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Priority to FI893548A priority Critical patent/FI893548L/fi
Publication of WO1989004866A1 publication Critical patent/WO1989004866A1/fr
Priority to DK367689A priority patent/DK367689A/da
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • 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
    • 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
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • This invention relates to cells and in particular to cells of the Bacillus genus.
  • the Bacillus cells of the present invention may be ' transformed with heterologous genetic material and used for industrial fermentations and production of heterologous polypeptides,
  • B.subtilis as a production host for recombinant products include its large secretory capacity, its regulatory acceptability, and its well developed fermentation and product recovery technologies. Like many Bacilli, B.subtilis can efficiently secrete large quantities of proteins directly into the growth medium. Such secretion has cost advantages in product recovery, since the protein is produced in a relatively pure form compared to an intracellular product.
  • B.subtilis as a production host for secreted recombinant products is the synthesis and secretion by this organism of a variety of proteases which degrade secreted heterologous proteins. About 90% of secreted protease activity is attributable to two major proteases, a neutral metalloprotease encoded by a gene "npr” and an alkaline serine protease (subtilisin) encoded by a gene "apr".
  • the present invention provides a Bacillus species of cells with a significantly decreased level of proteases.
  • Bacillus cells exhibiting, at least, the phenotype Apr , Npr , SpoOH .
  • the invention provides Bacillus cells exhibiting at least the phenotype Apr Npr and SpoOH .
  • the cells of the present invention include those carrying disabled apr, npr, and spoOH genes.
  • the genes may be disabled by mutation and the mutations may be of any type capable of deactivating the gene such that functional protein is not produced and may each be naturally occurring or produced by the methods of genetic engineering.
  • One or more of the genes may be deleted using techniques well known in the art.
  • Bacillus cells of the present invention exhibit significantly reduced levels of extracellular proteases and are therefore of considerable utility as host cells for the expression of recombinant heterologous polypeptides.
  • the cells of the invention exhibit a lower level of extracellular proteases than the Bacillus strains known in the art having the phenotype Apr , Npr , SpoOA .
  • the cells of the present invention may be prepared using the techniques described hereinafter e.g. by congression and using publicly available cell lines as starting materials.
  • a non-selectable marker such as spoOH
  • a selectable marker is co-transferred with a selectable marker into a recipient cell by transformation of competent cells of the recipient at high concentration of DNA prepared from the donor strain.
  • a high proportion of the transformants selected using the selectable marker e.g. removal of a nutritional requirement
  • the cells are preferably of the species Bacillus subtilis.
  • the cells are most preferably Bacillus subtilis strain CIMB7, CIMB14 and CIMB15.
  • the invention also provides B.subtilis CIMB7, CIMB14 and CIMB15.
  • Bacillus strains of the invention are the same as or similar to the strain CIMB7, CIMB14 or CIMB15 as hereinafter described.
  • the cells of the present invention may in addition lack one or more of the other naturally occurring extracellular proteases as a result of introducing other mutations e.g. spoOA.
  • the cells may lack one or more of the naturally occurring intracellular protease genes, such as the gene encoding intracellular serine protease (isp) .
  • the cells may be transformed with a vector capable of expressing a gene coding for a heterologous polypeptide using techniques known in the art.
  • the invention provides Bacillus cells exhibiting at least the phenotype Apr Npr SpoOH transformed with a vector capable of expressing a gene coding for a heterologous polypeptide.
  • the vector may be any vector which replicates within the host cell, such as for example a vector containing a Staphyloeoceus aureus replicon, a Bacillus replicon e.g. pBCl ⁇ and derivatives; a Lactobacillus replicon, or any shuttle vectors derived therefrom, and is especially a Bacillus vector as described in International Patent Application No. WO/8806622.
  • the cells of the invention may especially be useful as host cells for the expression of heterologous genes coding for, for example, any prokaryotic polypeptide such as for example a bacterial polypeptide such as an enzyme, e.g. ⁇ -amylase, ⁇ -amylase or ⁇ -galactosidase or any eukaryotic polypeptide such as for exmaple a mammalian polypeptide such as an enzyme e.g. chymosin or gastric lipase; an enzyme inhibitor e.g. tissue inhibitor of metalloproteinase (TIMP) ; a hormone e.g. growth hormone; a lymphokine e.g. an interferon; a plasminogen activator, e.g. tissue plasminogen activator (tPA) or prourokinase; or natural, modified or chimeric immunoglobulins or fragments thereof having dual acitivity such as antibody-enzyme or antibody-toxin chimeras.
  • the invention provides a process for the production of a polypeptide comprising culturing a Bacillus cell according to the second aspect of the invention and optionally recovering the polypeptide therefrom.
  • the invention provides a polypeptide produced by a process according to the third aspect of the invention.
  • Bacillus cells of the invention have very low secreted protease ' 5 . activities and are therefore of use as hosts for the expression of recombinant proteins.
  • Figure 1 is a photograph of a Coomassie stained gel of SPA incubated with supernatants of cultures of EMG50, CIMB12, CIMB13 and CIMB15 5 3 hrs and 15 hrs into stationary phase.
  • Figure 2 shows a graph of the results of a ⁇ -lactamase assay of samples of the supernatants of EMG50, CIMB12, CIMB13 and CIMB15, removed at time points throughout growth of the cultures.
  • the B.subtilis strains used are listed in Table 1. Representative strains carrying the npr, spoOH and spoOA mutations can be freely obtained from the Bacillus Genetic Stock Centre at the Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA. 0 Strains which carry the apr mutation may be prepared by methods known in the art, as described for example in Kawamura and Doi, 1984 (J. Bacteriol 160:424-444). Strains were routinely stored in L broth (see below) containing 10% glycerol at -20 C. Table 1 - strains used
  • L broth L broth or L broth solidified with 1.5% agar was used as the standard medium for cultivation of the strains. It contained per litre: lOgms tryptone: 5gms yeast extract; 5gms NaCl. Its pH was adjusted to 7 using concentrated NaOH.
  • M9 minimal salts was used as the standard medium.
  • M9 contained per litre: 12gms Na HPO ; 6gms KH PO ; Igm NaCl; 2gms NH Cl. Its pH was adjusted to 7.4. It was made up at twice final concentration, autoclaved, and mixed 1:1 (vol:vol) with 3% agar for use. After sterilisation separately sterilised glucose was added to a final concentration of 1% as carbon source, and amino acids to satisfy a final concentration of 1% as carbon source, and amino acids to satisfy auxotrophic requirements to a final concentration of 50ug/ml except where otherwise stated.
  • LGA LGA medium was used to distinguish sporulation-proficient
  • GM1 and GM2 were the media used in the nutritional step down procedure employed for preparing competent cells for B.subtilis transformations (see below). They were based on a minimal salts medium containing per litre: 14gms K HPO ; 6gms KH PO ; Igm tri-sodium citrate;2gms (NH ) SO . GM1 was minimal salts with the following added after sterilisation: 0.25% glucose; 5mM MgSO ; 0.02% Difco casamino acids; 0.1% yeast extract; 50ug/ml L-tryptophan; 200ug/ml of any other amino acid supplements to satisfy auxotrophic requirements of the strains.
  • GM2 was minimal salts with the following added after sterilisation: 0.25% glucose; lOmM MgSO ; 0.01% Difco casamino acids; 0.5mM CaCl ; 35ug/ml other amino acids needed to satisfy auxotrophic requirements.
  • the donor strain was first grown overnight in 200mls L-broth in a 21 flask with shaking at 37 C. The cells were removed by centrifugation, washed in 80mls TES (comprising per litre 6gms T IS, 1.9gms EDTA, 2.9gms NaCl, pH8.0), then resuspended in 7mls TESS (TES containing 25% sucrose) . 20mgs of lysozyme were added and the cell suspension was mixed well, then incubated at 37 C for 20 mins. After the addition of 1.5ml 0.5M EDTA and 2mls 12% SDS the mixture was incubated at 65 C for 7 mins.
  • TES comprising per litre 6gms T IS, 1.9gms EDTA, 2.9gms NaCl, pH8.0
  • Competent cells were prepared by a modification of the method of Dooley et al. 1971, J. Bacteriol 108:668. A large single colony of the strain to be transformed was inoculated into 25mls L-broth and o grown overnight with shaking at 30 C. The cells from lOmls were collected by centrifugation and inoculated into 25mls in a 250ml flask to an optical density at 600nm (o.D600) of 0.4. The culture was incubated with shaking at 37 C and its growth monitored by measuring 0D600 of samples taken at hourly intervals.
  • Protease activities in culture supernatants were assayed by a modification of the procedure described by Prestidge et al, 1971, J. Bacteriol 107:815-823.
  • Cultures were grown in 25mls twice normal concentration L-broth in 250ml flasks with shaking at 37 C, and growth of the cultures monitored by measuring 0.D600. In this medium the cultures reached stationary phase at 0.D6O0 of about 4.5. 1.5ml samples were taken at various time points from late in ° the growth phase until IShrs into stationary phase. These samples were spun for 10 mins at 12000rpm in a bench microcentrifuge, the cells discarded and the supernatant respun for a further 10 mins to competely remove the cells.
  • Protease activities in these supernatants were determined by the following azocasein assay, 5 150ul of supernatant was mixed with 50ul 1M Tris/HCl pH8, 50ul H O and 250ul 2% azocasein (Sigma Chemical Company). This mixture was incubated at 30 C for 60 mins and the reaction stopped by the addition of cold 7% perchloric acid. The mixture was then centrifuged for 30 mins at 12000rpm in a bench microcentrifuge. 0 lOOOul of the supernatant was then removed and thoroughly mixed with
  • Protease levels of strains were crudely assessed by comparing the sizes of clearing zones after 2-5 days at 37 C on L-agar plates 0 containing 1% Difco skim milk (SMLA plates).
  • Azocasein assays on culture supernatants were then performed (as described in Materials and Methods) for the strains identified by either test as showing reduced protease production, namely 0H46 (SpoOH ), 1S53 (SpoOA-), CIMB5 (Apr- Npr-) , and or a control strain 1A28 .
  • the results are given in Table 2, expressed as %age of activity displayed by the control strain 1A289.
  • the culture supernatants used in these assays were prepared from cultures two hours after their entry into stationary phase. At this time the strain carrying a mutation in the apr npr genes showed only 25% of the total protease activity found for 1A289.
  • the SpoOH strain showed a significant reduction in protease activity, (68% of that found for 1A289). Unexpectedly the strain carrying a mutation in the spoOA gene showed a somewhat higher protease level than 1A289 (107%) . In similar experiments Fahnestock and Fisher, 1987, Appl. Env. Microbiol 53:379-384, found reduced levels of protease activity were produced by the same SpoOA- strain (1S53) compared to a control strain. These results and other (see below) suggest that strain 1A289 itself is a low protease producer compared to most commonly used B.subtilis strains.
  • CIMB5 Competent cells of CIMB5 were prepared as described in Materials and Methods and transformed (separately) with chromosomal DNA of strains 0H46 and 1S53, at a DNA concentration of lOug/ml, also as described in Materials and Methods.
  • the transformation mixtures were plated onto minimal salts agar supplemented with lysine and tryptophan to select for Phe transformants. 100 such transformants were restreaked onto LGA to test for simultaneous acquisition of the spoOA or spoOH mutations. +
  • Representative Phe Spo strains were called CIMB7 in the case of the Apr- Npr- SpoOH- triple mutant and CIMB11 in the case of the Apr- Npr- SpoOA- triple mutant.
  • CIMB7 When streaked on SMLA plates neither CIMB7 nor CIMB11 produced a clearing zone, indicating they had both retained the apr and npr mutations.
  • CIMB5 In addition to carrying the mutations affecting levels of secreted proteases CIMB5, CIMB7 and CIMB11 all carry an insertion mutation affecting the major intracellular serine protease, encoded by the gene isp.
  • the fragment inserted in and inactivating isp carries the chloramphenicol resistance gene of plas id pC194.
  • the Cm gene is also carried on the plasmid pPOD2400, a plasmid expressing Eschericia coli B-lactamase in B.subtilis which was later to be used to compare degradation of a secreted heterologous protein in these r low protease strains (see below).
  • the presence of the same Cm gene in the chromosome of these strains would be likely to lead to integration of the plasmids following transformation. Derivatives
  • Competent cells of CIMB5 were tranformed with chromosomal DNA of strain 1S53 with selection on minimal salts agar supplemented with phenylalanine and tryptophan to select for Lys transformants. 100 such transformants were streaked onto L-agar containing chloramphenicol to identify those which had been simultaneously transformed to chloramphenicol sensitivity.
  • One such strain was shown to have retained the apr npr mutations by SMLA plate tests and was designated CIMB12.
  • Example 4 Degradation of Staphylococcal protein A by Apr Npr ⁇ , Apr Npr SpoOA and Apr Npr SpoOH strains.
  • SPA degradation tests were performed using culture supernatants of strains EMG50, CIMB12 (Apr “ Npr “ ) s CIMB13 (Apr- Npr " SpoOA-) and CIMB15 (a prototrophic derivative of the Apr- Npr " SpoOH- strain CIMB14).
  • Supernatants of cultures 3 hrs and 15 hrs into stationary phase were used.
  • Figure 1 is a photograph of the Coomassie stained SDS/acrylamide gel resulting from this experiment.
  • the samples electrophoresed were as follows: Lane 1, molecular weight markers; Lane 2, untreated SPA; Lane 3, SPA incubated with the 3 hr EMG50 supernatant; Lane 4 SPA incubated with the 3 hr CIMB12 supernatant; Lane 5, SPA incubated with the 3 hr CIMB15 supernatant; Lane 6, SPA incubated with the 3 hr CIMB13 supernatant; Lane 7, SPA incubated with the 15 hr EMG50 supernatant; Lane 8, SPA incubated with the 15 hr CIMB12 supernatant; Lane 9, SPA incubated with the 15 hr CIMB15 supernatant; Lane 10, SPA incubated with the 15 hr CIMB13 supernatant.
  • Example 5 Production and degradation of ⁇ -lactamase by EMG50. CIMB12. CIMB13 and CIMB15.
  • plasmid pPOD2400 was transformed into strains CIMB12, CIMB13, CIMB15 and EMG50.
  • pPOD2400 is derived from plasmid pCPP4 of Band et al, 1983, Gene 26:313-315, and carries the replication functions and kanamycin resistance of pUBHO, together with a hybrid gene comprising the mature coding region of the E.coli TEM beta-lactamase fused in frame with the signal sequence of a Bacillus alpha-amylase, such that the beta-lactamase is expressed in B.subtilis using the alpha-amylase transcription and translation initiation signals.
  • ⁇ POD2400 is thus very similar to plasmid pKTH78 described by Ulmanen et al, 1985, J.
  • Bacteriol 162:176-182 who found that the beta-lactamase is efficiently secreted from B.subtilis using the alpha-amylase signal sequence but is rapidly degraded at the end of the growth phase by the secreted proteases of B.subtilis even in low protease strains.
  • pPOD2400 was transformed into strains CIMB12, CIMB13, CIMB15 and EMG50 using the protoplast transformation procedure of Chang and Cohen, 1979, Molec. Gen. Genet 168:111-115, with selection on DM3 medium containing (in additon to the ingredients described by Chang and Cohen) 0.9% soluble starch, 0.4% gelatin, 0.04% bovine serum albumin and 300ug/ml kanamycin.
  • Plasmid minipreparations were performed on representative kanamycin resistant transformants as described in Maniatis et al, 1982, and agarose gel electrophoresis was used to confirm the integrity of the plasmid in these transformants.
  • One representative transformant of each strain carrying ⁇ POD2400 was then grown in a 25ml culture in a 200ml container in L-broth plus 3% glucose and lOug/ml kanamycin (to ensure retention of the plasmid), incubated with shaking at 37 C.
  • CIMB12 and CIMB13 carrying this plasmid produced significantly greater activities than EMG50, with activities being slightly greater for CIMB13 than CIMB12 up to the 9 hr time point.
  • the presence of the spoOA mutation therefore conferred only a slight reduction in beta-lactamase degradation on the Apr Npr strain.
  • the presence of the spoOH mutation led to a dramatic decrease in beta-lactamase degradation : beta-lactamase activities were found to be dramatically higher in supernatants of CIMB15 than in those of CIMB12 or CIMB13 at all time points tested.
  • the beta-lactamase activity in the CIMB15 supernatant was approximately fourfold higher than in those of any of the other strains.
  • dilutions of the cultures were plated out on both L-agar and L-agar containing lOug/ml kanamycin to determine the % of plasmid containing cells in each culture. The results indicated that the differences in beta-lactamase activities between CIM15 and the other strains cannot be explained by differences in plasmid stability.
  • Strains of B.subtilis with very low secreted protease activities have been constructed by introducing a spoOH mutation into an Apr- Npr double mutant, i.e. defective for both major secreted proteases.
  • the new triple mutant strains carrying mutations in the genes apr npr spoOH have been shown to have very low secreted protease activities in three separate tests, namely azocasein hydrolysis, degradation of Staphylococcal protein A, and degradation of E.coli TEM beta-lactamase.

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  • Health & Medical Sciences (AREA)
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Abstract

Est décrite une triple souche mutante de Bacillus subtilis. La cellule mutante comporte au moins le phénotype Apr-, Npr- SpoOH- et présente un niveau réduit de protéases extracellulaires. La réduction du niveau des protéases cellulaires secrétées par la cellule de bacille en fait un hôte utile pour l'expression de polypeptides hétérologues.
PCT/GB1988/001026 1987-11-27 1988-11-25 Cellules de bacille Ceased WO1989004866A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
FI893548A FI893548L (fi) 1987-11-27 1988-11-25 Bacillus-celler.
DK367689A DK367689A (da) 1987-11-27 1989-07-26 Bacillus celle, fremgangsmaade til fremstilling af et polypeptid ved dyrkning af denne, og det opnaaede polypeptid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8727772 1987-11-27
GB878727772A GB8727772D0 (en) 1987-11-27 1987-11-27 Host cells

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WO1989004866A1 true WO1989004866A1 (fr) 1989-06-01

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PCT/GB1988/001026 Ceased WO1989004866A1 (fr) 1987-11-27 1988-11-25 Cellules de bacille

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EP (1) EP0351427A1 (fr)
JP (1) JPH02502247A (fr)
AU (1) AU2727388A (fr)
FI (1) FI893548L (fr)
GB (1) GB8727772D0 (fr)
WO (1) WO1989004866A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997003185A1 (fr) * 1995-07-07 1997-01-30 Novo Nordisk A/S Production de proteines a l'aide de bacillus incapable de sporuler
WO1998012300A1 (fr) * 1996-09-19 1998-03-26 Novo Nordisk A/S Nouvelles cellules hotes et procedes de production de proteines
US6284490B1 (en) * 1990-12-21 2001-09-04 Eniricerche S.P.A. Asporogenous strain of bacillus subtilis and its use as a host for the preparation of heterologous products
US7981659B2 (en) 2005-10-13 2011-07-19 Kao Corporation Bacillus subtilis mutant strain

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986001825A1 (fr) * 1984-09-21 1986-03-27 Genex Corporation Souches de bacillus ayant des niveaux reduits de protease extracellulaire
FR2604726A1 (fr) * 1986-10-02 1988-04-08 Agency Ind Science Techn Souche de bacillus subtilis dont les activites de protease extra-cellulaire sont reduites, procede pour obtenir la souche et procede pour faire secreter des proteines en utilisant la souche
EP0130756B1 (fr) * 1983-06-24 1991-02-06 Genencor International, Inc. Carbonyl-hydrolases procaryotiques, méthodes, ADN, vecteurs et hôtes transformés pour leur production, et compositions des détergents contenant les dites hydrolases

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0130756B1 (fr) * 1983-06-24 1991-02-06 Genencor International, Inc. Carbonyl-hydrolases procaryotiques, méthodes, ADN, vecteurs et hôtes transformés pour leur production, et compositions des détergents contenant les dites hydrolases
WO1986001825A1 (fr) * 1984-09-21 1986-03-27 Genex Corporation Souches de bacillus ayant des niveaux reduits de protease extracellulaire
FR2604726A1 (fr) * 1986-10-02 1988-04-08 Agency Ind Science Techn Souche de bacillus subtilis dont les activites de protease extra-cellulaire sont reduites, procede pour obtenir la souche et procede pour faire secreter des proteines en utilisant la souche

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biochimie, vol. 55, no. 8, 1973 B.J. Dod et al.: "The kinetics of extracellular protease production in an abnormal sporulation mutant of B. subtilis", pages 1005-1006 *
Journal of Bacteriology, vol. 160, no. 1, October 1984. American Society for Microbiology (US) F. Kawamura et al.: "Construction of a Bacillus subtilis double mutant deficient in extracellular alkaline and neutral proteases", pages 442-444, *
Spores, vol. 6, 1975. J. Szulmajster et al.: "Isolation and properties of thermosensitive sporulation mutants of Bacillus subtilis deficient in intracellular protease activity", pages 271-278 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6284490B1 (en) * 1990-12-21 2001-09-04 Eniricerche S.P.A. Asporogenous strain of bacillus subtilis and its use as a host for the preparation of heterologous products
WO1997003185A1 (fr) * 1995-07-07 1997-01-30 Novo Nordisk A/S Production de proteines a l'aide de bacillus incapable de sporuler
WO1998012300A1 (fr) * 1996-09-19 1998-03-26 Novo Nordisk A/S Nouvelles cellules hotes et procedes de production de proteines
US6352841B1 (en) 1996-09-19 2002-03-05 Novozymes A/S Host cells and methods of producing proteins
US7981659B2 (en) 2005-10-13 2011-07-19 Kao Corporation Bacillus subtilis mutant strain

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EP0351427A1 (fr) 1990-01-24
JPH02502247A (ja) 1990-07-26
FI893548A0 (fi) 1989-07-24
FI893548A7 (fi) 1989-07-24
FI893548L (fi) 1989-07-24
GB8727772D0 (en) 1987-12-31
AU2727388A (en) 1989-06-14

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