WO2012175574A1 - Production accrue de pantothénate (vitamine b5) - Google Patents

Production accrue de pantothénate (vitamine b5) Download PDF

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Publication number
WO2012175574A1
WO2012175574A1 PCT/EP2012/061889 EP2012061889W WO2012175574A1 WO 2012175574 A1 WO2012175574 A1 WO 2012175574A1 EP 2012061889 W EP2012061889 W EP 2012061889W WO 2012175574 A1 WO2012175574 A1 WO 2012175574A1
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WO
WIPO (PCT)
Prior art keywords
production
pantothenate
medium
glucose
process according
Prior art date
Application number
PCT/EP2012/061889
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English (en)
Inventor
Ines GRUNER
Zoltan Pragai
Original Assignee
Dsm Ip Assets B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Priority to EP12730202.4A priority Critical patent/EP2723877A1/fr
Publication of WO2012175574A1 publication Critical patent/WO2012175574A1/fr

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Classifications

    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids

Definitions

  • the present invention relates to an improved production process for pantothenic acid (vitamin B5) using a Bacillus strain in a new medium containing a low amount of sugar such as, e.g. , glucose and a higher amount of slowly utilizable carbon source such as, e.g., raffinose.
  • This medium is particularly useful for high throughput (HTP) screening of Bacillus mutants.
  • Pantothenate is a member of the B complex of vitamins and is a nutritional requirement for mammals including humans, e.g. , from food sources, as a water-soluble vitamin supplement or as a feed additive. In cells, pantothenate is used primarily for the biosynthesis of coenzyme A and acyl carrier protein.
  • a method of producing pantothenate or pantoate by culturing genetically modified microorganisms of the genus Bacillus, particularly B. subtilis, in which at least one enzyme selected from the group consisting of PanB (ketopantoate hydroxymethyl transferase), PanC (pantothenate synthetase), PanD (aspartate- a-decarboxylase) and PanE (ketopantoate reductase) is overexpressed is known (WO 01 /21772).
  • the new medium composition is in particular useful for screening of pantothenate high producing strains, preferably the use of a medium with a concentration of glucose of 10 g/l or less for high throughput screening of pantothenate producing strains of Bacillus, more preferably Bacillus subtilis.
  • the increased production of pantothenate is proportional to the engineering level of the production strain.
  • raffinose mimetic the feed phase of the fermentation cultivation
  • a ratio range of 1 :2 to 1 :20 such as 1 :4, 1 :5, 1 :6, 1 :8, 1 :9, 1 : 10, 1 : 12, 1 : 15, preferably in a ratio of 1 :9, such as e.g. 1 g/l glucose and 9 g/l raffinose as starting concentration.
  • the glucose concentration varies preferably from 0 to 4 g/l, while the raffinose concentration may be in a range of 5-25 g/l, as long as the amounts fit with the ratios mentioned above.
  • pantothenate wherein a strain of Bad 11 us is cultivated in a medium comprising a sugar selected from fructose, ribose, sucrose or glucose, preferably glucose, mixed with raffinose and wherein the starting concentration of the sugar selected from fructose, ribose, sucrose or glucose is 10 g/l or less.
  • the pantothenate is recovered from the medium.
  • the ratio of sugar and raffinose is in the range of 1 :2 to 1 :20 or in any range described above, wherein the sugar is preferably glucose, but might also be selected from fructose, ribose or sucrose.
  • pantothenic acid includes but is not limited to pantothenic acid, precursors and/or derivatives thereof such as salts or esters thereof, i. e. pantothenate, in particular calcium pantothenate, or the alcohol form of pantothenic acid, i. e. pantothenol or panthenol.
  • pantothenic acid includes but is not limited to pantothenic acid, precursors and/or derivatives thereof such as salts or esters thereof, i. e. pantothenate, in particular calcium pantothenate, or the alcohol form of pantothenic acid, i. e. pantothenol or panthenol.
  • pantothenic acid pantothenate
  • vitamin B5 are used interchangeably herein.
  • Precursors/intermediates in the biosynthetic pathway of pantothenic acid which are included may be selected from e.g. pantoate, a-ketopantoate or a- ketoisovalerate.
  • the pantothenate production strain may be selected from Bacillus species represented by the Bacillus sensu stricto group, in particular Bacillus subtil is, Bacillus lent i morbus, Bacillus lent us, Bacillus ant hracis, Bacillus firmus, Bacillus pant othenticus, Bacillus cereus, Bacillus circu I ans, Bacillus coagu I ans, Bacillus megateri urn, Bacillus thuringiensis, Bacillus licheniformis, Bacillus amy I oliquef act ens, Bacillus pumi I us, Bacillus halodur ans (Zeigler and Perkins, 2008, Practical Handbook of Microbiology", Second Edition (E.
  • microorganism is selected from Bacillus subtil is. All microorganisms which can be used for the present invention may be publicly available from different sources, e.g. , Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenstr. 7B, D-38124 Braunschweig, Germany, American Type Culture Collection (ATCC), P.O.
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • ATCC American Type Culture Collection
  • microorganisms also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes.
  • the nomenclature of the microorganisms as used herein is the one officially accepted (at the filing date of the priority application) by the International Committee on Systematics of Prokaryotes and the Bacteriology and Applied Microbiology Division of the International Union of Microbiological Societies, and published by its official publication vehicle International Journal of Systematic and Evolutionary Microbiology (IJSEM).
  • the production strain e.g. a strain of Bacillus, preferably Bacillus subtil is, used for performing the present invention may be a non-modified or wild-type microorganism or may be a genetically modified/mutated or recombinant one, wherein at least one of the known pantothenate biosynthetic genes has been overexpressed.
  • a recombinant Bacillus strain is used. Modifications either on the DNA or protein level include all modification/mutations with a direct impact on the yield, production and/or efficiency of pantothenate production.
  • the person skilled in the art knows how to manipulate a Bacillus strain for said purpose. General techniques are described in Harwood, C.R. and Cutting, S.M. (1990). Molecular Bological Met hods for Bacillus. Chichester: John
  • mutants J j., Fritsch, E.F. and Maniatis, T. (1989). Molecular Qoning: a Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  • mutants may be obtained, e.g., by site- directed mutagenesis, saturation mutagenesis, random mutagenesis/directed evolution, chemical or UV mutagenesis of entire cells/organisms, and other methods which are known in the art.
  • mutants may also be generated, e.g., by designing synthetic genes, and/or produced by in vitro (cell-free) translation.
  • these mutants may be (over-) expressed by methods known to those skilled in the art with measurement of the pantothenate production. This and further methods leading to increased pantothenate production by, e.g., the replacement of the natural promoter of the pan genes are described in e.g. WO 2010/018196. Modification also includes manipulation or deregulation of the branch chain amino acid biosynthesis in the pantothenate production strain.
  • the term "overexpressing” or “overexpression” means expression of a gene product at a level higher than that expressed prior to modification of the microorganism or in a comparable microorganism which has not been modified.
  • the microorganism of the invention overexpresses one or more genes selected from the group consisting of panB, panQ panD, panE, ilvB, ilvC, ilvD, ilvN, glyA, serA, serC, and the gcv genes involved in the glycine cleavage pathway; as well as mutants thereof that result in the synthesis of encoded enzymes of improved catalytic properties.
  • the recombinant strain used for the purpose of the present invention carries at least two copies of panB and panD ⁇ panBD), more preferably 3 or 4 copies leading to increased pantothenate production.
  • the recombinant strain used for the purpose of the present invention is deregulated in the biosynthesis for branch chain amino acids such as e.g. leucine, valine, isoleucine.
  • Said modification is preferably combined with the overexpression of one or more genes selected from the group consisting of panB, panC, panD, panE, ilvB, ilvC, ilvD, ilvN, glyA, serA, serC, and the gcv genes involved in the glycine cleavage pathway.
  • further modification may also include deregulation of the branch chain amino acids biosynthesis (see reference strain 5 as further development of reference strain 4) and/or introduction of further copies of panBD, such as e.g. 2, 3 or 4 copies (see reference strain 6 as further development of reference strain 5).
  • deregulated means the alteration or modification of a gene in a microorganism such that the level or activity of the gene product in a microorganism is altered or modified.
  • at least one gene is altered or modified such that the gene product is enhanced/increased or
  • this deregulation is meant to be an overexpression of at least one of the genes.
  • the term "genetically engineered” or “genetically altered” means the scientific alteration of the structure of genetic material in a living organism. It involves the production and use of recombinant DNA. More in particular it is used to delineate the genetically engineered or modified organism from the naturally occurring organism. Genetic engineering may be done by a number of
  • microorganism is also often referred to as a recombinant organism, e.g.
  • the term "culturing a microorganism under suitable culturing conditions" refers to methods of maintaining and/or growing a living microorganism of the present invention which are well known in the art.
  • the microorganisms can be cultured in liquid, solid or semi-solid media.
  • the microorganism of the invention is cultured in liquid media comprising nutrients essential or beneficial to the maintenance and/or growth of the microorganism.
  • nutrients include, but are not limited to, carbon sources or carbon substrates, such as alcohols, sugars, sugar alcohols, complex carbohydrates such as starches, hydrocarbons, fatty acids, other organic acids, oils, fats; nitrogen sources, e.g.
  • the new medium as described herein is useful for screening purpose, e.g. for HTP screening of Bacillus strains, preferably Bacillus subtilis, which are in particular useful for high production of pantothenate.
  • HTP screening as well as production of pantothenate using the medium as of the present invention under conditions described herein can be performed according to any method known to the skilled person, including e.g. the use of shake-flask, deep-well assay or micro-fermentation such as e.g. use of a BioLector ® (m2p-l_abs, 52499 Baesweiler, Germany).
  • Useful protocols for shake-flask assay, micro-fermentation using the BioLector ® or deep-well assays are given in the Examples and are furthermore known by the skilled person.
  • the chloramphenicol-resistance gene (cat) cassette was obtained from plasmid pC194 (GenBank M19465, Cat# 1 E17 Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio 43210 USA).
  • the S aureus erythromycin resistance gene (GenBank V01278) was amplified from plasmid pDG646 (Guerout-Fleury et al. , 1995).
  • the S aureus spectinomycin resistance gene (X03216) was amplified from plasmid pDG1726 (Guerout-Fleury et al.
  • Deep well assay Single colonies grown on selective TBAB plates or frozen glycerol working cell banks were used to inoculate in 24-DW plate containing 3 ml of VY medium per well. The bacteria were grown overnight (about 17 h) at 39°C with an agitation of 550 rpm and humidity of 85 % (Infors HT microtron shaker). Next day a new 24-DW plate was prepared containing 3 ml of the different production media per well. For inoculation 30 ⁇ of overnight cultures were used. Plates were covered with a gas-permeable sealing film that supports the oxygen exchange. The cultures grew for 48 h in Infors HT microtron shaker as described above.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé perfectionné de production pour l'acide pantothénique (vitamine B5) à l'aide d'une souche de Bacillus dans un nouveau milieu contenant une faible quantité de sucre tel que, par exemple, du glucose, et une quantité supérieure d'une source carbonée lentement utilisable telle que, par exemple, le raffinose. Ce milieu est particulièrement utile pour un criblage à haut débit (HTP) de mutants de Bacillus.
PCT/EP2012/061889 2011-06-21 2012-06-20 Production accrue de pantothénate (vitamine b5) WO2012175574A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP12730202.4A EP2723877A1 (fr) 2011-06-21 2012-06-20 Production accrue de pantothénate (vitamine b5)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH10592011 2011-06-21
CH01059/11 2011-06-21

Publications (1)

Publication Number Publication Date
WO2012175574A1 true WO2012175574A1 (fr) 2012-12-27

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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5518906A (en) 1992-09-25 1996-05-21 Takeda Chemical Industries, Ltd. Production of d-pantoic acid and d-pantothenic acid
WO2001021772A2 (fr) 1999-09-21 2001-03-29 Basf Aktiengesellschaft Procedes et micro-organismes de production de composes panto
WO2002057474A2 (fr) 2001-01-19 2002-07-25 Basf Aktiengesellschaft Production amelioree de pantothenate
WO2002061108A2 (fr) 2001-01-19 2002-08-08 Basf Aktiengesellschaft Micro-organismes et procedes de production amelioree de pantothenate
WO2002066665A2 (fr) 2001-02-21 2002-08-29 Basf Aktiengesellschaft Procede pour la production d'acide d-pantothenique ou de ses sels comme additif a des aliments pour animaux
WO2004005527A1 (fr) 2002-07-03 2004-01-15 Basf Aktiengesellschaft Micro-organismes et procedes de production amelioree de pantothenate
WO2004111214A1 (fr) 2003-06-12 2004-12-23 Dsm Ip Assets B.V. Mevalonate kinases resistant a la retro-inhibition
WO2004113510A2 (fr) 2003-06-18 2004-12-29 Dsm Ip Assets B.V. Production de pantothenate au moyen de micro-organismes inaptes a la sporulation
WO2007065602A1 (fr) 2005-12-05 2007-06-14 Dsm Ip Assets B.V. PROCÉDÉ DE STABILISATION d'ARNm
WO2007131750A1 (fr) 2006-05-16 2007-11-22 Dsm Ip Assets B.V. Procédé de production du panthénol
WO2008148575A2 (fr) 2007-06-07 2008-12-11 Dsm Ip Assets B.V. Production accrue d'un produit cible par stabilisation d'arnm
WO2010018196A1 (fr) 2008-08-12 2010-02-18 Dsm Ip Assets B.V. Augmentation de la production de pantothénate (vitamine b5)

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5518906A (en) 1992-09-25 1996-05-21 Takeda Chemical Industries, Ltd. Production of d-pantoic acid and d-pantothenic acid
WO2001021772A2 (fr) 1999-09-21 2001-03-29 Basf Aktiengesellschaft Procedes et micro-organismes de production de composes panto
US7220561B2 (en) 2001-01-19 2007-05-22 Basf Aktiengesellschaft Processes for enhanced production of pantothenate
WO2002057474A2 (fr) 2001-01-19 2002-07-25 Basf Aktiengesellschaft Production amelioree de pantothenate
WO2002061108A2 (fr) 2001-01-19 2002-08-08 Basf Aktiengesellschaft Micro-organismes et procedes de production amelioree de pantothenate
WO2002066665A2 (fr) 2001-02-21 2002-08-29 Basf Aktiengesellschaft Procede pour la production d'acide d-pantothenique ou de ses sels comme additif a des aliments pour animaux
WO2004005527A1 (fr) 2002-07-03 2004-01-15 Basf Aktiengesellschaft Micro-organismes et procedes de production amelioree de pantothenate
WO2004111214A1 (fr) 2003-06-12 2004-12-23 Dsm Ip Assets B.V. Mevalonate kinases resistant a la retro-inhibition
WO2004113510A2 (fr) 2003-06-18 2004-12-29 Dsm Ip Assets B.V. Production de pantothenate au moyen de micro-organismes inaptes a la sporulation
WO2007065602A1 (fr) 2005-12-05 2007-06-14 Dsm Ip Assets B.V. PROCÉDÉ DE STABILISATION d'ARNm
WO2007131750A1 (fr) 2006-05-16 2007-11-22 Dsm Ip Assets B.V. Procédé de production du panthénol
WO2008148575A2 (fr) 2007-06-07 2008-12-11 Dsm Ip Assets B.V. Production accrue d'un produit cible par stabilisation d'arnm
WO2010018196A1 (fr) 2008-08-12 2010-02-18 Dsm Ip Assets B.V. Augmentation de la production de pantothénate (vitamine b5)

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
"Ullman's Encyclopedia of Industrial Chemistry", 2007
CHICHESTER: JOHN WILLEY; SAMBROOK, J.; FRITSCH, E.F.; MANIATIS, T.: "Molecular Cloning: a Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
HARWOOD, C.R.; CUTTING, S.M., MOLECULAR BIOLOGICAL METHODS FOR BACILLUS, 1990
HÜMBELIN ET AL., J. IND. MICROBIOL. BIOTECH., vol. 22, 1999, pages 1 - 7
LEE ET AL., MOL. GEN. GENET., vol. 180, 1980, pages 57 - 65
PERKINS ET AL., J. IND. MICROBIOL. BIOTECH., vol. 22, 1999, pages 8 - 18
ZEIGLER; PERKINS: "Practical Handbook of Microbiology", 2008, CRC PRESS, pages: 301 - 329

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