EP1242612A1 - PROCEDE POUR LA CULTURE PERMANENTE DE MICRO-ORGANISMES DE L'ESPECE i EREMOTHECIUM /i - Google Patents

PROCEDE POUR LA CULTURE PERMANENTE DE MICRO-ORGANISMES DE L'ESPECE i EREMOTHECIUM /i

Info

Publication number
EP1242612A1
EP1242612A1 EP00990744A EP00990744A EP1242612A1 EP 1242612 A1 EP1242612 A1 EP 1242612A1 EP 00990744 A EP00990744 A EP 00990744A EP 00990744 A EP00990744 A EP 00990744A EP 1242612 A1 EP1242612 A1 EP 1242612A1
Authority
EP
European Patent Office
Prior art keywords
range
flow rates
microorganism
eremothecium
produced
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP00990744A
Other languages
German (de)
English (en)
Inventor
Stephan Freyer
Henning ALTHÖFER
Klaus-Peter Stahmann
Andreas Wiesenburg
Cornelia Gaetgens
Hermann Sahm
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Forschungszentrum Juelich GmbH
Original Assignee
BASF SE
Forschungszentrum Juelich GmbH
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 BASF SE, Forschungszentrum Juelich GmbH filed Critical BASF SE
Publication of EP1242612A1 publication Critical patent/EP1242612A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • 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
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a process for the continuous cultivation of microorganisms of the genus Eremothecium and products produced in this process.
  • yeasts or yeast-like fungi such as, for example, Saccharomyces or Candida species
  • Saccharomyces or Candida species have been described in large numbers in the literature. This applies to batch or fed-batch processes as well as to continuous fermentations.
  • the advantages of continuous biotechnological processes include in particular the reduction of dead times, often higher productivity and continuous product acquisition.
  • the filamentous fungus Eremothecium gossypii (synonym: Ashbya gossypii; Kurtzman, CP, J. Ind. Microbiol., 1995, 14: 523-530) has so far been of biotechnological importance due to its ability to produce riboflavin (Vandamme EJ, J. Chem. Tech Biotechnol., 1992, 53: 313-327).
  • the production of ribofiavin by fermentation of Eremothecium gossypii and the closely related fungus Eremothecium ashbyi is known (The Merck Index, Windholz et al,. Eds. Merck & Co., 1983, page 1183; Bacher A.
  • the present invention is a method for culturing a microorganism of the genus Eremothecium, wherein the cells in continuous fermentation at flow rates in the range of greater than zero are cultured to 0.8 h ".
  • the process is preferably carried out at flow rates in the range from 0.001 to 0.8 h "1.
  • Flow rates in the range from 0.01 to 0.7 h “ 1 are preferred, particularly preferably from 0.05 to 0.6 h “1 .
  • a fermentation in batch mode is characterized in a classic manner in that the microorganism, starting from a start-up phase, goes through an exponential phase, a stationary phase and a dying phase. This means that there are different cultural conditions at all times, so the cultural conditions are constantly changing.
  • continuous fermentation is characterized in that an equilibrium state (flow equilibrium) is established so that the same culture conditions prevail over the long term.
  • filamentous fungi such as, for example, Penicillium or Fusarium species
  • values in the range from 0.15 to 0.25 h "1 are described for glucose-containing medium for the maximum specific growth rate (Christensen et al., J. Biotechnol., 1995, 42: 95-107; Wiebe et al., Microbiology, 1994, 140: 3015-3021).
  • the method according to the invention is distinguished in that a maximum specific growth rate of a microorganism of the genus Eremothecium is permanent in the range from 0.5 to 0.6 h "1 , in particular from 0.55 h " 1 .
  • the present invention also relates to a method in which the flow rate is alternately set to constant higher or lower values at defined time intervals.
  • the time interval between the changes in the flow rates is to be selected in such a way that on the one hand the stability of the system (settling into steady state) and on the other hand a desired maximum productivity is ensured. However, this depends on the respective objective of the cultivation process carried out.
  • Values in the range from 0.1 to 0.8 h "1 , preferably from 0.12 to 0.5 h " 1, are to be mentioned as examples of the constantly set higher flow rates.
  • Value ranges for constantly set lower flow rates are, for example, 0.01 to 0.2 h '1 , ranges from 0.02 to 0.1 h "1 are preferred, and a flow rate of approximately 0.05 h " 1 is particularly preferred.
  • the flow rate is reduced to a value of approximately 0.05 h "1.
  • TBM dry biomass
  • the dry biomass increased from 1.63 g / l to 3.93 g / l within about 8 hours.
  • the flow rate is reduced in the same way, starting from an initial flow rate of approximately 0.20 h "1 or approximately 0.30 h " 1 .
  • this method is particularly suitable for increasing the riboflavin production during the continuous cultivation of the fungus.
  • FIG. 2 A graphic representation of the course of the dry biomass and the riboflavin concentration is shown in FIG. 2.
  • the cells are stable continuously over a period of several weeks, preferably from 2 to 8 weeks, particularly preferably from 2 to 4 weeks, in particular from more than 2.5 weeks (corresponding to more than 400 hours) be cultivated.
  • the cells predominantly grow at flow rates of greater than zero to 0.20 h "1 , preferably from 0.01 to 0.20 h " 1
  • the method according to the invention is characterized in that the microorganism predominantly grows as a hyphen-shaped mycelium at flow rates in the range of> 0.20-0.70 h -1 and / or loose aggregations of this mycelium are formed in the form of mycelial pellets Mycelial pellets with a size of 1 to 3 mm in diameter At this stage of the culture, growth-linked product formation predominantly takes place.
  • a fungus of the species Eremothecium gossypii is preferably used in the process according to the invention.
  • an ATCC 10895 genetically modified compared to the wild-type Eremothecium gossypii (synonym: Ashbya gossypii) is used.
  • genetic changes are to be understood as natural, ie spontaneously occurring or artificially generated mutations.
  • Artificially created mutations can be caused, for example, by treating the fungus with mutagenic agents or by irradiation, especially UV radiation. These mutations are also called undirected mutations.
  • the present invention also encompasses genetically modified microorganisms which are produced in a targeted manner by genetic engineering methods.
  • mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues, which can be of homologous or heterologous origin. These mutations can affect gene expression or affect the activity of the gene product, which can be weakened or enhanced.
  • the mutations can be chromosomally coded or have a multiple copy number on so-called extrachromosomal gene structures (vectors).
  • changes to several genes are simultaneously included within an organism. For example, the metabolism of the microorganism can be steered in the direction of a desired product in this way (metabolic design).
  • the present method uses the cell's own and / or non-cell products, for example polysaccharases, lipases or proteases, as well as sugars or organic acids, etc. Amino acids. It is also possible to produce heterogeneous gene products which consist of a mixture of homologous and heterologous components, such as fusion proteins.
  • the present invention further relates to a microorganism of the genus Eremothecium, which is produced in the process according to the invention.
  • the invention also relates to primary metabolic products or end products of energy metabolism, including, for example, ethanol, acetate, lactate, acetone or butanol, produced in the process according to the invention described above.
  • ethanol production takes place at flow rates in the range from 0.06 to 0.40 h " 1 , preferably from 0.09 to 0.32 h " ⁇ particularly preferably from 0.25 to 0.32 h "1 (Table 1) ,
  • the present invention allows a filamentous microorganism to experience a crabtree effect, i.e. the formation of ethanol from glucose with an excess supply of oxygen (aerobic fermentation) can be observed.
  • the present invention also relates to secondary metabolites, including e.g. antibiotics or gibberellins, intermediate metabolites, etc. e.g. amino acids, citric acid or vitamins, energy reserves, etc. for example lipids, polysaccharides, such as, for example, glycogen, dextran or xanthan or polyhydroxybutyric acid, and also extracellular or intracellular enzymes, including, for example, amylases, proteases, cellulases or ⁇ -galactosidase, which are produced by the process according to the invention.
  • secondary metabolites including e.g. antibiotics or gibberellins, intermediate metabolites, etc. e.g. amino acids, citric acid or vitamins, energy reserves, etc. for example lipids, polysaccharides, such as, for example, glycogen, dextran or xanthan or polyhydroxybutyric acid, and also extracellular or intracellular enzymes, including, for example, amylases, proteases, cellulases or
  • the present invention furthermore relates to the use of the microorganism produced by the process according to the invention or of the aforementioned metabolic products according to the invention in areas of the chemical industry, pharmacy, medicine, food and / or feed industry, as well as agriculture and / or crop protection.
  • the present invention also relates to the use of the metabolic products according to the invention for Manufacture of medicines for the treatment of diseases in the aforesaid fields.
  • culture broth was filtered (paper circular filter, Schleicher & Schuell) and the glucose content in the filtrate was determined with the UV test for the determination of D-glucose (Röche Diagnostics).
  • the method is based on the enzymatic conversion of D-glucose and ATP to D-glucose-6-phosphate and ADP, (enzyme: hexokinase).
  • the enzyme glucose-6phosphate dehydrogenase oxidizes D-glucose-6-phosphate to D-gluconate-6-phosphate.
  • NADP + to NADPH takes place.
  • the amount of NADPH formed is equivalent to the amount of D-glucose used.
  • the formation of NADPH was measured in the UV spectrometer (UV-160, Shimadzu) at 340 nm.
  • Ethanol / acetate standard 0.81 g / l ethanol 1.64 g / l sodium acetate (corresponds to 1.18 g / l acetate)
  • the oxygen partial pressure (pO 2 ) was measured with a pO electrode (Mettler Toledo) (Demain & Solomon, Manual of Industrial Microbiology and Biotechnology, Washington, DC, 1986).
  • a combined pH electrode (pH combination electrode) from Ingold was used to measure the pH.
  • the calibration was carried out with standard commercial solutions (pH 4 and pH 7) before autoclaving.
  • Nile Red staining was carried out to detect fat deposits in the hyphae (Stahmann et al, Appl. Microbiol. Biotechnol., 1994, 42: 121-127). In contrast to an aqueous medium, this dye has good solubility in a hydrophobic environment and shows a strong fluorescence capacity under hydrophobic conditions (e.g. lipid deposits). The excitation takes place at 450 to 500 nm and the emission has a wavelength of> 528 nm.
  • Nile-Red solution 1 mg of Nile-Red in 1 ml of acetone
  • fluorescence microscope photo and fluorescence microscope, Zeiss
  • the shake flask including the medium, was sterilized by Autoclaving. Inoculation was carried out with mycelium from the stock holding plate, which was crushed intensively with the help of glass beads (diameter 5 mm). The cultivation took place overnight on a shaker at 30 ° C. and 120 rpm. To prepare the inoculum, the preculture was likewise homogenized with the aid of glass beads immediately before inoculating the fermenter or the shake flask series.
  • the LABFORS system from Infors was available for the cultivation experiments in the laboratory fermenter.
  • the glass stirred kettle had a maximum working volume of 5 liters.
  • a two-day disc stirrer with 6 blades ensured the mixing of the culture.
  • the risk of contamination, for example a mechanical seal, could be reduced by using a magnetic coupling.
  • the fermenter was provided with four baffles. It was ventilated with adjustable aeration rate with compressed air via a gassing pipe.
  • the pO 2 was controlled by varying the stirrer speed using an integrated control device.
  • the culture was tempered via the double jacket of the fermenter.
  • An exhaust air cooler (4 ° C) was used to reduce the discharge of volatile substances (e.g. ethanol) with the gas flow.
  • a standpipe was used for sampling and harvesting in continuous operation.
  • the tube opening for sampling was stored in ethanol (96% (v / v)). Due to its length, the harvesting line used in continuous operation did not require any additional measures to maintain sterility.
  • a culture sample was checked under the microscope at regular intervals for possible contamination. If necessary, an anti-foaming agent could be added using an infusion pump (PRECIDOR TYPE 5003, Infors AG, flow: 2> 0.4 ml / h).
  • PRECIDOR TYPE 5003, Infors AG, flow: 2> 0.4 ml / h The online measured values for pO 2 , pH, temperature and speed were measured with the data acquisition software MEDUSA 1.2 (Institute for Biotechnology 2, Anlagenstechnik GmbH) recorded and saved, the values of the CO 2 exhaust air analysis were recorded with a recorder.
  • FIG. 1 The experimental setup for continuous cultivation in the laboratory fermenter is shown in FIG. 1.
  • a peristaltic pump (4) (Watson Marlow) continuously pumped sterile nutrient medium from the storage bottle (2) (NALGENE, 20 liter volume) into the fermenter (1).
  • the flow rate could be set by selecting the pump output [ml / h].
  • the culture volume strictly speaking the weight of the fermenter, was kept constant by the harvesting pump (3) (B. Braun AG), which was controlled by a balance (5) (Bioengineering AG) on which the fermenter was stored.
  • the storage container (2) was mixed with a magnetic stirrer (15).
  • the fermenter system consisting of the filtration section for the medium, the storage bottle, the fermenter and the harvesting section, was sterilized by autoclaving.
  • the medium was pumped through the sterile filter (7) (SARTOBRAN-P CAPSULE, Sartorius AG) into the storage bottle (2) and the fermenter (1) was filled with 3 liters of medium.
  • Hose connections and the complete filling of the reactor jacket were tared (5) and the weight to be kept constant was determined. Inoculation was carried out with approximately 90 ml of homogenized preculture. Cultivation started in batch mode. Taking advantage of the favorable physiological state of the culture in the exponential growth phase, the system was switched to continuous operation after about 8 hours and a dry biomass content of approx. 1 g / l.
  • the cultivation conditions were: Working volume: 3 1
  • Figure 1 Schematic representation of the experimental setup for the continuous cultivation of a microorganism of the genus Eremothecium in the laboratory fermenter
  • FIG. 2 Graphical representation of the course of the dry biomass (TBM) and the riboflavin concentration of Eremothecium gossypii ATCC 10895 after lowering the flow rate (D) to 0.05 h "1 from steady state at a) 0.16 h " 1 , b) 0.2 h “1 and c) 0.3 h " 1 as a function of time (t).
  • Table 1 Overview of various parameters measured during the continuous fermentation of Eremothecium gossypii ATCC 10895
  • TBM dry biomass [g / l] nd: undetectable ( ⁇ 0.1 mg / l)

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour la culture permanente d'un micro-organisme de l'espèce Eremothecium, ainsi que les produits fabriqués sur cette base et leur utilisation.
EP00990744A 1999-12-16 2000-12-14 PROCEDE POUR LA CULTURE PERMANENTE DE MICRO-ORGANISMES DE L'ESPECE i EREMOTHECIUM /i Withdrawn EP1242612A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE19960703 1999-12-16
DE19960703A DE19960703A1 (de) 1999-12-16 1999-12-16 Verfahren zur kontinuierlichen Kultivierung von Mikroorganismen der Gattung Eremothecium
PCT/EP2000/012696 WO2001044491A1 (fr) 1999-12-16 2000-12-14 Procede pour la culture permanente de micro-organismes de l'espece eremothecium

Publications (1)

Publication Number Publication Date
EP1242612A1 true EP1242612A1 (fr) 2002-09-25

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EP00990744A Withdrawn EP1242612A1 (fr) 1999-12-16 2000-12-14 PROCEDE POUR LA CULTURE PERMANENTE DE MICRO-ORGANISMES DE L'ESPECE i EREMOTHECIUM /i

Country Status (6)

Country Link
US (1) US20030064500A1 (fr)
EP (1) EP1242612A1 (fr)
JP (1) JP2003516758A (fr)
CN (1) CN1409769A (fr)
DE (1) DE19960703A1 (fr)
WO (1) WO2001044491A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009032987A1 (fr) * 2007-09-05 2009-03-12 Microbia, Inc. Isolation de microorganismes formant des granules

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RO60041A2 (fr) * 1973-11-13 1976-06-15
DD248031A3 (de) * 1984-11-09 1987-07-29 Jenapharm Veb Verfahren zur pruefung der degenerationsgeschwindigkeit von antibiotikumbildenden selektanten
US5837528A (en) * 1989-06-22 1998-11-17 Hoffmann La Roche, Inc. Bacterial strains which overproduce riboflavin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0144491A1 *

Also Published As

Publication number Publication date
WO2001044491A1 (fr) 2001-06-21
DE19960703A1 (de) 2001-07-19
CN1409769A (zh) 2003-04-09
US20030064500A1 (en) 2003-04-03
JP2003516758A (ja) 2003-05-20

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