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WO2009138994A1 - Procédé de fermentation pour la fabrication de métabolites secondaires - Google Patents

Procédé de fermentation pour la fabrication de métabolites secondaires Download PDF

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Publication number
WO2009138994A1
WO2009138994A1 PCT/IN2008/000412 IN2008000412W WO2009138994A1 WO 2009138994 A1 WO2009138994 A1 WO 2009138994A1 IN 2008000412 W IN2008000412 W IN 2008000412W WO 2009138994 A1 WO2009138994 A1 WO 2009138994A1
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WO
WIPO (PCT)
Prior art keywords
fermentation
production
iodate
dissolved oxygen
medium
Prior art date
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Ceased
Application number
PCT/IN2008/000412
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English (en)
Inventor
Saurabh Garg
Kumar Chittnalli Ramegowda Naveen
Bimal Kumar
Sanjay Tiwari
Anuj Goel
Harish Iyer
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Biocon Ltd
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Biocon Ltd
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Filing date
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Publication of WO2009138994A1 publication Critical patent/WO2009138994A1/fr
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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms

Definitions

  • the present invention demonstrates the utilization of certain forms of halide anions such as iodates, periodates, bromates, chlorates, perchlorates and other forms of halides as additives in fermentation media for the production of secondary metabolites employing fungal, actinomycetes or bacterial systems such as systems Aspergillus, Rhizopus, Streptomyces etc.
  • the significant aspects of the invention specifically relate to a noticeably reduced oxygen uptake rate without any observed growth inhibition of the fermenting organism.
  • a typical fungal or bacterial fermentation consist of a growth medium in which the micro-organism is propagated and then transferred to a production medium which is designed to produce the product of interest. Even after extensive optimization of the medium and operating conditions, scale-up of processes using fungal cultures and other mycelia forming cultures is a difficult task.
  • the major limitation being the high oxygen demand, which cannot be satisfied in very large scale production fermenters due to limitations in the oxygen transfer coefficients.
  • the dissolved oxygen levels as measured by the online dissolved oxygen probes are almost zero (below 5%) for high cell density fermentation processes utilizing the above mentioned cultures. Achieving high dissolved oxygen levels in fermenter is desirable for better productivities.
  • Fermenting cells cultured under various aeration conditions require an optimal dissolved oxygen concentration level, and this impacts several factors of the fermentation process which are of major significance a) yield of the desired fermentation product from the fermentation substrate (expressed in grams product/grams substrate consumed), b) growth rates of the fermenting organism c) specific productivity of the desired fermentation product (expressed in weight product/weight dry cell/unit time) and d) substrate consumption rates (expressed in weight of substrate consumed/unit time).
  • dissolved oxygen levels are below the desired values as a result of poor oxygen mass transfer coefficients because of which achieving high cell densities becomes a matter of concern.
  • the lower dissolved oxygen levels also reduce the specific productivity of the cell mass because of oxygen starvation.
  • the objective of the invention is to provide a method of reducing the oxygen uptake by the fermenting micro-organism.
  • the objective is also to provide a method wherein a more nutritious medium can be used to achieve higher cell growth and thus higher productivities without compromising on the dissolved oxygen levels in the submerged fermentation processes thereby leading to higher yields of the desired end product.
  • the main objective of the present invention is to provide a process of fermentation for production of secondary metabolites.
  • Another object of the present invention is to provide a method of reducing oxygen uptake by the fermenting organism without hindering the growth of fermenting organism thereby resulting higher productivities.
  • the present invention provides for a fermentation process for the production of secondary metabolites, characterized in that the addition of halide anions inhibits reduction of dissolved oxygen content in the fermentation medium.
  • FIG 1 Effect of the supplementation of Iodate on the production of Mycophenolic acid
  • FIG 2 Effect of the supplementation of iodate on the production of mycophenolic acid in high nutrient medium.
  • FIG 3 Effect of the supplementation of Iodate on the production of Compactin.
  • FIG 4 Effect of the supplementation of Iodate on the production of Lovastatin.
  • FIG 5 Effect of the supplementation of Iodate on the production of Pneumocandin.
  • FIG 6 Effect of the supplementation of Iodate on the production of Tacrolimus.
  • FIG 7 Biomass- MPA fermentation run with iodate supplementation.
  • FIG 8 Titer and DO profile - MPA fermentation run with iodate supplementation.
  • FIG 9 Effect of other oxidative forms of halogen on mycophenolic acid production. DETAILED DESCRDPTION OF THE INVENTION
  • the present invention is in relation to a fermentation process for the production of secondary metabolites, characterized in that the addition of halide anions inhibits reduction of dissolved oxygen content in the fermentation medium.
  • the addition of halide ions reduces oxygen uptake of the micro-organism in the fermentation medium.
  • the halide anion is utilized as a salt in combination with its cationic metal component.
  • the halide anion is selected from a group comprising iodate, chlorate, bromate, fiorate perchlorate, perbromate and metaperiodate or their combination thereof or any of the oxidative forms of halides.
  • the cationic component is selected from a group comprising alkali metals, alkaline earth metals and/or their mixtures thereof. In still another embodiment of the present invention the cationic component is
  • the concentration of halide anion is ranging from of 0.1 - 50g/L.
  • the concentration of halide anion is ranging from of 0.1 - 1 Og/L.
  • the addition of halide anion decreases the dissolved oxygen uptake by the fermenting microorganism.
  • the residual dissolved oxygen level is increased by at least 10%.
  • the fermenting organism is a fungal species.
  • the fermenting organism is a bacterial species.
  • the fermenting organism is an Actinomycete species.
  • the productivity is increased by at least 20% It is an objective of the present invention to provide a method of reducing oxygen uptake by the fermenting organism without hindering the growth of the fermenting organism thereby resulting in higher productivities.
  • the use of certain forms of halide anions significantly reduces the oxygen uptake rates by the fermenting organism acting as a process control parameter, thereby allowing the fermentation process to be optimized, balancing high yields to the desired fermentation product with good production rates that is not hindered by slow growth rates of the organism, the dissolved oxygen content no more acting as a limiting factor.
  • Utilization of oxidative forms of halide anions as supplements in the fermentation medium can have a positive impact on a) specific productivity of the desired fermentation product (expressed in weight product/weight dry cell/unit time) and b) substrate consumption rates (expressed in weight of substrate consumed/unit time) without hindering the growth rates of the fermenting organism.
  • these ions that may be utilized in context of the present invention include, for example and without limitation, iodates, chlorates, bromates, perchlorates, periodates and the like.
  • the concentration of the halide anions used is in the range of 0.1 - 50g/L.
  • the fermentation medium can be supplemented with said halide anions initially or periodically during the course of fermentation.
  • Yield co-efficient of biomass produced over substrate utilized is herein understood to mean the amount of biomass produced in grams dry weight over the amount of substrate utilized in grams.
  • the yield coefficient of fermentation product produced over substrate may be expressed as units or grams of product produced per Kg of substrate used.
  • dissolved oxygen content or DO shall mean and refer to the ' dissolved oxygen content of the medium.
  • Secondary metabolites are organic compounds that are not directly involved in the normal growth, development or reproduction of organisms. A compound that is not necessary for the growth or maintenance of cellular functions but is synthesized, generally for the protection of a cell or microorganism during the stationary phase of the growth cycle.
  • the rate of consumption of oxygen per unit weight of the product titer can be determined from the amount of dissolved oxygen that is consumed, conveniently determined by measuring the amount of oxygen supplied to and consumed from the fermentation vessel per unit time. The amount of supplied oxygen can be measured by monitoring aeration rates.
  • the salt anion may be any suitable anion that results in a water soluble oxidizable salt when combined with the cation-containing component.
  • Suitable anions include, for example and without limitation, inorganic halide anions such as iodates, chlorates, perchlorates, periodates and the like. Other suitable anions would be well known to those skilled in the art in view of this disclosure.
  • halide anions that may be used in the present invention may be used in their salt forms such as hydrogen, alkali and alkali earth salts of chlorate, bromate, iodate, perchlorate, perbromate and metaperiodate and the like.
  • Particularly preferred metals include sodium, potassium, calcium, and magnesium, alone or in combination.
  • limiting nutrient source refers to a source of a nutrient (including the nutrient itself) essential for the growth of a microorganism in that, when the limiting nutrient is depleted from the growth medium, its absence substantially limits the microorganism from growing or producing further. However, since the other nutrients are still in abundance, the organism can continue to make and accumulate intracellular and/or extracellular products. By choosing a specific limiting nutrient, one can control the type of products that are accumulated. Therefore, providing a limiting nutrient source at a certain rate allows one to control both the rate of growth of the microorganism and the production or accumulation of desired products
  • microorganisms are selected from the group comprising, fungi (including yeasts), actinomycetes, protists, bacteria, or mixtures thereof, wherein the desired fermenting microorganism selected are capable of converting the fermentation substrates under suitable fermentable conditions to produce the desired end product.
  • the process of the invention can be conducted continuously, batch-wise, or some combination thereof.
  • the invention provides a medium which allows important secondary metabolites to be produced commercially, in large quantity, using a biological conversion process.
  • the invention provides a low cost alternative to prior art media and methods for its use that can be easily prepared and sterilized and most importantly results in the production of the desired end product at high yields. It is surprising that the myriad of components with a defined addition of anions such as iodates, chlorates, perchlorates, bromates yields high productivity attributed to the optimal maintenance of the dissolved oxygen levels without sacrificing the quality of microbial growth.
  • the fermentation medium of the present invention contains in aqueous solution: (a) from about 10 g/1 to about 60 g/1, most preferably from about 20 g/1 to about 40 g/1 of a source of metabolizable carbon and energy, preferably glucose; sucrose; dextrose and the like (b) about 0.1% - 10% of a preferable nitrogen source; (c) from about 1 g/1 to about 10 g/1, preferably from about 1 g/1 to about 7 g/1, of a source of phosphates, sulphates, chlorates, carbonates in their salt forms (d) from about 0.01 g/1 to about 10 g/1, preferably from about 2 g/1 to about 10 g/1 of at least one anion selected from the group consisting of iodate, chlorate, bromate, perchlorate, periodate, perbromate and other halides.
  • the anions are incorporated in the medium as their salt form in combination with a suitable cationic component, such components are selected from the group comprising al
  • the fermentation medium can be supplemented with said halide anions initially or periodically during the course of fermentation.
  • a foam inhibitor is added to the fermentation media in order to prevent the accumulation and build up of foam caused by oxygen sparging of the fermentation broth contained therein.
  • the process may be operated over any pH or temperature range where the fermenting microorganism can grow and catalyze the desired conversion reaction.
  • the process of this invention may be carried out at a temperature of about 22° C. to about 37° C, preferably about 27°C. Shifts in pH are prevented by addition of bases such as NaOH or acids such as H 2 SO 4 or HCl.
  • the regulating agent is typically a hydroxide or an organic or inorganic acid. Examples of suitable pH regulating agents are potassium hydroxide, sodium hydroxide and hydrochloric acid.
  • An important aspect of the present invention is the control of a number of process parameters to favor the desired reaction products.
  • the process or segments of the process can be conducted as continuous operations or various distinct unit operations. The length of time which the fermentation process is allowed to continue depends upon the composition of the fermentation medium, temperature, quantity of inoculum, quantity of product desired, etc.
  • increase in the rate of productivity is at least 10%, at least 20%, preferably at least 25%, preferably at least 30%, preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, most preferably at least 60%, most preferably at least 65% and most preferably at least 70%.
  • yield conversion obtainable is about 100%.
  • EXAMPLE 1 Supplementation of iodate in penicillium fermentation for the production of Mycophenolic Acid.
  • the spore vial for the Penicillium brevicompactum stored at -85 0 C was thawed and inoculated into the growth medium consisting of sucrose 10%, soya peptone 1%, soya flour 2%, magnesium sulphate 0.1% , sodium nitrate 0.2% and small quantities of antifoam.
  • the flask were incubated at 24 0 C for 48 h and then transferred to the production medium. Different levels of iodate ion in the form of potassium iodate were added to flasks.
  • the concentration of potassium iodate tested in this experiment was 0.0, 0.1, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 7.5 and 10.0 g/L.
  • the production medium consisted of Sucrose 10%, Casein hydrolysate 3.6%, cotton seed flour 2.0%, Potassium phosphate 0.45%, Magnesium Sulphate 0.1%, Potassium chloride 0.15%, calcium carbonate 0.6% and antifoam.
  • EXAMPLE 2 Supplementation of iodate in penicillium fermentation for the production of compactin.
  • Example 1 and 2 The similar experiment as mentioned in Example 1 and 2 was carried out for the production of Compactin.
  • the growth medium for the experiment was same as that in Example 1.
  • the production medium consisted of sucrose 35%, soya peptone 2%, soya flour 4%, magnesium sulphate 0.1%, sodium nitrate 0.2% and small quantities of antifoam. It was observed that KIO3 doesn't have any deleterious effect on the growth or productivity of compactin using the strain of penicillium rather results in higher productivity of compactin in the concentrated medium wherein otherwise the titers achieved were much lower.
  • the results are graphically represented in FIG. 3 EXAMPLE 3: Supplementation of iodate in Aspergillus fermentation for the production of Lovastatin.
  • the production medium for Lovastatin consist of starch 2.5%, dextrose mohohydrate 15%, soya flour 13%, peptone 1%, malt extract 1%, citric acid 1.05%, sodium acetate 0.88%, calcium carbonate 0.63%, sodium chloride 0.32%, potassium dihygrogen phosphate 0.075%, magnesium sulphate 0.075% and antifoam.
  • Pneumocandin BO is an antibiotic produced by submerged fermentation using the culture Zalerion arboricola.
  • the growth medium used for this fungal culture consisted of maltodextrin 2.5%, soya flour 1.2%, tryptone 0.5%, peptone 0.1%, yeast extract 0.2% , potassium phosphate 0.9% and antifoam.
  • the culture inoculated in the growth medium was incubated at 22 - 26 0 C for 3-4 days for proper growth of the culture.
  • the production medium consisted of the following components - Mannitol 10.0%, Casein 0.825%, potassium phosphate 0.2%, lactic acid 0.75%, corn steep powder 0.15%, soya flour 1.1%, dextrin 2.5%, glycine 0.2%, proline 1.65%, tryptone 0.825% and antifoam.
  • the flasks were incubated for 14-16 days.
  • EXAMPLE 5 Effect of iodate on Streptomyces sp in submerged fermentation for the production of Tacrolimus.
  • Tacrolimus is produced by fermentation using Streptomyces sp. in submerged cell cultivation.
  • Potassium iodate was found to have similar effects on Streptomyces cultures as in Fungal cultures and the productivities were found to increase with the increase in iodate concentration and then stabilized.
  • the growth medium used in this experiment consisted of the following components - Starch 1.0%, Dried Yeast 0.5%, Dextrose 0.5 %, glycerol 1.0%, Cotton seed flour 1.0%, corn steep liquor 0.5%, calcium carbonate 0.1% and antifoam.
  • the culture was grown for 48-56 hours in the growth medium at 22-30 0 C and then transferred to the production medium containing Starch 10.0%, Dried Yeast 4.5% , Calcium Carbonate 0.1% and antifoam.
  • the concentration of iodate at which the effect stabilized, was found to be much less in Streptomyces as compared to in fungal strains (shown in Example 1, 2 and 3). The results are shown in FIG 6.
  • EXAMPLE 6 Two 50 L fermentor batches were run using Penicillium aerenicola for the production of Mycophenolic acid.
  • One batch (Experiment #1) is run as a reference batch where iodate was not supplemented in the medium. The maximum product achieved was about 4.8 g/L and the maximum biomass concentration achieved was about 48% v/v when centrifuged at 10000 rpm for 10 min.
  • the second trial (Experiment #2) was taken with 3 g/L iodate ion added to the production medium.
  • the fermenter was operated under the exactly similar condition as of Experiment #1.
  • the experimental run #2 resulted in the final titre of about 6.29 g/L which was about 30% higher than that in the control trial.

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Abstract

La présente invention porte sur l'utilisation d'anions halogénures (iodates, periodates, bromates, chlorates, perchlorates et autres formes d'halogénures) comme suppléments dans des milieux de fermentation utilisés pour la fabrication de métabolites secondaires employant des systèmes fongiques tels que les systèmes Aspergillus, Rhizopus, Streptomyces, etc., des actinomycètes ou des systèmes bactériens. Certains aspects significatifs de la présente invention portent spécifiquement sur un taux d'absorption d'oxygène sensiblement réduit sans qu’aucune inhibition de croissance ne soit observée dans l'organisme en train de fermenter.
PCT/IN2008/000412 2008-05-14 2008-06-26 Procédé de fermentation pour la fabrication de métabolites secondaires Ceased WO2009138994A1 (fr)

Applications Claiming Priority (2)

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IN1175CH2008 2008-05-14
IN01175/CHE/2008 2008-05-14

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WO2009138994A1 true WO2009138994A1 (fr) 2009-11-19

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB712547A (en) * 1951-03-26 1954-07-28 Parke Davis & Co Antibiotic and methods of obtaining same
GB764198A (en) * 1953-10-23 1956-12-19 Konink Nl Gisten Spiritusfabri The antibiotic vengicide and processes for the preparation of the antibiotics oxytetracycline and vengicide
GB782125A (en) * 1953-12-04 1957-09-04 Bristol Lab Ltd Production of the antibiotic tetracycline by fermentation
WO2000001840A1 (fr) * 1998-07-03 2000-01-13 Dsm N.V. Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres
WO2000073489A1 (fr) * 1999-06-01 2000-12-07 Indena S.P.A. Procede de preparation de derives de glycosides steroidiens de ruscus aculeatus

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB712547A (en) * 1951-03-26 1954-07-28 Parke Davis & Co Antibiotic and methods of obtaining same
GB764198A (en) * 1953-10-23 1956-12-19 Konink Nl Gisten Spiritusfabri The antibiotic vengicide and processes for the preparation of the antibiotics oxytetracycline and vengicide
GB782125A (en) * 1953-12-04 1957-09-04 Bristol Lab Ltd Production of the antibiotic tetracycline by fermentation
WO2000001840A1 (fr) * 1998-07-03 2000-01-13 Dsm N.V. Processus de fermentation visant la production d'acide clavulanique a faible concentration d'acides amines libres
WO2000073489A1 (fr) * 1999-06-01 2000-12-07 Indena S.P.A. Procede de preparation de derives de glycosides steroidiens de ruscus aculeatus

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