WO2005014840A1 - Procede de preparation de l-threonine - Google Patents

Procede de preparation de l-threonine Download PDF

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Publication number
WO2005014840A1
WO2005014840A1 PCT/EP2004/008383 EP2004008383W WO2005014840A1 WO 2005014840 A1 WO2005014840 A1 WO 2005014840A1 EP 2004008383 W EP2004008383 W EP 2004008383W WO 2005014840 A1 WO2005014840 A1 WO 2005014840A1
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Prior art keywords
process according
threonine
removal
source
culture
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PCT/EP2004/008383
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English (en)
Inventor
Daniela Kruse
Thomas Hermann
Mechthild Rieping
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Degussa Ag
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Priority claimed from DE102004030417A external-priority patent/DE102004030417A1/de
Application filed by Degussa Ag filed Critical Degussa Ag
Priority to EP04763520A priority Critical patent/EP1649031A1/fr
Publication of WO2005014840A1 publication Critical patent/WO2005014840A1/fr

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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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the invention relates to an improved process for the fermentative preparation of L-threonine using bacteria of the Enterobacteriaceae family.
  • L-Threonine is used in animal nutrition, in human medicine and in the pharmaceuticals industry.
  • L-threonine can be prepared by fermentation of strains of the Enterobacteriaceae family, in particular Escherichia coli. Because of the great importance of this amino acid, work is constantly being undertaken to improve the preparation processes . Improvements to the process can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form, by e.g. ion exchange chromatography, or the intrinsic output properties, i.e. those of genetic origin, of the microorganism itself.
  • fermentation measures such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form, by e.g. ion exchange chromatography, or the intrinsic output properties, i.e. those of genetic origin, of the microorganism itself.
  • One variant of the fed batch process is the repeated fed batch process.
  • some of the culture broth is removed from the fermenter at a certain point in time, e.g. when the fermenter is full, so that the feed of nutrients can be prolonged.
  • US6562601 describes such a process for the production of L-threonine.
  • a nutrient medium is fed to the culture continuously, while culture broth is removed continuously or semi-continuously, so that the volume of the culture broth in the fermenter remains approximately constant.
  • a continuous fermentation can be operated without limitation. Prolonging the fermentation has a significantly positive effect on the overall productivity of a fermenter (average amount of product produced per hour) , since the influence of the time between two fermentations on the overall productivity of a fermenter is reduced.
  • Industrial utilization of continuous processes for the preparation of amino acids is described in only isolated cases. The main reason for this lies in the degeneration of the culture often observed. Amino acids are often produced with genetically optimized microorganisms which have been selected on the basis of high substrate/product yields.
  • US5763230 describes a continuous process for the preparation of amino acids, and in particular for L-lysine. In this process the growth of the cells is limited by the supplying of phosphate with simultaneous limitation of sugar; or in EP 0796616 with limitation of phosphate and / or sugar. A process was stabilized for up to 850 hours by the double limitation of growth. In the process described, a Corynebacterium glutamicum which overproduces L-lysine and is not characterized in more detail was used. It is said that the process can also be used with similar microorganisms for production of other amino acids, e.g. glutamate or threonine.
  • other amino acids e.g. glutamate or threonine.
  • US Patent 6025169 and FR 2669935 disclose that amino acids can be prepared by aerobic fed batch/continuous or continuous culturing with cell recycling of microorganisms. A process such that the concentration of the source of C in the medium can be kept below 5 g/1 or below 3 g/1 is described.
  • L-threonine it is described therein how such a process leads to an increased formation of threonine with a lower formation of acetate in the fed batch process with Brevibacterium flavum FERM BP-1173.
  • JP62-289192 describes the improvement of a continuous process for the fermentative preparation of amino acids with Corynebacterium glutamicum . Improvements were achieved by varying the carbon concentration in the nutrient solution fed in, the stirrer speed, the redox potential, the oxygen concentration and the intensity of the aeration.
  • JP62-289192 describes a continuous process for the fermentative preparation of amino acids with bacteria which produce amino acids. The authors found that the process can be improved if the concentration of the source of carbon of the nutrient solution fed in continuously is above 10% and the redox potential in the culture is more than -200 mV. It is also disclosed here that the use of several fermenters can improve the utilization of the source of carbon if relatively large amounts of the starting substances remain in the culture broth removed. As examples, lysine is produced with Corynebacterium glutamicum and arginine with Corynebacterium acetoacidophilum. Miwa Harufumi describes in JP62048394 a specific continuous process for the fermentative preparation of L-glutamate.
  • Substrate is fed to the reactor and culture broth is removed.
  • the producing molds are separated off from the removal stream.
  • the product is obtained from the supernatant and the cells are recycled into the process .
  • the object of the invention is to provide new measures for improved fermentative preparation of L-threonine.
  • the invention provides a fermentation process, which is characterized in that
  • a bacterium of the Enterobacteriaceae family which produces L-threonine is inoculated and cultured in at least a first nutrient medium
  • At least a further nutrient medium or several further nutrient media is/are then fed continuously to the culture in one (Fl) or several feed streams (Fl+) , the further nutrient medium or the further nutrient media comprising at least one source of carbon, at least one source of nitrogen and at least one source of phosphorus, under conditions which allow the formation of L-threonine, and at the same time culture broth is removed from the culture with at least one (F2) or several removal streams (F2+) which substantially corresponds/correspond to the feed stream (Fl) or the total of the feed streams (Fl+) , wherein
  • the cells are completely or partly separated off from the removal stream or the removal streams (F4 or F9) with a return flow ratio, R, of 0 ⁇ R ⁇ 1 by appropriate processes and are recycled into the culturing step (b) (F5 or F10) , ⁇ . ) the threonine formed is removed, either 1) directly from the removal stream or streams (F7) or 2) after complete or partial separating off of the cells from the removal stream or the removal streams (F3), completely or partly from this or these removal streams ,
  • the concentration of the source (s) of carbon during the culturing is adjusted to not more than 30 g/1.
  • the product threonine is removed a) directly from the removal stream or streams (F7) or b) after complete or partial separating off of the cells from the removal stream or the removal streams (in the following, derivatives of the removal stream (F3)) completely (100%) or partly from this removal stream or from the derivatives of the removal stream.
  • the culture broth remaining after removal of the threonine (with or with depleted biomass) is then fed back to the culture vessel (P8 or Fll) . If a complete or partial separating off of the biomass has taken place before the removal of the threonine, this can be combined again with the threonine- depleted stream.
  • the combining can furthermore take place with previously complete or partial separating off of the biomass in the culture vessel by recycling at least two streams (one with enriched biomass (F10) and one with a reduced threonine content (Fll)) into the culture vessel.
  • FIGs 1 and 2 are diagrams which represent the plant components and feed and the removal and return flow streams of processes according to the invention.
  • the symbols denote the following:
  • Fl a nutrient medium feed stream according to the invention
  • F1+ several nutrient media feed streams according to the invention
  • F2 a removal stream according to the invention
  • F2+ several removal streams according to the invention
  • F3 cell-depleted removal stream or F4 - F5
  • F4 one or more removal streams for separating off cells
  • F5 the completely recycled removal stream
  • F6 stream with removed threonine or F7 - F8
  • F7 one or more removal streams according to the invention for separating off threonine
  • F8 the threonine-depleted recycled stream
  • X separating off, according to the invention, of cells
  • T separating off, according to the invention, of threonine.
  • F9 one or more removal streams according to the invention for separating off cells and/or threonine;
  • F10 cell- enriched stream or F9-F3; and
  • Fll stream with depleted threonine .
  • the culture broth remaining after removal of the threonine ( with or with depleted biomass) is then fed back to the culture vessel (F8 or Fll) .
  • this (FlO) can be combined again with the threonine-depleted stream (Fll) .
  • the combining can furthermore take place with previously complete or partial separating off of the biomass in the culture vessel by recycling at least two streams (one with concentrated biomass F5 or FlO and one with a reduced threonine content F8 or Fll) into the culture vessel.
  • cells can be completely or partly separated off from the removal stream (F4 or F9) with a return flow ratio, R, of 0 ⁇ R ⁇ 1 by appropriate processes and can be recycled as F5 or FlO into the culturing step (b) .
  • This return flow ratio is equivalent to a recycling of 0% up to and including 100% of the removed cells, preferably of 10% up to and including 100%, of 20% up to and including 100%, of 30% up to and including 100%, of 40% up to and including 100%, of 50% up to and including 100%, of 60% up to and including 100%, of 70% up to and including 100%, of 80% up to and including 100%, or of 90% up to and including 100%.
  • continuous means that the feed stream or the feed streams are substantially uninterrupted. That is to sa-Y. nutrient medium or nutrient media is/are added to the culture with at most short, individual pauses. The individual interruptions or pauses are up to a maximum of 0.5, a maximum of 1, a maximum of 2 or a maximum of
  • the sum of the individual interruptions or pauses in the culturing according to step b) is a maximum of 10%, a maximum of 8%, a maximum of 6%, a maximum of 4%, a maximum of 2% or a maximum of 1% of the total time of the culturing according to step b) .
  • threonine is only partly removed from the removal stream or streams (F7) or the derivatives of the removal stream F3, this is separated off from 0 to less than or equal to ( ⁇ ) 10%, from 0 to ⁇ 20%, from 0 to ⁇ 30%, from 0 to ⁇ 40%, from 0 to ⁇ 50%, from 0 to ⁇ 60%, from 0 to ⁇ 70%, from 0 to ⁇ 80%, from 0 to ⁇ 90%, or from 0 to ⁇ 100%.
  • the separating off of the threonine can take place here in the presence, partial presence or absence of the biomass, by using chemical or physical processes or employing combinations of the two.
  • Chemical processes include extractions with chemical substances in which threonine dissolves. These include toluene, acids, e.g. hydrochloric acid, sulfuric or phosphoric acid, long-chain alcohols, e.g. butanol, pentanol or hexanol, and also carboxylic acid esters, such as e.g. ethyl acetate.
  • the physical methods of separating off threonine include crystallization, for example by vacuum or cooling, filtration (for example micro-, ultra- or nanofiltration) and purification with chromatographic processes, such as, for example, ion exchange chromatography or, for example, ion exclusion chromatography. Physical extraction processes are preferred according to the invention.
  • the volumetric productivity, also called the space/time yield, of a process depends not only on the output capacity of the microorganism chosen, characterized by its specific productivity, but also on the biomass concentration available in the culture system.
  • the specific productivity describes the ratio of the product formed and the cell mass and is therefore a measure of the activity of the cells for product formation.
  • the biomass concentration in a free culture system remains less than 50 g/1, less than 40 g/1, less than 30 g/1, or even less than 20 g/1.
  • free culture system means a process without cell retention, that is to say with a free overflow.
  • the cells By immobilization or cell retention systems, the cells can be utilized for product formation for longer than they are available in a free culture system.
  • the specific growth rate By cell recycling the specific growth rate can be uncoupled from the dilution rate, and in particular such that the process can be operated with considerably higher dilution rates compared with a free culture system.
  • the specific growth rate or specific growth speed is the ratio of the growth speed and the biomass concentration.
  • the dilution rate is described by the ratio of the amount flowing through and the volume of liquid in the reactor (Horst Chmiel, Bioreatechnik: Einf ⁇ hrung in die Biovonstechnik, Gustav Fischer Verlag, Stuttgart 1991) .
  • Centrifuges, separators and settlers (after flocculation) as well as cross-stream, micro- and ultra-filters are suitable for continuous separating off of cells ( Biotechnologie, by H. Weide, J. Paca and . A. Knorre,
  • the return flow ratio R is defined as the ratio of the flow of the biomass-depleted permeate or sedimenter overflow or cell-depleted stream F3 to the total feed streams of the fermentation (Fl and F1+) .
  • the return flow ratio should be in the range of 0 ⁇ R ⁇ 1, preferably between 0.1 ⁇ R ⁇ 1, 0.2 ⁇ R ⁇ 1, 0.3 ⁇ R ⁇ 1, 0.4 ⁇ R ⁇ 1, 0.5 ⁇ R ⁇ 1, 0.6 ⁇ R ⁇ 1, 0.7 ⁇ R ⁇ 1, 0.8 ⁇ R ⁇ 1, 0.9 ⁇ R ⁇ 1.
  • the biomass concentrations are increased compared with a free culture system. That is to say, if the same microorganism is employed, according to the invention the biomass concentrations are increased by at least 5%, preferably 10%, 15%, 20%, 25%, 30%, 50%, 80%, 100%, 150%, or even by 200% or more.
  • the separating off of cells can take place here, for example, by micro- or ultrafiltration in one step, or sequentially by various centrifugation or separation steps. If the separating off of the biomass takes place in one step to 100%, some of the biomass retained or the complete biomass can be recycled back into the culture vessel/the fermenter, and in particular corresponding to a return flow ratio R of 0 ⁇ R ⁇ 1.
  • the return flow ratio R can be determined by a particular fraction of the centrifugate or several of or all the fractions of the centrifugates being recycled. Particular fractions can furthermore be mixed with one another in order to establish a desired return flow ratio.
  • the return flow ratio is adjusted by determining the biomass concentration in the culture vessel/fermenter and/or determining the biomass concentration in the return flow.
  • the biomass concentration is determined by the techniques conventionally used, such as, for example, counting the cell titer in a calibrated counting chamber, cytometrically with or without staining of the cells, determination of the optical density of the culture or by determination of the dry biomass.
  • counting of the cell titer can also be automated, for example by determining the biomass concentration in a filtrate or centrifugate stage by optical methods, such as photometry or cyto etry, or by using physical methods, such as, for example, determination of the conductivity or the absorption of radiation of the near infra-red or middle infra-red range.
  • the plant output of a fermentation unit which produces L-threonine can be increased by culturing by the batch process or fed batch process in the first culturing step a) described above, at least one additional nutrient medium being employed if the fed batch process is used.
  • at least one further nutrient medium or several further nutrient media are fed continuously to the culture in one (Fl) or several feed streams (F1+) and at the same time culture broth is removed from the culture with at least one (F2) or several removal streams (F2+) , which substantially corresponds/correspond to the feed stream or the total of the feed streams.
  • plant output is understood as meaning that in a plant, such as e.g. a fermenter, the weight or amount of a product, e.g. L-threonine, is prepared with a certain yield and with a certain space/time yield. These parameters largely determine the costs or the profitability of a process .
  • the bacterium is inoculated in at least a first nutrient medium and cultured by the batch process or fed batch process. If the fed batch process is used, an additional nutrient medium is fed in after more than 0 to not more than 10 hours, preferably after 1 to 10 hours, preferentially after 2 to 10 hours and particularly preferably after 3 to 7 hours.
  • the first nutrient medium comprises as the source of carbon one or more of the compounds chosen from the group consisting of sucrose, molasses from sugar beet or cane sugar, fructose, glucose, starch hydrolysate, lactose, galactose, cellulose hydrolysate, arabinose, maltose, xylose, acetic acid, ethanol and methanol, in concentrations of 1 to 50 g/kg, preferably 10 to 45 g/kg, particularly preferably 20 to 40 g/kg.
  • Starch hydrolysate is understood according to the invention as the hydrolysis product of starch from maize, cereals, potatoes or tapioca.
  • Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea
  • inorganic compounds such as ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, potassium nitrate and potassium sodium nitrate, can be used as the source of nitrogen in the first nutrient medium.
  • the sources of nitrogen can be used individually or as a mixture in concentrations of 1 to 40 g/kg, preferably 10 to 30 g/kg, particularly preferably 10 to 25 g/kg.
  • Phosphoric acid alkali metal or alkaline earth metal salts of phosphoric acid, in particular potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts, polymers of phosphoric acid or the hexaphosphoric acid ester of inositol, also called phytic acid, can be used as the source of phosphorus in the first nutrient medium in concentrations of 0.1 to 5 g/kg, preferably 0.3 to 3 g/kg, particularly preferably 0.5 to 1.5 g/kg.
  • the culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth.
  • the additional nutrient medium which is used in a fed batch process in general comprises merely as the source of carbon one or more of the compounds chosen from the group consisting of sucrose, molasses from sugar beet or cane sugar, fructose, glucose, starch hydrolysate, lactose, galactose, cellulose hydrolysate, arabinose, maltose, xylose, acetic acid, ethanol and methanol, in concentrations of 300 to 700 g/kg, preferably 400 to 650 g/kg, and optionally an inorganic source of nitrogen, such as e.g.
  • these and other components can also be fed in separately.
  • the constituents of the further nutrient medium can be fed to the culture in the form of a single further nutrient medium and in a plurality of further nutrient media.
  • the further nutrient medium or the further nutrient media are fed to the culture in at least one (1) feed stream or in a plurality of feed streams of at least 2 to 10, preferably 2 to 7 or 2 to 5 feed streams .
  • the further nutrient medium or the further nutrient media comprises/comprise as the source of carbon one or more of the compounds chosen from the group consisting of sucrose, molasses from sugar beet or cane sugar, fructose, glucose, starch hydrolysate, maltose, xylose, acetic acid, ethanol and methanol, in concentrations of 20 to 700 g/kg, preferably 50 to 650 g/kg.
  • the further nutrient medium or the further nutrient media furthermore comprises or comprise a source of nitrogen consisting of organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate and/or potassium nitrate or potassium sodium nitrate.
  • the sources of nitrogen can be used individually or as a mixture in concentrations of 5 to 50 g/kg, preferably 10 to 40 g/kg.
  • the further nutrient medium or the further nutrient media furthermore comprises or comprise a source of phosphorus consisting of phosphoric acid, alkali metal or alkaline earth metal salts of phosphoric acid, in particular potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts, polymers of phosphoric acid or the hexaphosphoric acid ester of inositol, also called phytic acid.
  • the sources of phosphorus can be used individually or as a mixture in concentrations of 0.3 to 3 g/kg, preferably 0.5 to 2 g/kg.
  • the culture medium must furthermore comprise salts of metals, such as e.g.
  • magnesium sulfate or iron sulfate which are necessary for growth, in concentrations of 0.003 to 3 g/kg, preferably in concentrations of 0.008 to 2 g/kg.
  • essential growth substances such as amino acids (e.g. homoserine) and vitamins (e.g. thiamine) , are employed in addition to the above-mentioned substances.
  • Antifoams such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam.
  • a single further nutrient medium is used, this is typically fed to the culture in one feed stream. If a plurality of further nutrient media are used, these are fed in a corresponding plurality of feed streams. If a plurality of further nutrient media are used, it should be noted that these in each case can comprise only one of the sources of carbon, nitrogen or phosphorus described, or also a mixture of the sources of carbon, nitrogen or phosphorus described.
  • the nutrient medium fed in or the nutrient media fed in is/are adjusted such that there is a phosphorus to carbon ratio (P/C ratio) of not more than 4; of not more than 3; of not more than 2; of not more than 1.5; of not more than 1; of not more than 0.7; of not more than 0.5; of not more than 0.48; of not more than 0.46; of not more than 0.44; of not more than 0.42; of not more than 0.40; of not more than 0.38; of not more than 0.36; of not more than 0.34; of not more than 0.32; or of not more than 0.30 mmol of phosphorus / mol of carbon.
  • P/C ratio phosphorus to carbon ratio
  • the feed stream (Fl) or the total of the feed streams (F1+) in the process according to the invention are fed in at a rate corresponding to an average residence time of less than 30 hours, preferably less than 25, very particularly preferably less than 20 hours.
  • the average residence time here is the theoretical time the particles remain in a continuously operated culture.
  • the average residence time is described by the ratio of the volume of liquid in the reactor and the amount flowing through (Biotechnologie; H. Weide, . Paca and W. A. Knorre; Gustav Fischer Verlag Jena; 1991) .
  • Intensive growth at the start of culturing is usually a logarithmic growth phase.
  • the logarithmic growth phase is in general followed by a phase of less intensive cell growth than in the logarithmic phase.
  • At least one further nutrient medium or several further nutrient media are fed continuously to the culture in one or several feed streams and at the same time culture broth is removed from the culture with at least one or several removal streams, which substantially corresponds/correspond to the feed stream or the total of the feed streams.
  • substantially here means that the speed of the removal stream or the removal streams corresponds to 80% - 120%, 90% - 110% of the feed stream or of the total of the feed streams.
  • the removal can be realized industrially by pumping off and/or by draining off the culture broth.
  • the concentration of the source of carbon during the culturing according to step b) is in general adjusted to not more than 30 g/1, to not more than 20 g/1, to not more than 10 g/1, preferably to not more than 5 g/1, particularly preferably not more than 2 g/1. This concentration is maintained for at least 75%, preferably for at least 85%, particularly preferably for at least 95% of the culturing time according to step b) and/or c) .
  • the concentration of the source of carbon is determined here with the aid of methods which are prior art.
  • ⁇ -D-Glucose is determined e.g. in a YSI 02700 Select glucose analyzer from Yellow Springs Instruments (Yellow Springs, Ohio, USA) .
  • the culture broth removed can be provided with oxygen or an oxygen-containing gas, optionally with stirring, until the concentration of the source of carbon falls below 2 g/1; below 1 g/1; or below 0.5 g/1.
  • the yield is at least 31%, at least 33%, at least 35%, at least 37%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46% or at least 48%.
  • the yield is defined here as the ratio of the total amount of L-threonine formed in a culturing to the total amount of the source of carbon employed or consumed.
  • L-threonine is formed with a space/time yield of at least 1.5 to 2.5 g/1 per h, of at least 2.5 to 3.5 g/1 per h, of at least 2.5 to more than 3.5 g/1 per h, of at least 3.5 to 5.0 g/1 per h, of at least 3.5 to more than 5.0 g/1 per h, or of at least 5.0 to 8.0 g/1 per h or more, such as, for example, at least 9 g/1 per h.
  • the space/time yield is defined here as the ratio of the total amount of threonine formed in a culturing to the volume of the culture over the total period of time of culturing.
  • the space/time yield is also called the volumetric productivity.
  • the product is of course prepared in a certain yield and in a certain space/time yield (volumetric productivity) .
  • L-threonine can be prepared in a yield of at least 31 % and a space/time yield of at least 1.5 to 2.5 g/1 per h. Further couplings of yield with space/time yield, such as, for example, a yield of at least 37% and a space/time yield of at least 2.5 g/1 per h, automatically result from the above statements.
  • the culturing in steps a) and b) is carried out under conditions which allow the formation of L-threonine: During the culturing the temperature is adjusted in a range from 29 to 42 °c, preferably 33 to 40 9 C. The culturing can be carried out under normal pressure or optionally under increased pressure, preferably under an increased pressure of 0 to 1.5 bar.
  • the oxygen partial pressure is regulated at 5 to 50%, preferably approx. 20% atmospheric saturation. During this procedure the culture is stirred and supplied with oxygen. Regulation of the pH to a pH of approx. 6 to 8, preferably 6.5 to 7.5, can be effected with 25% aqueous ammonia .
  • the process according to the invention is operated for at least 72 hours, preferably 100 to 300 hours, particularly preferably 200 to > 300 hours.
  • the volume of the culture is exchanged at least by half, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 12 times .
  • the L-threonine can be isolated, collected or concentrated and optionally purified.
  • Separation methods such as, for example, centrifugation, filtration, decanting, flocculation or a combination thereof are employed for the removal or separating off of the biomass .
  • the broth obtained is then thickened or concentrated by known methods, such as, for example, with the aid of a rotary evaporator, thin film evaporator, falling film evaporator, by reverse osmosis, by nanofiltration or a combination thereof .
  • This concentrated broth is then worked up by methods of freeze drying, spray drying, spray granulation or by other processes to give a preferably free-flowing, finely divided powder.
  • This free-flowing, finely divided powder can then in turn be converted by suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product.
  • the water is removed in total to the extent of more than 90% by this means, so that the water content in the product is less than 10%, less than 5%.
  • L-threonine and other amino acids can be carried out by anion exchange chromatography with subsequent ninhydrin derivation, as described by Spackman et al. (Analytical Chemistry, 30: 1190-1206 (1958)) or it can be carried out by reversed phase HPLC, as described by Lindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)) .
  • Bacteria of the Enterobacteriaceae family which produce L-threonine chosen from the genera Escherichia, Erwinia, Providencia and Serratia are suitable for carrying out the process according to the invention.
  • the genera Escherichia and Serratia are preferred.
  • the species Escherichia coli and of the genus are preferred.
  • Serratia in particular the species Serratia marcescens are to be mentioned.
  • the bacteria contain at least one copy of a thrA gene or allele which codes for a threonine-insensitive aspartate kinase I - homoserine dehydrogenase I.
  • a thrA gene or allele which codes for a threonine-insensitive aspartate kinase I - homoserine dehydrogenase I.
  • Such bacteria are typically resistant to the threonine analogue ⁇ -amino- ⁇ - hydroxyvaleric acid (AHV) . If appropriate, the thrA gene or allele is overexpressed.
  • Strains from the Enterobacteriaceae family which produce L-threonine preferably have, inter alia, one or more genetic or phenotypic features chosen from the group consisting of: resistance to ⁇ -amino- ⁇ -hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to ⁇ -methylserine, resistance to diaminosuccinic acid, resistance to ⁇ -aminobutyric acid, resistance to borrelidin, resistance to cyclopentane-carboxylic acid, resistance to rifampicin, resistance to valine analogues, such as, for example, valine hydroxamate, resistance to purine analogues, such as, for example, 6-dimethylaminopurine, a need for L-methionine, optionally a partial and compensable need for L-isoleucine, a need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptide
  • Escherichia in particular of the species Escherichia coli, are, for example
  • Bacteria of the Enterobacteriaceae family which contain a stop codon chosen from the group consisting of opal, ochre and amber, preferably amber in the rpoS gene, and a t-RNA suppressor chosen from the group consisting of opal suppressor, ochre suppressor and amber suppressor, preferably amber suppressor, are moreover suitable.
  • the amber mutation preferably lies at position 33 according to the amino acid sequence of the RpoS gene product.
  • supE is preferably employed as the amber suppressor.
  • the nucleotide sequence of the rpoS gene can be found in the prior art.
  • the nucleotide sequence of the rpoS gene corresponding to Accession No. AE000358 is shown as SEQ ID NO. 1.
  • the amino acid sequence of the associated RpoS gene product or protein is shown in SEQ ID NO. 2.
  • the nucleotide sequence of an rpoS allele which contains a stop codon of the amber type at the position of the nucleotide sequence corresponding to position 33 of the amino acid sequence of the RpoS gene product or protein, corresponding to SEQ ID NO. 1 or SEQ ID NO. 2 respectively, is reproduced in SEQ ID NO. 3.
  • the suppressor supE is also described in the prior art and is shown as SEQ ID NO. 4.

Abstract

L'invention concerne un procédé amélioré de préparation par fermentation de L-thréonine à l'aide de bactéries de la famille des Enterobacteriaceae produisant la L-thréonine.
PCT/EP2004/008383 2003-08-01 2004-07-27 Procede de preparation de l-threonine WO2005014840A1 (fr)

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DE10335251.1 2003-08-01
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US49195903P 2003-08-04 2003-08-04
US60/491,959 2003-08-04
DE102004030417A DE102004030417A1 (de) 2003-08-01 2004-06-24 Verfahren zur Herstellung von L-Threonin
DE102004030417.3 2004-06-24

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DE102007051024A1 (de) 2007-03-05 2008-09-11 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von Stämmen der Familie Enterobacteriaceae
EP1975241A1 (fr) 2007-03-29 2008-10-01 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2036979A1 (fr) 2007-09-15 2009-03-18 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2055785A1 (fr) 2007-11-02 2009-05-06 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2060636A1 (fr) 2007-11-14 2009-05-20 Evonik Degussa GmbH Procédé de fabrication d'acides aminés L en utilisant des souches de la famille des Enterobacteriaceae améliorées
EP2098597A1 (fr) 2008-03-04 2009-09-09 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102008002309A1 (de) 2008-06-09 2009-12-10 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
DE102008040352A1 (de) 2008-07-11 2010-01-14 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Tryptophan unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
DE102008044768A1 (de) 2008-08-28 2010-03-04 Evonik Degussa Gmbh Verfahren zur Herstellung von organisch-chemischen Verbindungen unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP3385275A1 (fr) 2017-04-07 2018-10-10 Evonik Degussa GmbH Procédé de fabrication d'aminoacides l aromatiques à l'aide de troncs améliorés de la famille des enterobacteriaceae
WO2019011946A1 (fr) * 2017-07-11 2019-01-17 Adisseo France S.A.S. Levure produisant de la thréonine
CN109355325A (zh) * 2018-10-18 2019-02-19 许传高 颗粒苏氨酸和颗粒蛋白的共生产工艺
CN109439702A (zh) * 2018-10-18 2019-03-08 许传高 处理苏氨酸高浓度发酵废水的工艺
EP3608409A1 (fr) 2018-08-09 2020-02-12 Evonik Operations GmbH Procédé de préparation d'acides aminés l au moyen de souches améliorées de la famille des enterobacteriaceae
EP3677594A1 (fr) 2019-01-07 2020-07-08 Evonik Operations GmbH Procédé de production de l-tryptophane utilisant des souches améliorées de la famille des entérobactéries
US11053526B2 (en) 2018-08-09 2021-07-06 Evonik Operations Gmbh Process for preparing L amino acids using improved strains of the enterobacteriaceae family

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007051024A1 (de) 2007-03-05 2008-09-11 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von Stämmen der Familie Enterobacteriaceae
EP1975241A1 (fr) 2007-03-29 2008-10-01 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2036979A1 (fr) 2007-09-15 2009-03-18 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102007044134A1 (de) 2007-09-15 2009-03-19 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2465869A1 (fr) 2007-11-02 2012-06-20 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP2055785A1 (fr) 2007-11-02 2009-05-06 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102007052270A1 (de) 2007-11-02 2009-05-07 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2060636A1 (fr) 2007-11-14 2009-05-20 Evonik Degussa GmbH Procédé de fabrication d'acides aminés L en utilisant des souches de la famille des Enterobacteriaceae améliorées
EP2098597A1 (fr) 2008-03-04 2009-09-09 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102008002309A1 (de) 2008-06-09 2009-12-10 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Aminosäuren unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2133420A1 (fr) 2008-06-09 2009-12-16 Evonik Degussa GmbH Procédé de fabrication d'aminoacides L en utilisant des troncs améliorés de la famille des enterobacteriaceae
DE102008040352A1 (de) 2008-07-11 2010-01-14 Evonik Degussa Gmbh Verfahren zur Herstellung von L-Tryptophan unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2147972A1 (fr) 2008-07-11 2010-01-27 Evonik Degussa GmbH Procédé de fabrication de tryptophan L en utilisant des souches améliorées de la famille des enterobacteriaceae
DE102008044768A1 (de) 2008-08-28 2010-03-04 Evonik Degussa Gmbh Verfahren zur Herstellung von organisch-chemischen Verbindungen unter Verwendung von verbesserten Stämmen der Familie Enterobacteriaceae
EP2163613A2 (fr) 2008-08-28 2010-03-17 Evonik Degussa GmbH Procédé de fabrication de liaisons organo-chimiques en utilisant des troncs améliorés de la famille des enterobacteriaceae
EP3385275A1 (fr) 2017-04-07 2018-10-10 Evonik Degussa GmbH Procédé de fabrication d'aminoacides l aromatiques à l'aide de troncs améliorés de la famille des enterobacteriaceae
WO2019011946A1 (fr) * 2017-07-11 2019-01-17 Adisseo France S.A.S. Levure produisant de la thréonine
US11535830B2 (en) 2017-07-11 2022-12-27 Adisseo France S.A.S. Threonine-producing yeast
EP3608409A1 (fr) 2018-08-09 2020-02-12 Evonik Operations GmbH Procédé de préparation d'acides aminés l au moyen de souches améliorées de la famille des enterobacteriaceae
US11053526B2 (en) 2018-08-09 2021-07-06 Evonik Operations Gmbh Process for preparing L amino acids using improved strains of the enterobacteriaceae family
CN109355325A (zh) * 2018-10-18 2019-02-19 许传高 颗粒苏氨酸和颗粒蛋白的共生产工艺
CN109439702A (zh) * 2018-10-18 2019-03-08 许传高 处理苏氨酸高浓度发酵废水的工艺
EP3677594A1 (fr) 2019-01-07 2020-07-08 Evonik Operations GmbH Procédé de production de l-tryptophane utilisant des souches améliorées de la famille des entérobactéries

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