EP1649031A1

EP1649031A1 - Process for the preparation of l-threonine

Info

Publication number: EP1649031A1
Application number: EP04763520A
Authority: EP
Inventors: Daniela Kruse; Thomas Hermann; Mechthild Rieping
Original assignee: Degussa GmbH
Current assignee: Evonik Operations GmbH
Priority date: 2003-08-01
Filing date: 2004-07-27
Publication date: 2006-04-26
Also published as: WO2005014840A1

Abstract

The invention relates to an improved process for the fermentative preparation of L-threonine using bacteria of the Enterobacteriaceae family which produce L-threonine.

Description

Process for the Preparation of L-Threonine

Field of the Invention

The invention relates to an improved process for the fermentative preparation of L-threonine using bacteria of the Enterobacteriaceae family.

Background of the Invention

L-Threonine is used in animal nutrition, in human medicine and in the pharmaceuticals industry.

It is known that 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.

It is known from the prior art, such as is described, for example, in US-A-5, 538, 873 and in EP-B-0593792 , WO014525 or by Okamoto et al . (Bioscience, Biotechnology, and Biochemistry 61 (11), 1877 - 1882, 1997), that threonine is prepared by fermentation in the batch process or fed batch process, in the batch process, all the nutrients are initially introduced directly at the start of the fermentation. In the fed batch process one or more nutrients is/are fed continuously to the culture. This feed/these feeds can start directly from the start of the culturing or after a certain culturing time. Alternatively, however, the feed / the feeds can also be started when the nutrients in the culture broth which are fed in are consumed.

One variant of the fed batch process is the repeated fed batch process. In this, 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.

In another type of fermentation, continuous fermentation or chemostat culturing, 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. In theory, 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. In contrast to their precursors, they often have poorer growth properties here. During long-lasting culturing, a reversion to variants which produce less may occur. This leads to a loss in the productivity of the culture. 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.

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. For 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 . By the removal of culture broth and the recycling of cells it is ensured that the cells are not exposed to high glutamate concentrations and a high productivity can be achieved.

Object of the Invention

The object of the invention is to provide new measures for improved fermentative preparation of L-threonine.

Summary of the Invention

The invention provides a fermentation process, which is characterized in that

a) a bacterium of the Enterobacteriaceae family which produces L-threonine is inoculated and cultured in at least a first nutrient medium,

b) 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

c) 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) , σ.⁾ 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 ,

e) the culture broth remaining after removal of the threonine (with (F8) or with depleted biomass (Fll) ) is fed back to the culture vessel, and

f) the concentration of the source (s) of carbon during the culturing is adjusted to not more than 30 g/1.

Detailed Description of the Invention

According to the invention, 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. Brief Description of the Figures

Figures 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. In Figure 1 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 F4 or F4-F3; F6 = stream with removed threonine or F7 - F8; F7 = one or more removal streams according to the invention for separating off threonine; and F8 = the threonine-depleted recycled stream; X = separating off, according to the invention, of cells; T = separating off, according to the invention, of threonine.

In Figure 2 the symbols denote the following:

Fl, F1+, F2, F2+, F3 , X and T as described in Figure 1.

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 .

As shown in Figure 1 and Figure 2, according to the invention, the product threonine is furthermore removed a) directly from the removal stream or streams (F7 in Figure 1) , 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 in Figure 2)) completely or partly (> 0 to = 100%) 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 (F8 or Fll) . If a complete or partial separating off of the biomass has taken place before the removal of the threonine (that is to say relates only to Figure 2), 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.

As Figure 1 and Figure 2 show, according to the invention 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%.

The term "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

3 hours . 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) . If 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. In many cases, however, 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. According to the invention, "free culture system" means a process without cell retention, that is to say with a free overflow. These different biomass concentrations in a free culture system depend on the microorganism employed and the process employed.

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. 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, Bioprozesstechnik: Einfϋhrung in die Bioverfahrenstechnik, 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,

Gustav Fischer Verlag, Jena, 1991) . 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+) . At R = i the cells are completely retained and growth tends towards zero, while R = 0 represents normal chemostatic operation (Horst Chmiel, Bioprozesstechnik: Einfϋhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart 1991) .

According to the invention, 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. According to the invention, by the cell retention 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. In the case of sequential separating off of the biomass, for example by sequential centrifugation steps or sequential sedimentation steps, 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. However, 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. In the culturing step b) described, 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.

The term 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 .

During the culturing step (a) , 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, or 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. These substances are present in concentrations of 0.003 to 3 g/kg. Finally, 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.

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. ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, potassium nitrate or potassium sodium nitrate. Alternatively, these and other components can also be fed in separately.

It has been found that 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. According to the invention, 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. Finally, 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.

If 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.

According to the invention, 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.

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.

After 10 to 72 hours, preferably 15 to 48 hours, or during or after the logarithmic growth phase, as described above 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. According to the invention, 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. β-D-Glucose is determined e.g. in a YSI 02700 Select glucose analyzer from Yellow Springs Instruments (Yellow Springs, Ohio, USA) .

If appropriate, 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.

In a process according to the invention, 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.

In a process according to the invention, 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. In a fermentation process like that according to the invention, the product is of course prepared in a certain yield and in a certain space/time yield (volumetric productivity) . In a process according to the invention, 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 40⁹C. 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. In the process according to the invention, 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 .

From the culture broth removed, the L-threonine can be isolated, collected or concentrated and optionally purified.

It is also possible to prepare a product from the culture broth removed (= fermentation broth) by removing the bacterium biomass contained in the culture broth completely (100%) or almost completely, i.e. more than or greater than (>) 90%, >95%, >97%, >99% and leaving the other constituents of the fermentation broth largely, i.e. to the extent of 30% - 100%, 40% - 100%, 50% - 100%, 60% - 100%, 70% - 100%, 80% - 100%, or 90% - 100%, preferably greater than or equal to (>) 50%, >60%, >70%, >80%, ≥90% or >95% or also completely (100%) in the product.

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%.

The process steps mentioned do not necessarily have to be carried out in the sequence stated here, but can optionally be combined in an industrially appropriate manner.

The analysis of 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. Of the genus Escherichia in particular the species Escherichia coli and of the genus

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. Such bacteria are typically resistant to the threonine analogue α-amino-β- 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 α-amino-β-hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to α-methylserine, resistance to diaminosuccinic acid, resistance to α-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 dipeptides, resistance to L-threonine, resistance to threonine raffinate, resistance to L-homoserine, resistance to L-lysine, resistance to L-methionine, resistance to L-glutamic acid, resistance to L-aspartate, resistance to L-leucine, resistance to L-phenylalanine, resistance to L-serine, resistance to L-cysteine, resistance to L-valine, sensitivity to fluoropyruvate, defective threonine dehydrogenase, optionally an ability for sucrose utilization, enhancement of the threonine operon, enhancement of ho oserine dehydrogenase I-aspartate kinase I, preferably of the feed back resistant form, enhancement of homoserine kinase, enhancement of threonine synthase, enhancement of aspartate kinase, optionally of the feed back resistant form, enhancement of aspartate semialdehyde dehydrogenase, enhancement of phosphoenol pyruvate carboxylase, optionally of the feed back resistant form, enhancement of phosphoenol pyruvate synthase, enhancement of transhydrogenase, enhancement of the RhtB gene product, enhancement of the RhtC gene product, enhancement of the YfiK gene product, enhancement of a pyruvate carboxylase, and attenuation of acetic acid formation.

Suitable L-threonine-producing strains of the genus

Escherichia, in particular of the species Escherichia coli, are, for example

- Escherichia coli KY10935 (Bioscience Biotechnology and Biochemistry 61 (11) =1877-1882 (1997),

- Escherichia coli BKIIM B-3996 (US-A-5, 175, 107) ,

- Escherichia coli kat 13 (WO 98/04715), and

- Escherichia coli KCCM-10132 (WO 00/09660⁾

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. These bacteria are described in PCT/EP02/02055.

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.

Claims

What is claimed is:

1. Process for the preparation of L-threonine using bacteria of the Enterobacteriaceae family which produce L-threonine, wherein a) the bacterium is inoculated and cultured in at least a first nutrient medium, h) at least a further nutrient medium or further nutrient media is/are then fed continuously to the culture in one (Fl) or several feed streams (F1+) , the nutrient medium or the 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 c) 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 FlO) , d) 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, e) the culture broth remaining after removal of the threonine (with (F8) or with depleted (Fll) biomass) is fed back to the culture vessel, and f) the concentration of the source of carbon during the culture is adjusted to not more than 30 g/1.

2. Process according to claim 1, wherein the culturing step (a) is carried out by the batch process.

3. Process according to claim 1, wherein the culturing step (a) is carried out by the fed batch process, at least one additional nutrient medium being employed.

4. Process according to claim 1, 2 or 3, wherein the product threonine is completely separated off directly from the removal stream or streams.

5. Process according to claim 1, 2 or 3, wherein the product threonine is partly separated off directly from the removal stream or streams .

6. Process according to claim 1 or 2 , wherein the further nutrient medium or the further nutrient media is (are) fed in after >0 to 20 hours, with respect to the start of the batch process .

7. Process according to claim 1 or 3 , wherein the further nutrient medium or the further nutrient media is (are) fed in after >0 to 80 hours, with respect to the start of the fed batch process .

8. Process according to claim 1 , 2 or 3 , wherein the product threonine is completely separated off after complete or partial separating off of cells from the removal stream or the removal streams.

9. Process according to claim 1, 2 or 3 , wherein the product threonine is partly separated off after complete or partial separating off of cells from the removal stream or the removal streams.

10. Process according to claim 1, 2 or 3 , wherein the L- threonine formed is purified.

11. Process according to claim 1, 2 or 3 , wherein, in step (b) , the biomass is first removed to the extent of at least 90% from the culture removed, and the water is then removed to the extent of at least 90%.

12. Process according to claim 1, wherein the source of carbon is one or more of the compounds chosen from the group consisting of sucrose, molasses from sugar beet or cane sugar, fructose, glucose, starch hydrolysate, cellulose hydrolysate, arabinose, maltose, xylose, acetic acid, ethanol and methanol.

13. Process according to claim 1, wherein the source of nitrogen is one or more organic nitrogen-containing substances or substance mixtures chosen from the group consisting of peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor, soya bean flour and urea and/or one or more of the inorganic compounds chosen from the group consisting of ammonia, ammonium- containing salts and salts of nitric acid.

14. Process according to claim 13, wherein the ammonium- containing salts and salts of nitric acid are ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, potassium nitrate and potassium sodium nitrate.

15. Process according to claim 1, wherein the source of phosphorus is phosphoric acid or alkali metal or alkaline earth metal salts thereof or polymers thereof or phytic acid.

16. Process according to claim 15, wherein the alkali metal salts of phopsphoric acid are potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts .

17. Process according to claim 1, wherein the speed of the removal stream (F2) or the removal streams (F2+) corresponds to 80% - 120%, 90% - 110% of the feed stream (Fl) or of the total of the feed streams (Fl+) .

18. Process according to claim 1, wherein the bacteria of the Enterobacteriaceae family are the species Escherichia coli.

19. Process according to claim 1, wherein the bacterium of the Enterobacteriaceae family contains at least one thrA gene or allele which codes for a threonine- insensitive aspartate kinase I - homoserine dehydrogenase I.

20. Process according to claim 1, wherein the bacterium of the Enterobacteriaceae family contains 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.

21. Process according to claim 1, wherein the feed stream (Fl) or the total of the feed streams (Fl+) is fed in at a rate corresponding to an average residence time of less than 30 hours, less than 25, less than 20 hours .

22. Process according to claim 1, wherein in the nutrient medium fed in or the nutrient media fed in 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 is established.

23. Process according to claim 1, wherein the culture broth removed is provided with oxygen or an oxygen- containing gas until the concentration of the source of carbon falls below 2 g/1; below 1 g/1; below 0.5 g/1.

24. Process according to claim 21, wherein the L-threonine formed is purified.

25. Process according to claim 21, wherein in step (b) the biomass is first removed to the extent of at least 90% from the culture removed, and the water is then removed to the extent of at least 90%.

26. Process according to claim 1, 2 or 3, wherein the concentration of the source of carbon during the culture is adjusted to not more than 20, 10 or 5 g/1.

27. Process according to claim 1, 2 or 3 , wherein the concentration of the source of carbon during the culture is adjusted to not more than 5 or 2 g/1.

28. Process according to claim 26 or 27, wherein the concentration of the source of carbon during the culture is adjusted to not more than 5 g/1.

29. Process according to claim 26 or 27, wherein the concentration of the source of carbon during the culture is adjusted to not more than 2 g/1.

30. Process according to claim 1, 2 or 3, wherein the yield of L-threonine formed, based on the source of carbon employed, is at least 31%.

31. Process according to claim 1, 2 or 3 , wherein the yield of L-threonine formed, based on the source of carbon employed, is at least 37%.

32. Process according to claim 1, 2 or 3 , wherein the yield of L-threonine formed, based on the source of carbon employed, is at least 42%.

33. Process according to claim 1, 2 or 3 , wherein the yield of L-threonine formed, based on the source of carbon employed, is at least 48%.

34. Process according to claim 1, 2 or 3 , wherein L- threonine is formed with a space/time yield of 5.0 to more than 8.0 g/1 per h.

35. Process according to claim 1, 2 or 3 , wherein L- threonine is formed with a space/time yield of 3.5 to more than 5.0 g/1 per h.

36. Process according to claim 1, 2 or 3 , wherein L- threonine is formed with a space/time yield of 2.5 to more than 3.5 g/1 per h.

37. Process according to claim 1, 2 or 3 , wherein L- threonine is formed with a space/time yield of 1.5 to more than 2.5 g/1 per h.

38. Process according to claim 1, wherein a fed batch process is used in the culturing step (a) , and wherein L-threonine is formed with a space/time yield of at least 2.5 to 5.0 g/1 per h.