ZA200301636B

ZA200301636B - Fermentation process for the preparation of L-threonine.

Info

Publication number: ZA200301636B
Application number: ZA200301636A
Authority: ZA
Inventors: Thomas Hermann; Mechthild Rieping
Original assignee: Degussa
Priority date: 2000-08-31
Filing date: 2003-02-27
Publication date: 2004-02-03
Also published as: DE10103778A1; KR20030033039A

Description

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Fermentation Process for the Preparation of L-~Threonine . Field of the Invention

The invention provides a new process for the fermentative preparation of L-threonine with Enterobacteriaceae.

Prior Art

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 or by

Okamoto et al. (Bioscience, Biotechnology, and Biochemistry 61 (11), 1877 - 1882, 1997), that threonine is prepared by fermentation in the batch process (batch) or feed process (fed batch).

Object of the Invention ‘The inventors had the object of providing new measures for } improved fermentative preparation of L-threonine.

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Summary of the Invention , The invention provides a fermentation process, which is characterized in that a) an L-threonine-producing microorganism of the

Enterobacteriaceae family is cultured by the feed process (fed batch) in a known manner, subsequently b) a portion of the fermentation broth is separated off, 1 to 90 vol.%, in particular 1 to 50 vol.%, preferably 1 to 25 vol.% and particularly preferably 5 to 50 vol.$ of the total volume of the fermentation broth remaining in the fermentation tank, subsequently c) the remaining fermentation broth is topped up with growth medium and, preferably after a growth phase, a further fermentation is carried out by the feed process (fed batch) mentioned, d) steps b) and c) are optionally carried out several times, and e) the L-threonine is isolated from the fermentation broths collected.

Detailed Description of the Invention

The microorganisms with which the process according to the invention can be carried out can prepare L-threonine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, or from glycerol and ethanol, the preparation from glucose, sucrose or molasses being preferred. They are representatives of Enterobacteriaceae, in particular of the ) genera Escherichia, Serratia and Providencia. Of the genus

Escherichia the species Escherichia coli and of the genus

Serratia the species Serratia marcescens are to be mentioned in particular.

i WO 02/18543 PCT/EP01/08603

Suitable L-threonine-producing strains of the genus

Escherichia, in particular of the species Escherichia coli, : are, for example . Escherichia coli TF427

Escherichia coli H-4225

Escherichia coli H-4226

Escherichia coli H-4257

Escherichia coli H-4258

Escherichia coli H-4435

Escherichia coli H-4436

Escherichia coli H-4578

Escherichia coli H-7256

Escherichia coli H-7263

Escherichia coli H-7293

Escherichia coli H-7294

Escherichia coli H-7700 ’

Escherichia coli H-7729

Escherichia coli H-8309 : Escherichia coli H-8311

Escherichia coli H-9244

Escherichia coli KY10935

Escherichia coli EL1003

Escherichia coli VNIIgenetika MG-442

Escherichia coli VNIIgenetika VL334/pYN7

Escherichia coli VNIIgenetika M1

Escherichia coli VNIIgenetika 472T23

Escherichia coli VNIIgenetika TDH-6

Escherichia coli BKIIM B-3996

Escherichia coli BKIIM B-5318

Escherichia coli B-3996-C43 . Escherichia coli B-3996-C80

Escherichia coli B-3996/pTWV-pps , Escherichia coli B-3996 (pMW: : THY)

Escherichia coli B-3996/pBP5

Escherichia coli Ferm BP-3756

Escherichia coli Ferm BP-4072 i WO 02/18543 PCT/EP01/08603 v 4

Escherichia coli Ferm BP-1411

Escherichia coli kat 13 ~ Escherichia coli KCCM-10132

Escherichia coli KCCM-10133. 5S Suitable L-threonine-producing strains of the genus

Serratia, in particular of the species Serratia marcescens, are, for example

Serratia marcescens HNr21l

Serratia marcescens TLrl56

Serratia marcescens T2000

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 a-amino-f-hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to a- methylserine, resistance to diaminosuccinic acid, resistance to a-aminobutyric acid, resistance to borrelidin, 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 compensatable need for L-isoleucine, a need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, 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, ’ 30 sensitivity to fluoropyruvate, defective threonine dehydrogenase, optionally an ability for sucrose : utilization, enhancement of the threonine operon, enhancement of homoserine dehydrogenase I-aspartate kinase

I, preferably of the feed back resistant form, enhancement of homoserine kinase, enhancement of threonine synthase,

v 5 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, i 5 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.

Thus, for example, the strain 472T23 (US-A-5,631,157) has, inter alia, an enhanced, "feed back" resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to at least 5 g/1 L- threonine and the ability to utilize sucrose as a source of carbon.

Thus, for example, the strain B-3996 (US-A-5,175,107) has, inter alia, an enhanced, "feed back" resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, an attenuated threonine dehydrogenase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.

Thus, for example, the strain kat-13 (US-A-5,939,307) has, inter alia, an enhanced, "feed back" resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated : threonine dehydrogenase, resistance to borrelidin and the ability to utilize sucrose as a source of carbon.

Thus, for example, the strain KCCM-10132 (WO 00/09660) has a resistance to a-methylserine, a resistance to . diaminosuccinic acid, sensitivity to fluoropyruvate, a resistance to L-glutamic acid and a resistance to at least 7% L-threonine. The strain is also in need of the amino acids L-methionine and L-isoleucine.

The term "enhancement" in this connection describes the increase in the intracellular activity of one or more ) enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of ' 5 copies of the gene or allele or of the genes or alleles, using a potent promoter or using a gene or allele which codes for a corresponding enzyme having a high activity, and optionally combining these measures.

By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on the starting microorganism.

The term "attenuation" in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele which codes for a corresponding enzyme with a low activity or inactivates the corresponding gene or enzyme (protein), and optionally combining these measures.

By attenuation measures, the activity or concentration of the corresponding protein is in general reduced to 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein.

According to the invention, the system output of a fermentation unit producing L-threonine is increased by a procedure in which after a first fermentation step a . portion of the fermentation broth obtained in this way remains in the production fermenter and serves as the inoculum for one or more further fermentation steps (batches).

According to the invention, 1 to 90 vol.%, preferably 1 to 50 vol.%, preferentially 1 to 25 vol.%, 1 to 20 vol.%, 1 to ’ 15 vol.% or 1 to 10 vol.%, and particularly preferably 5 to 20 vol.%, 5 to 15 vol.% or 1 to 10 vol.% of the total ‘ 5 volume of the fermentation broth remains in the fermentation tank.

The broth remaining in the fermentation tank is preferably topped up with a growth medium. After optionally > 0 to not more than 10 hours, preferably after 1 to 10 hours, preferentially 2 to 10 hours and particularly preferably 3 to 7 hours a production medium is fed in. Alternatively, the components of this medium can also be fed in separately. After 20 to 72 hours, preferably 20 to 48 hours, the batch is ended and a portion of the fermentation broth, as described above, is separated off. A new fermentation stage is then optionally started with the remainder. The process can be repeated at least once, preferably approx. 2 to 6 times, depending on the stability of the strain used. Repetitions of approx. 2 to 8 times or 200 2 to 10 times or 2 to 4 times are also possible.

Appropriately stable strains which do not lose their production properties in the course of the process are particularly suitable for the process described.

The growth medium typically comprises sugars, such as e.g. glucose, starch hydrolysate, sucrose or molasses, as the source of carbon. 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 ammonium sulfate, ammonium ) 30 chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. ’ The sources of nitrogen can be used individually or as a mixture. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium- containing salts can be used as the source of phosphorus.

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The culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate, manganese sulfate : or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids (e.g. ‘ 5 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.

In general, the production medium comprises only one sugar, such as e.g. sucrose or glucose, and optionally an inorganic source of nitrogen, such as e.g. ammonium sulfate. Alternatively, these and other components can also be fed in separately.

During the growth or production phase, the temperature is established in a range from 29°C to 42°C, preferably 33°C to 40°C. Temperatures in a range from 27°C to 39°C are also possible. The fermentation 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. 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 distinguished with respect to conventional processes above all by an increased space/time yield or productivity.

The present invention is explained in more detail in the following with the aid of embodiment examples.

The isolation of plasmid DNA from Escherichia coli and all techniques of restriction, Klenow and alkaline phosphatase treatment were carried out by the method of Sambrook et al. (Molecular Cloning. A laboratory manual (1989) Cold Spring

Harbor Laboratory Press). Unless described otherwise, the transformation of Escherichia coli was carried out by the method of Chung et al. (Proceedings of the National Academy ) of Sciences of the United States of America USA (1989) 86: 2172-2175). 5S Example 1

Preparation of the Escherichia coli K-12 strain DM1265

A plasmid-free variant of the E. coli strain 472T23 was obtained from the American Type Culture Collection (Manasas, VA., USA) as ATCC98082. The strain ATCC98082 is described in the patent specification US-A-5,631,157. The

E. coli strain VL334/pYN7 was obtained from the Russian

National Collection of Industrial Microorganisms (VKPM,

Moscow, Russia) as CMIM B-1684. The strain CMIM B-1684 is described in the patent specification US-A-4,278,765.

The plasmid pYN7 was isolated from the strain VL334/pYN7. A

DNA fragment 6.25 kbp long which carries the thrABC operon was isolated from plasmid pYN7 by preparative agarose gel electrophoresis with the aid of the restriction enzymes

HindIII and BamHI.

The plasmid pBR322 (Bolivar et al., Gene 2, 95-113 (1977)) was obtained from Pharmacia Biotech (Uppsala, Sweden) and treated with the restriction enzymes HindIII and BamHI. The

DNA fragment 4.3 kbp long was isolated by preparative agarose gel electrophoresis. The two DNA fragments were mixed, treated with T4 DNA ligase, and the strain DHS5a was transformed with the ligation mixture. After selection on ampicillin-containing (50 pg/mL) LB agar, transformants which contained a plasmid which corresponded in its structure to the plasmid pYN7 were obtained.

The plasmid was isolated from a transformant, cleaved partly with the enzyme EcoRI and completely with the enzyme

HindIII and ligated with the parB gene region isolated. For this, the plasmid pKG1022 (Gerdes, Biotechnology (1988)

©:1402-1405) was cleaved with the enzymes EcoRI and

HindIII, the cleavage batch was separated in 1% agarose gel ) and the parB fragment 629 bp in size was isolated with the aid of the QIAquick Gel Extraction Kit (QIAGEN, Hilden,

Germany). The ligation mixture was employed for transformation of strain ATCC98082. Selection of plasmid- carrying cells was carried out on LB agar (Lennox, Virology 1:190 (1955)), to which 50 pg/ml ampicillin had been added.

Successful cloning of the parB gene region could be detected after isolation of the plasmid DNA, control cleavage with EcoRI and HindIII and analysis of the cleavage batch by agarose gel electrophoresis. The plasmid was designated pYN7parB.

A transformant of the type ATCC98082/pYN7parB has been designated DM1265 and deposited in the form of a pure culture on 30th April 1999 at the Deutsche Sammlung fur

Mikroorganismen und Zellkulturen (German Collection of

Microorganisms and Cell Cultures = DSM, Braunschweig,

Germany) as DSM12790 in accordance with the Budapest

Treaty.

The strain DM1265 has, inter alia, an enhanced, "feed back" resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.

This strain is distinguished by a high stability, in particular segregation stability.

Comparative Example A

Preparation of L-threonine with the aid of the Escherichia coli K-12 strain DM1265 by conventional fermentation

An individual colony of the strain DM1265 was transinoculated on to minimal medium with the following composition: 3.5 g/1 Na HPO4*2H,0, 1.5 g/1 KH,PO4, 1 g/1

NH,C1l, 0.1 g/1 MgSO4*7H,0, 2 g/l sucrose, 20 g/l agar, : 50 mg/l ampicillin. The culture was incubated at 37°C for ’ approx. 5 days. 10 ml preculture medium with the following composition: 2 g/l yeast extract, 10 g/l (NH4)2S04, 1 g/1

KHPO4, 0.5 g/1 MgSO0,4*7H;0, 15 g/l CaCO;, 20 g/l sucrose, 50 mg/l ampicillin were inoculated with an inoculating loop and incubated for 16 (h) at 37°C and 180 rpm on an ESR incubator from Kihner AG (Birsfelden, Switzerland).

A volume of 1 ml of this first preculture was inoculated into 1402 g of the nutrient medium Al1-144. The culturing fermentation was carried out in 2 1 stirred reactor fermenters from B. Braun (BBI, Germany, Melsungen, Biostat

MD model). The nutrient medium Al1-144 contained the constituents listed in Table 1. This second preculture was cultured for 22.5 h at a temperature of 37°C, a volume- specific gassing of 0.71 vvm (volume per volume per minute), an oxygen partial pressure of 10% of the atmospheric saturation and a pH of pH 7.0 until an optical density (OD) (660 nm) of 16.3 was reached.

For inoculation of 1233 g of the growth medium M1-463, which was contained in 2 1 stirred reactor fermenters from

B. Braun (BBI, Germany, Melsungen, Biostat MD model), 157.6 g of the second preculture in nutrient medium Al1-144 were added. The growth medium M1-463 contained the constituents listed in Table 2. The culture was cultured at a temperature of 37°C, an aeration of 1 l/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until a residual sugar concentration of approx. 3 g/l was reached.

The broth obtained in this way was subsequently cultured for a further 30 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 33.4 was reached.

During this time, 450 g of a production medium comprising a

) sucrose solution with a concentration of 650 g/l was fed in continuously. :

The optical density (OD) was then determined with a- digital photometer of the LP1W type from Dr. Bruno Lange GmbH (Berlin, Germany) at a measurement wavelength of 660 nm and the concentration of L-threonine formed was determined by ion exchange chromatography and post-column reaction with ninhydrin detection with an amino acid analyzer from

Eppendorf~-BioTronik (Hamburg, Germany),

After 39.5 h, an L-threonine concentration of 69.6 g/l was found in the final fermentation sample. The space/time yield in this experiment was thus 1.76 g/1 - h.

Table 1

Composition of nutrient medium Al-144

EE EC

Table 2 : Composition of growth medium M1-463

Yeast extract 1.87 g

MgSQ4 - THO 0.38 ¢

Example 2 :

Preparation of L-threonine with the aid of the strain

DM1265 with 2 subsequent feed processes and 10% inoculum in each case

An individual colony of the strain DM1265 was transinoculated on to minimal medium with the following . 10 composition: 3.5 g/1 Na,HPO,*2H,0, 1.5 g/1 KH;PO4, 1 g/1

NH4.C1l, 0.1 g/1 MgSO,*7H,0, 2 g/l glucose, 20 g/l agar, : 50 mg/l ampicillin. The culture was incubated at 37°C for approx. 5 days. 10 ml preculture medium with the following composition: 2 g/l1 yeast extract, 10 g/l (NH4),SO,, 1 g/1

KHyPO4, 0.5 g/l MgSO,*7H,0, 15 g/1 CaCOs, 20 g/l glucose,

50 mg/l ampicillin were inoculated with an inoculating loop and incubated for 16 h at 37°C and 180 rpm on an ESR incubator from Kihner AG (Birsfelden, Switzerland). . A volume of 1 ml of this first preculture was inoculated into 1402 g of the nutrient medium Al1-144. The culturing fermentation was carried out in 2 1 stirred reactor fermenters from B. Braun (BBI, Germany, Melsungen, Biostat

MD model). The nutrient medium Al-144 contained the constituents listed in Table 1. This second preculture was cultured for 22.5 h at a temperature of 37°C, a volume- specific gassing of 0.71 vvm, an oxygen partial pressure of 10% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 16.3 was reached.

B. Braun (BBI, Germany, Melsungen, Biostat MD model), 157.6 g of the second preculture in nutrient medium Al-144 were added. The growth medium M1-463 contained the constituents listed in Table 2. The culture was cultured as described in Comparative Example A at a temperature of 37°C, an aeration of 1 l/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until a residual sugar concentration of approx. 3 g/l was reached after 9.5 h. The fermentation broth obtained in this way was then cultured for a further 30 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 33.4 was reached.

During this time, 450 g of a production medium comprising a sucrose solution with a concentration of 650 g/l was fed in continuously. After the feed solution had been consumed and the residual sugar in the fermentation broth of this first run had been consumed, 90% of the fermentation broth (1656 g) of the fermenter contents was removed by pumping off.

The remaining 10% of the volume (184 g) was topped up with 1200 g of the growth medium M1-474 and the fermentation was ’ started again. The growth medium M1-474 contained the constituents listed in Table 3. The culture of this second ) 5 run was cultured as described in Comparative Example A at a temperature of 37°C, an aeration of 1 l/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until a residual sugar concentration of approx. 3 g/l was reached after 5 h. The broth was then cultured for a further 31.25 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 35.7 was reached. During this time, 450 g of a production medium comprising a sucrose solution with a concentration of 650 g/l was fed in continuously. After the feed solution had been consumed and the residual sugar in the fermentation broth had been consumed, 90% of the fermentation broth (1656 g) was removed from the fermenter by pumping off.

The remaining 10% of the total amount (184 g) was topped up with 1200 g of the growth medium M1-474 and the fermentation was started again. The growth medium M1-474 contained the constituents listed in Table 3. The culture of this third run was cultured as described in Comparative

Example A at a temperature of 37°C, an aeration of 1 1/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until a residual sugar concentration of approx. 3 g/l was reached after 5.25 h. The culture was then cultured for a further 30.5 h at a temperature of 37°C, an oxygen partial pressure . of 20% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 32.5 was reached. During this time, 450 g of a production medium comprising a sucrose solution with a concentration of 650 g/1 was fed in.

At the end of each fermentation the OD and the concentration of L-threonine formed were determined as in

Comparative Example A. The results of the particular runs are shown in Table 4.

The term "space/time" yield here describes the volumetric productivity, i.e. the quotient of the concentration of L- threonine at the end of the fermentation and the fermentation time.

Table 3

Composition of growth medium M1-474

Yeast extract 1.68 g

MgSO, - 7H,0 0.38 g

Table 4

Results from Example 2

Run | Time [h] | L-Threonine OD Space/time (g/1] (660 yield nm) | [g/1 : hI

HEE EEE

Example 3

Preparation of L-threonine with the aid of the strain

DM1265 with 4 subsequent feed processes and 25% inoculation in each case

An individual colony of the strain DM1265 was transinoculated on to minimal medium with the following composition: 3.5 g/l Na,HPO4*2H,0, 1.5 g/l KH;PO4, 1 g/1

NH4C1l, 0.1 g/1 MgSO.*7H;0, 2 g/l glucose, 20 g/l agar, 50 mg/l ampicillin. The culture was incubated at 37°C for approx. 5 days. 10 ml preculture medium with the following composition: 2 g/l yeast extract, 10 g/1 (NH4).SO4, 1 g/l.

KHzPO4, 0.5 g/1 MgSO4*7H,0, 15 g/l CaCOs, 20 g/l glucose, 50 mg/l ampicillin were inoculated with an inoculating loop and incubated for 16 h at 37°C and 180 rpm on an ESR incubator from Kihner AG (Birsfelden, Switzerland).

A volume of 1 ml of this first preculture was inoculated into 1402 g of the nutrient medium Al-144. The culturing fermentation was carried out in 2 1 stirred reactor fermenters from B. Braun (BBI, Germany, Melsungen, Biostat

MD model). The nutrient medium Al-144 contained the constituents listed in Table 1. This second preculture was cultured for 22.5 h at a temperature of 37°C, a volume- specific gassing of 0.71 vvm, an oxygen partial pressure of il 10% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 16.3 was reached. 5S For inoculation of 1233 g of the growth medium M1-463, which was contained in 2 1 stirred reactor fermenters from

B. Braun (BBI, Germany, Melsungen, Biostat MD model), 157.6 g of the second preculture in nutrient medium Al-144 were added. The growth medium M1-463 contained the constituents listed in Table 2. The culture was cultured as described in Comparative Example A at a temperature of 37°C, an aeration of 1 1/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until a residual sugar concentration of approx. 3 g/l was reached after 9.5 h. The fermentation broth obtained in this way was then cultured for a further 32 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 7.0 until an OD (660 nm) of 35.8 was reached.

During this time, 450 g of a production medium comprising a sucrose solution with a concentration of 650 g/l was fed in continuously. After the feed solution had been consumed and the residual sugar in the fermentation broth of this first run had been consumed, 75% of the fermentation broth of the fermenter contents was removed by pumping off. The first fermentation (first run) was ended after 41.5 h and reached a titre of 67.1 g/l threonine.

The remaining 25% of the total amount (453 g) was topped up with 700 g of the growth medium M1-527 and the fermentation was started again. The growth medium M1-527 contained the constituents listed in Table 5. The culture of this second run was cultured as described in Comparative Example A at a temperature of 37°C, an aeration of 1 1/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until a

~ residual sugar concentration of approx. 3 g/l was reached.

The culture was then cultured for a further 30 h at a ) temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 7.0 until an OD ) 5 (660 nm) of 36.2 was reached. During this time, 450 g of a production medium comprising a sucrose solution with a concentration of 650 g/l was fed in as in the first run.

The draining off of the fermentation broth to 25% and the topping up of the fermenter with M1-527 was repeated a total of four times.

Comparative Example A.

Table 6 shows the results of the particular runs. The term "Total L-threonine formed" relates to the L-threonine effectively formed or produced during the fermentation run.

To calculate the total L-threonine formed, the amount of L- threonine introduced by the inoculum is subtracted from the amount of L-threonine present in the fermentation tank at the end of the run. The term "Productivity" designates the quotient of the total L-threonine formed per fermentation run and the fermentation time per run.

Table 5 : Composition of growth medium M1-527

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Table 6 . Results from Example 3

Run Time [h] | L-Threonine OD Total L- Product- [g/1] (660 Threonine ivity nm) formed [g/h] lg]

IE CE

Comparative Example B

S Preparation of L-threonine with the aid of the Escherichia coli K-12 strain kat-13 by conventional fermentation

The L-threonine-producing E. coli strain kat-13 is described in US-A-5,939,307 and deposited at the

Agriculture Research Service Patent Culture Collection (Peoria, Illinois, USA) as NRRL B-21593.

The strain kat-13 has, inter alia, an enhanced, "feed back" resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine dehydrogenase, resistance to borrelidin and the ability to utilize sucrose as a source of carbon.

An individual colony of the strain kat-13 was transinoculated on to minimal medium with the following composition: 3.5 g/1 NapHPO4*2H,0, 1.5 g/l KHzPO,, 1 g/1

NH4Cl, 0.1 g/1 MgS0,*7H,0, 2 g/l glucose, 20 g/l agar. The culture was incubated at 37°C for approx. 5 days. 10 ml preculture medium with the following composition: 2 g/l

Cl yeast extract, 10 g/l (NH4)2S04, 1 g/l KHPO,, 0.5 g/1

MgSO4*7H,0, 15 g/l CaCOs, 20 g/1 glucose, were inoculated with an inoculating loop and incubated for 16 h at 37°C and 180 rpm on an ESR incubator from Kiihner AG (Birsfelden,

Switzerland).

A volume of 0.45 ml of this first preculture was inoculated into 1500 g of the nutrient medium Al1-158. The culturing fermentation was carried out in 2 1 stirred reactor fermenters from B. Braun (BBI, Germany, Melsungen, Biostat

MD model). The nutrient medium A1-158 contained the constituents listed in Table 7. This second preculture was cultured for 19.75 h at a volume-specific gassing of 1.16 vvm, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 6.9 until all the glucose had been consumed. The fermentation was started at a temperature of 39°C, and after a fermentation time of 18 h the temperature was lowered to 37°C.

For inoculation of 725 g of the growth medium M1-530, which was contained in 2 1 stirred reactor fermenters from B.

Braun (BBI, Germany, Melsungen, Biostat MD model), 110 g of the second preculture in nutrient medium Al1-158 were added.

The growth medium M1-530 contained the constituents listed in Table 8. The culture was cultured at a temperature of 37°C, an aeration of 1.3 1/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until all the glucose initially introduced had been consumed after 8 h. The fermentation broth obtained in this way was then cultured for a further 57 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation, an aeration of 1.5 1/min and a pH of pH 7.0. During this time, 1000 g of a production medium comprising a glucose-+H,0

° 23 solution with a concentration of 550 g/1 was fed in continuously.

The OD and the concentration of L-threonine formed were . then determined as in Comparative Example A.

S After 65 h, an L-threonine concentration of 101.3 g/l was found in the final fermentation sample. The space/time yield in this experiment was thus 1.56 g/l - h.

Table 7

Composition of nutrient medium Al-158

Table 8 : : Composition of growth medium M1-530

Example 4

Preparation of L-threonine with the aid of the strain kat- 13 with a subsequent feed process and 10% inoculation

An individual colony of the strain kat-13 was transinoculated on to minimal medium with the following composition: 3.5 g/l Na;HPO4*2H,0, 1.5 g/l KH;PO,, 1 g/l : 10 NH, Cl, 0.1 g/l MgSOs*7H20, 2 g/1 glucose, 20 g/l agar. The culture was incubated at 37°C for approx. 5 days. 10 ml preculture medium with the following composition: 2 g/1 yeast extract, 10 g/l (NH4)2S0,, 1 g/1 KH,PO4, 0.5 g/1

MgS04*7H,0, 15 g/1 CaCOsz, 20 g/l glucose, were inoculated with an inoculating loop and incubated for 16 h at 37°C and

180 rpm on an ESR incubator from Kiihner AG (Birsfelden,

Switzerland).

A volume of 0.45 ml of this first preculture was inoculated . into 1500 g of the nutrient medium A1-158. The culturing fermentation was carried out in 2 1 stirred reactor fermenters from B. Braun (BBI, Germany, Melsungen, Biostat

Braun (BBI, Germany, Melsungen, Biostat MD model), 110 g of the second preculture in nutrient medium A1-158 were added.

The growth medium M1-530 contained the constituents listed in Table 8. The culture was cultured at a temperature of 37°C, an aeration of 1.3 1/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation until all the glucose initially introduced had been consumed after 8 h. The fermentation broth obtained in this way was then cultured for a further 57 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation, an aeration of 1.5 1/min and a pH of pH 7.0 until an OD (660 nm) of 46.4 was reached. During this time, 1000 g of a production medium comprising a glucose-H,O solution with a concentration of 550 g/1 was fed in continuously. After the feed solution had been consumed and the residual sugar in the fermentation broth of this first run had been consumed, 90% of the fermentation broth (1651 g) of the fermenter contents was removed by pumping off.

The remaining 10% of the volume (184 g) was topped up with 650 g of the growth medium M1-531 and the fermentation was started again. The growth medium M1-531 contained the constituents listed in Table 9. The culture was cultured at a temperature of 37°C, an aeration of 1.5 1/min, a minimum stirring of 800 rpm and a pH of 7.0 and an oxygen partial pressure of 20% of the atmospheric saturation. During this _ time, 1000 g of a production medium comprising a glucose-H,0 solution with a concentration of 550 g/1 was fed in continuously.

At the end of each fermentation the OD and the : concentration of L-threonine formed were determined as in

Comparative Example A.

The results of the two runs are shown in Table 10. The term "Total L-threonine formed" relates to the L-threonine effectively formed or produced during the fermentation run.

Table 9 : Composition of growth medium M1-531

Table 10 5S Results of Example 4

Run | Time [h] | L-Threonine OD total L- Product- {g/1] (660 Threonine ivity nm) formed [g/h]

CTE EEE ree [oro Jue] ae | ose 2 [ee | ene [eo] wre | oem

’ | 28

Comparative Example C ~ Preparation of L-threonine with the aid of the Escherichia coli K-12 strain B-3996 by conventional fermentation

The L-threonine-producing E. coli strain B-3996 is described in US-A-5,175,107 and deposited at the Russian

National Collection for Industrial Microorganisms (VKPM,

Moscow, Russia).

The strain B-3996 has, inter alia, an enhanced, "feed back" resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, an attenuated threonine dehydrogenase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.

An individual colony of the strain B-3996 was transinoculated on to minimal medium with the following composition: 3.5 g/l NayHPO,*2H,0, 1.5 g/l KH,PO4, 1 g/1

NHsCl, 0.1 g/1 MgSO4*7H;0, 2 g/l sucrose, 20 g/l agar, pg/ml streptomycin. The culture was incubated at 37°C for approx. 5 days. 10 ml preculture medium with the following composition: 2 g/l yeast extract, 10 g/l 20 (NH4)2S04, 1 g/1 KHpPO,, 0.5 g/1 MgSO,*7H,0, 15 g/l CaCOs, 20 g/1 sucrose, 20 pg/ml streptomycin were inoculated with an inoculating loop and incubated for 16 h at 37°C and 180 rpm on an ESR incubator from Kiihner AG (Birsfelden,

Switzerland).

A volume of 20 ml of this first preculture was inoculated into 1000 g of the nutrient medium Al1-160. The culturing fermentation was carried out in 2 1 stirred reactor fermenters from B. Braun (BBI, Germany, Melsungen, Biostat

MD model). The nutrient medium A1-160 contained the constituents listed in Table 11. This second preculture was cultured for 14 h at a temperature of 37°C, a volume- specific gassing of 1.00 vvm, an oxygen partial pressure of

10% of the atmospheric saturation and a pH of pH 6.9 until all the sucrose had been consumed.

For inoculation of 1000 g of the growth medium M1-546, . which was contained in 2 1 stirred reactor fermenters from 5S B. Braun (BBI, Germany, Melsungen, Biostat MD model), 100 g of the second preculture in nutrient medium Al1-160 were added. The growth medium M1-546 contained the constituents listed in Table 12. The culture was cultured at a temperature of 37°C, an aeration of 1.0 l/min, a minimum stirring of 800 rpm and a pH of 6.9 and an oxygen partial pressure of 20% of the atmospheric saturation until all the sucrose initially introduced had been consumed after 7 h.

The fermentation broth obtained in this way was then cultured for a further 29 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation, an aeration of 1.0 1/min and a pH of pH 6.9.

During this time, a production medium comprising a sucrose solution with a concentration of 600 g/kg was fed in such that the sucrose concentration was always above 0.5 g/l.

The optical density (OD) was then determined with a digital photometer of the LPIW type from Dr. Bruno Lange GmbH (Berlin, Germany) at a measurement wavelength of 660 nm and the concentration of L-threonine formed was determined by ion exchange chromatography and post-column reaction with ninhydrin detection with an amino acid analyzer from

Eppendorf-BioTronik (Hamburg, Germany), 63.8 g L-threonine were produced in the fermentation in 36 h. The productivity in this experiment was thus 1.77 g/h.

co 30

Table 11 : . Composition of nutrient medium —

EE RC

EE IC

EE ECR i 31

Table 12 } Composition of growth medium M1-546

Ea ICR

Example 5

Preparation according to the invention of L-threonine with the aid of the Escherichia coli K-12 strain B-3996 with 5 subsequent feed processes and 10% inoculation in each case

For inoculation of 1000 g of the growth medium M1-546, which was contained in 2 1 stirred reactor fermenters from

B. Braun (BBI, Germany, Melsungen, Biostat MD model), 100 g of the second preculture in nutrient medium A1l-160 were : added, as described in Example 7. The growth medium M1-546 contained the constituents listed in Table 12. The culture : was cultured at a temperature of 37°C, an aeration of 1.0 1/min, a minimum stirring of 800 rpm and a pH of 6.9 and an oxygen partial pressure of 20% of the atmospheric . saturation until all the sucrose initially introduced had been consumed after 7 h. The fermentation brotH obtained in this way was then cultured for a further 29 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation, an aeration of 1.0 l/min and a ) 5 pH of pH 6.9. During this time, 411.8 g of a production medium comprising a sucrose solution with a concentration of 600 g/kg was fed in continuously.

The fermentation broth was then drained off to 10% of the total amount. The remaining 10% of the total amount was topped up with growth medium M1-546 to the starting weight of 1100 g and the fermentation was started again. The growth medium M1-546 contained the constituents listed in

Table 13. The culture of this third run was cultured as described in Example 7 at a temperature of 37°C, an aeration of 1 1/min, a minimum stirring of 800 rpm and a pH of 6.9 and an oxygen partial pressure of 20% of the atmospheric saturation until all the sucrose initially introduced had been consumed after 8 h. The culture was then cultured for a further 28 h at a temperature of 37°C, an oxygen partial pressure of 20% of the atmospheric saturation and a pH of pH 6.9. During this time, a . production medium comprising a sucrose solution with a concentration of 600 g/kg was fed in such that the sucrose concentration in the fermenter was always above 0.5 g/l.

After a total of 36 h, the fermentation run was ended. The draining off of the fermentation broth to 10% and the topping up of the fermenter with M1-546 was repeated a total of five times.

Comparative Example A. ‘ Table 13 shows the results of the particular runs. The term "Total L-threonine formed" relates to the L-threonine effectively formed or produced during the fermentation run.

To calculate the total L-threonine formed, the amount of L-

threonine introduced by the inoculum is subtracted from the amount of L-threonine present in the fermentation tank at . the end of the run. The term "Productivity" designates the quotient of the total L-threonine formed per fermentation

S 5 run and the fermentation time per run.

Table 13:

Results of Example 5

Run Time [h] | L-Threonine ~ OD Total L- Product- : yield (660 | Threonine ivity (g/g nm) formed [g/h] sucrose] (g]

IE I 2 I 1 I IN KC) a

IE EC NC Ca CO NC

L IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession number given by the

INTERNATIONAL DEPOSITARY AUTHORITY:

DM1265

DSM 12790

I. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under 1. above was accompanied by: (3) a scientific description - CC ’ (X) a proposed taxonomic designation (Mark with a cross where applicable).

This International Depositary Authority accepts the microorganism identified under 1. above, which was received by ton 1999-04-29 (Date of the original deposit)’.

IV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under | above was received by this Intemational Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by iton (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY h Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the

MIKROORGANISMEN UND ZELLKULTUREN GmbH Intemational Depositary Authority or of authorized official(s):

Address: Mascheroder Weg 1b -

D-38124 Braunschweig ) Co Ae

Date: 1999-04-30 ' Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary suthority was acquired.

Form DSMZ-BP/4 (soc page) 0196

1. DEPOSITOR 11. IDENTIFICATION OF THE MICROORGANISM

Name: Degussa-Huls AG . Accession number given by the

Kantstr. 2 INTERNATIONAL DEPOSITARY AUTHORITY:

Address: DSM 12790 33790 Halle i.W.

Date of the deposit or the transfer’: 1899-04-29

Jil. VIABILITY STATEMENT

The viability of the microorganism identified under 11 above was testedon 1999-04-29 2.

On that date, the said microorganism was (X)* viable : ( ) no longer viable

IV. CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED'

V. INTERNATIONAL DEPOSITARY AUTHORITY ’

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the

MIKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized official(s):

Address: Mascheroder Weg 1b 4 (/ - 4 3 D-38124 Braunschweig Ps yr

Dae: 1999-04-30 ! Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer). h In the cases referred to in Rule 10.2(2) (ii) and (iii), refer to the most recent viability test. > Mark with a cross the applicable box. ‘ Fill in if the information has been requested and if the results of the test were negative,

Form DSMZ-BP/ (sole page) 0196

Claims

What is claimed is: . 1. Process for the fermentative preparation of L- threonine, wherein AS a) an L-threonine-producing microorganism of the Enterobacteriaceae family is cultured by the feed process (fed batch), subsequently b) a portion of the fermentation broth is separated off, 1 to 90 vol.%, in particular 1 to 50 vol.$, preferably 1 to 25 vol.% of the total volume of the fermentation broth remaining in the fermentation tank, subsequently c) the remaining fermentation broth is topped up with growth medium and, preferably after a growth phase, a further fermentation is carried out in accordance with a), d) steps b) and c) are optionally carried out several times, and e) the L-threonine is isolated from the fermentation broths collected.
2. Process according to claim 1, wherein steps b) and c) are carried out two to six times and the L-threonine is isolated from the fermentation broths collected.
3. Process according to claims 1 and 2, wherein . microorganisms of the species Escherichia coli are employed. :
’ 4. L-Threonine-producing and -secreting microorganisms of the Entercbacteriaceae family which have an enhanced ‘ resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to at least 5 g/l threonine and the ability to utilize he 37 sucrose as a source of carbon, and the parB gene region.
5. Transformants according to claim 4, deposited under © number DSM 12790 at the DSMZ [German Collection of Microorganisms and Cell Cultures], Braunschweig.
6. Process according to claims 1 and 2, wherein L- threonine-producing and -secreting microorganisms of the Enterobacteriaceae family which have one or more of the features chosen from the group consisting of: an -10 enhanced, feed back resistant aspartate kinase I- homoserine dehydrogenase I, an attenuated threonine deaminase, an attenuated threonine dehydrogenase, a resistance to at least 5 g/l threonine, a resistance to borrelidin, a resistance to a-methylserine, a resistance to diaminosuccinic acid, a sensitivity to fluoropyruvate, a resistance to L-glutamic acid, a need for L-methionine, and the ability to utilize sucrose as a source of carbon are used.
7. Process according to claim 6, wherein L-threonine- producing and ~secreting microorganisms of the Enterobacteriaceae family which have an enhanced resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to : at least 5 g/l threonine and the ability to utilize sucrose as a source of carbon are used.
8. Process according to claims 1 and 2, wherein L- threonine-producing and -secreting microorganisms of the Enterobacteriaceae family which have one or more of ; the features chosen from the group consisting of: an enhanced, feed back resistant aspartate kinase I-

. homoserine dehydrogenase I, an attenuated threonine deaminase, an attenuated threonine dehydrogenase, a resistance to at least 5 g/l threonine and the ability to utilize sucrose as a source of carbon are used.
9. Process according to claims 1 and 2, wherein L- threonine-producing and -secreting microorganisms of ’ the Enterobacteriaceae family which have one or more of the features chosen from the group consisting of: an v 5 enhanced, feed back resistant aspartate kinase I- homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to borrelidin and the ability to utilize sucrose as a source of carbon are used.
10. Process according to claims 1 and 2, wherein L- threonine-producing and -secreting microorganisms of the Enterobacteriaceae family which have one or more of the features chosen from the group consisting of: a resistance to a-methylserine, a resistance to diaminosuccinic acid, a sensitivity to fluoropyruvate, a resistance to L-glutamic acid and a resistance to at least 7% L-threonine, a need for L-methionine and a need for L-isoleucine are used.
11. Process according to claims 1 and 2, wherein L- threonine-producing and -secreting microorganisms of “the Enterobacteriaceae family which have one or more of the features or characteristics of the strains chosen from the group consisting of: DSM12790, B-3996, kat 13, KCCM-1032 and KCCM~1033 are used.
12. Process according to claims 1 and 2, wherein one or more strains chosen from the group consisting of DSM12790, B-3996, kat 13, KCCM-1032 and KCCM-1033 are used. *