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WO2005021772A1 - Process for the preparation of l-lysine - Google Patents

Process for the preparation of l-lysine Download PDF

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Publication number
WO2005021772A1
WO2005021772A1 PCT/EP2004/008882 EP2004008882W WO2005021772A1 WO 2005021772 A1 WO2005021772 A1 WO 2005021772A1 EP 2004008882 W EP2004008882 W EP 2004008882W WO 2005021772 A1 WO2005021772 A1 WO 2005021772A1
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WIPO (PCT)
Prior art keywords
lysine
concentration
product
fermentation broth
led
Prior art date
Application number
PCT/EP2004/008882
Other languages
French (fr)
Inventor
Marc Gerigk
Thomas Hermann
Brigitte Bathe
Ralf Kelle
Original Assignee
Degussa Ag
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Publication date
Priority claimed from DE2003139847 external-priority patent/DE10339847A1/en
Application filed by Degussa Ag filed Critical Degussa Ag
Publication of WO2005021772A1 publication Critical patent/WO2005021772A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

Definitions

  • the invention relates to an improved process for the fermentative preparation of L-lysine using coryneform bacteria.
  • L-Lysine is used in human medicine, in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
  • L-lysine can be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. 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 bacterium itself.
  • fermentation measures such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product' form, by e.g. ion exchange chromatography, or the intrinsic output properties, i.e. those of genetic origin, of the bacterium itself.
  • the inventors had the object of providing new measures for improved fermentative preparation of L-lysine.
  • the invention provides a fermentation process, which comprises a procedure in which a) a coryneform bacterium which produces L-lysine 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 or several feed streams, the futher 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-lysine, 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, wherein c) over the entire period of time of step (b) a concentration of the source (s) of carbon of not more than 10 g/1.
  • the plant output of a fermentation unit which produces L-lysine can be increased by culturing by the batch process (batch) or feed process (fed batch) in the first culturing step (a) described above, at least one additional nutrient medium being employed if the feed process is used.
  • 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 5 or several removal streams, which substantially corresponds/correspond to the feed stream or the total of the feed streams .
  • the bacterium is inoculated 10 in at least a first nutrient medium and cultured by the batch process (batch) or feed process (fed batch) . If the feed 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, preferably after 2 to 10 hours and 15 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
  • Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep 30 liquor, soya bean flour and urea
  • inorganic compounds such as ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, potassium nitrate and potassium sodium nitrate, can be used as the source of nitrogen in the first nutrient medium.
  • the sources of nitrogen can be used individually, or as a mixture in concentrations of 1 to 50 g/kg, preferably 3 to 40 g/kg, particularly preferably 5 to 30 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 2,0 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.
  • essential growth substances such as amino acids (e.g. homoserine) and vitamins (e.g. thi,.amine) , ,. can be employed in addition to the , abovementioned 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 feed process (fed batch) 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, 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.
  • the constituents of the further nutrient medium can be fed to the culture in the form of a single further nutrient medium and in a plurality of further nutrient media.
  • the 10 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
  • the further nutrient medium or the further nutrient media furthermore comprises or comprise a source of nitrogen
  • organic nitrogen-containing compounds such as peptones, yeast extract,- meat extract, malt extract, corn steep liquor, soya bean flour and urea
  • inorganic compounds such as ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium
  • the sources of nitrogen can be used individually or as a mixture in concentrations of 5 to 500 g/kg, preferably 25 to 400 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 0.0003 to 15 g/kg, preferably in concentrations of 0.008 to
  • a single further nutrient medium is used, this is typically fed to the culture in one feed stream. If a plurality of further nutrient media are used, these. are fed in a corresponding plurality of feed streams. If a plurality of further nutrient media are used, it should be noted that these in each case can comprise only one of the sources of carbon, nitrogen or phosphorus described, or also a mixture of the sources of carbon, nitrogen or • phosphorus described.
  • the feed stream or the sum of the feed streams in the process according to the invention are added at a rate corresponding to an average residence time of less than 60 hours, less than 55 hours, less than 50 hours, less than 45 hours, less than 40 hours, preferably less than 35 hours, very particularly preferably less than 30 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 [Biotechnology] ; H. Weide, J. Paca and . A. Knorre; Gustav Fischer Verlag Jena; 1991) .
  • Intensive growth at the start of culturing is usually a logarithmic growth phase.
  • the logarithmic growth phase is in general followed by a phase of less intensive cell growth than in the logarithmic phase.
  • At least one further nutrient medium or several further nutrient media are fed continuously .to the culture in. one . or several feed streams and at the same time culture broth is removed from the culture with at least one or several removal streams, which substantially corresponds/correspond , to the feed stream or the total of the feed streams .
  • the rate of the removal stream or the removal streams corresponds to 80% - 120%, 90% - 110% of the feed stream or of the sum of the feed streams.
  • the removal can be realized industrially by pumping off and/or by draining off the culture broth.
  • the concentration of the source of carbon over the entire period of time of step (b) and/or in the case of a prior culturing in the feed process (fed batch) , also during the feeding of the additional .nutrient medium is adjusted to not more than 10 g/1, not more than 5 g/1 * preferably not more than 3 g/1, particularly preferably not more than 1 g/1.
  • the concentration of the source of carbon is determined here with the aid of methods which are prior art.
  • ⁇ -D-Glucose is determined e.g. in a YSI 02700 Select glucose analyzer from Yellow Springs Instruments (Yellow Springs, Ohio, USA) .
  • the culture broth removed can be provided with oxygen or an oxygen-containing gas until the concentration of the source of carbon falls below 2 g/1; below 1 g/1; or below 0.5 g/1.
  • the yield (Yp / s) is at least. 43 wt.%; at least 45 wt.%; at least 48 wt.%; at least 50 wt.%; at least 52 wt.%.
  • the yield Y P/S is defined here as the ratio of the total amount of L-lysine formed in a culturing to the total amount of the source of carbon employed or• consumed.
  • L-lysine is formed with a space/time yield (STY) of at least 2.5 g/1, preferably at least 3.5 g/1, in particular at least 2.5 to 3.0 g/1 per h, at least 3.0 to more than 4.0 g/1, at least 4.0 to
  • the space/time yield is defined here as the ratio of the total amount of L-lysine formed in a culturing to the actively producing volume of the culture over the total period of time of culturing.
  • the space/time yield is also called the volumetric productivity.
  • the L-lysine concentration (c) based on lysine HCl, in the fermentation broth led off is at least 100 g/1, at least 110 g/1, at least 120 g/1, preferably more than 130 g/1, particularly preferably more than 140 g/1.
  • STY space/time yield
  • Yp / s yield
  • c L-lysine concentration
  • the output index based on the process according to the invention, reaches at least 130 g 2 /(l 2 *h), at least 140 g 2 ,/(l 2 ,*h), at least 150 g 2 ./(l 2 *h), at least ... 160 g a /(l 2 *h), at least 170 g 2 /(l 2 *h), at least 190 g 2 /(l 2 *h), at least 210 g 2 /(l 2 *h), at least 230 g 2 /(l 2 *h), at least 250 g 2 /(l 2 *h).
  • the temperature is adjusted in a range from 28°C to 40°C, preferably 30 to 35°C.
  • the fermentation can be carried out under normal pressure or optionally under increased pressure, preferably under an increased pressure of 0 to 2.5 bar, particularly preferably under 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 ammonia gas or 25% aqueous ammonia.
  • the culturing conditions can remain constant during the culturing or can be changed. Likewise, the culturing conditions in step (a) and (b) can be identical or can differ.
  • the process as claimed in claim 1 comprises a procedure in which the oxygen uptake rate (OUR) established in step b) is not more than 350 mmol/ (l*h) , not more than 325 mmol/(l*hj, not more than 300 mmol/ (l*h) , not more than 275 mmol/ (l*h) , not more than 250 mmol/ (l*h) , not more than 225 mmol/ (l*h) , not more than 200 mmol/ (l*h) , not more than 175 mmol/ (l*h) , not more than 150 mmol/ (l*h) .
  • the oxygen uptake rate (OUR) established in step b) is not more than 350 mmol/ (l*h) , not more than 325 mmol/(l*hj, not more than 300 mmol/ (l*h) , not more than 275 mmol/ (l*h) , not more than 250 mmol/ (l
  • the oxygen uptake rate OUR here describes the specific oxygen absorption rate by the microorganisms in mmol 0 2 per litre of fermentation broth and hour (Biotechnologie [Biotechnol.ogy]; D. Schlee and H.-P. Kleber, Gustav. Fischer Verlag Jena; 1991) .
  • the process according to the invention is operated for at least 100 hours, preferably more than 150 hours, in particular more than 200 hours, preferably more than 250 hours, particularly preferably more than 300 hours.
  • the volume of the culture is exchanged at least 1 time, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 12 times, at least 20 times.
  • the streams described for the first nutrient media and further nutrient media or the sum of the streams of first nutrient media and further nutrient media comprise, complex constituents.
  • Sources of carbon or nitrogen which have a purity of less than 95% in the form employed are described as complex constituents.
  • Such a complex constituent is one or more compounds from the group consisting of peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor and soya bean flour.
  • the content of complex constituents in the nutrient media employed is less than 10 wt.%, less than 5 wt.%, less than 2.5 wt.%, less than 1.0 wt.%, less than 0.5 wt.%.
  • the osmolarity of the L-lysine- containing fermentation broth led off is less than 2,100 mosm/1, preferably less than 1,800 mosm/1, in particular less than 1,500 mosm/1, preferably less than 1,200 mosm/1.
  • the osmolarity is defined as the concentration of osmotically active particles in 1 litre of liquid volume.
  • a 1 molar glucose solution corresponds to 1,000 mosm/1 (Biotechnologie [Biotechnology] ;. H. Wei.de, J. Paca and W. A. Knorre; Gustav
  • coryneform bacteria in particular of the genus Corynebacterium, are suitable for carrying out the process according to the invention.
  • the coryneform bacteria are, in particular, the genus Corynebacterium.
  • the species Corynebacterium glutamicum and furthermore the species Brevibacterium flavum and Corynebacterium thermoaminogenes are to be mentioned in particular. These are known among experts for their ability to produce L-lysine.
  • Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild-type strains
  • the coryneform bacteria contain at least one copy of an lysC gene or allele, which codes for an aspartate kinase, which is insensitive towards the inhibition of lysine or mixtures of lysine and threonine (lysC fbr ) .
  • Such bacteria are typically resistant to the lysine analogue S-(2- aminoethyl) -cysteine . (AEC) .
  • L-Lysine-producing coryneform bacteria which one or more of the features chosen from the group consisting of lysC allele (lysC fbr ) , horn allele (hom leaky ) , zwf allele, coding for an NADPH-insensitive glucose 6-phosphate dehydrogenase, and the pyc allele which codes for pyruvate carboxylase are furthermore suitable.
  • the pyc allele is described in EP 1 108 790.
  • L-Lysine-producing coryneform bacteria which have one or more resistances chosen from the group consisting of azauracil r (Aza r ) , rifamycin r (Rif r ) , streptomycin r (Strep r ) are also suitable.
  • L-Lysine-producing coryneform bacteria which include. at least the following properties are furthermore suitable: two (2) copies of an lysC allele, which codes for a lysine- resistant aspartate kinase (lysC fbr ) , a hom allele, which codes for an attenuated homoserine dehydrogenase (hom leay ) and two (2) copies of a zwf allele, which codes for an NADPH-insensitive glucose 6-phosphate dehydrogenase.
  • L-Lysine-producing coryneform bacteria which contain one or more of the properties chosen from the group consisting of three (3) , four (4) or five (5) copies of an lysC allele (lysC fbr ) , two (2).. copies of an lysE gene, two (2) copiea ⁇ of a zwal gene are furthermore suitable.
  • diaminopimelic acid analogues includes compounds such as 4-fluoro-diaminopimelic acid, 4-hydroxy-diaminopimelic acid, 4-oxo-diaminopimelic acid or 2,4, 6-triaminopimelic acid.
  • Mutagenesis methods are used to produce the coryneform bacteria according to the invention which are sensitive towards 4-hydroxy-diaminopimelic acid.
  • Conventional in vivo mutagenesis methods using mutagenic substances such as, for example, N-methyl-N' -nitro-N-nitrosoguanidine, or ultraviolet light (Miller, J. H. : A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992) can be used for the mutagenesis.
  • the coryneform bacteria which are sensitive towards 4-hydroxy-diaminopimelic acid can be identified by plating out on nutrient medium plates containing 4-hydroxy- diaminopimelic acid. Final concentrations of approx. 5 to 15 g/1, for example approx. 10 g/1 4-hydroxy-diaminopimelic acid in the nutrient medium are particularly suitable for this. At this concentration, mutants which are sensitive towards 4-hydroxy-diaminopimelic acid can be distinguished from the unchanged parent strains by a slowed-down growth. After selection has taken place, the mutants which are sensitive towards 4-hydroxy-diaminopimelic acid show an improved L-lysine production. A . , ⁇ «-
  • the L-lysine-producing coryneform bacteria have the ability to divert the carbon flow through the oxidative pentose phosphate pathway with a percentage content of more than 75%, more than 85%, more than 95%, more than 105%, more than 115%, more, than 125%, more than 135%, more than 145%.
  • the L-lysine-producing coryneform bacteria furthermore have the ability to divert the carbon flow through the tricarboxylic acid cycle, based on the acetyl radicals which are transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction, with a percentage content of at least 1% but not more than 20%, at least 2% but. not more than 18%, at least 3% but not more than 16%..
  • the tricarboxylic acid cycle also serves for synthesis, of compounds which are essential precursors of the amino acid synthesis pathway.
  • Oxaloacetate serves, for example, as a precursor of lysine. synthesis.
  • the withdrawal of these precursors from the tricarboxylic acid cycle is compensated by replenishing reactions, so-called anaplerotic reactions- Depending on the nature of the source of carbon, growth rate and product formation of the coryneform bacteria, these reactions can proceed forwards or backwards. Forwards in this connection means that the carbon flow takes place from glycolysis in the direction of the tricarboxylic acid cycle (e.g.
  • L-Lysine-producing coryneform bacteria which are particularly suitable according to the invention are those which have the ability to divert the carbon flow through the anaplerotic reactions, based on the sum of pyruvate and phosphoenol pyruvate (PEP) , which are converted into oxaloacetate by PEP carboxylase and pyruvate carboxylase respectively, coded by ppc and pyc respectively, with a percentage content of more than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, more than 37%. According to the definition, this corresponds to a net flow through the anaplerotic reactions of more than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, more than 37%.
  • PEP phosphoenol pyruvate
  • L-Lysine-producing coryneform bacteria which are particularly suitable according to the invention are those which have the ability to divert the carbon flow through aspartate kinase, coded by lysC, with a percentage content of at least 28% but not more than 60%, at least 30% .but not more than 57%, at least 32% but not more than 53%, at least 33% but not more than 50%. .> . . .. - • .
  • L-Lysine-producing coryneform bacteria which are furthermore suitable according to the invention are those which have the ability to divert the carbon flow through diaminopimelate dehydrogenase, coded by ddh, with a percentage content of at least 49% but not more than 98%, at least 53% but not more than 95%, at least 56% but not more than 91%, at least 58% but not more than 87%.
  • the L-lysine containing fermentation broth from the process according to the invention which is led off has a solids content of at least 10 wt.%, at least 12.5 wt.%, at least 15 wt.%, at least 17.5 wt.%.
  • the L-lysine produced can then be purified from the fermentation broth. 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 L-lysine- containing broth is then purified by known methods, such as, for example, by ion exchange chromatography, ion exclusion chromatography, extraction, crystallization, precipitation or a combination thereof.
  • the L-lysine-containing fermentation broth led off can also be dewatered.
  • the fermentation broth is 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.
  • the water is removed by this means to the extent of 10% to 90%.
  • the pH can be changed into the acidic (pH 2 to 5) or alkaline (pH 9 to 12) range by addition of acid or alkali.
  • 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.
  • the water is removed by this means to the extent of 10% to 90%.
  • Concentrated broth furthermore can then be 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 by converted by suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product.
  • the water is then removed to the extent in total of more than 90% by this means, so that the water content in the product is less than 10%, less than 5%.
  • the process according to the invention is distinguished with respect to the conventional fed batch process above all by an increased space/time yield.
  • An L-lysine-containing product of the following composition is preferably obtained from the fermentation broth by removal of water: Lysine 35 - 80 wt Protein max. 7 wt . Carboxylic aci .ds ma . 7 wt . Total sugars ma . 9 wt . Fats and oils max. 5 wt . Minerals 3 -30 wt.%
  • this L-lysine- containing product after dewatering this L-lysine- containing product has a water content of at least 0.5 wt . %, but. not more than 5.0 wt . % .
  • the fermentation broth led off should furthermore adhere to the following by-product concentrations.
  • the L-lysine-containing fermentation broth for the preparation of an L-lysine-containing product it is preferable for the L-lysine-containing fermentation broth to have a total by-product concentration of not more than 5.0%, preferably not more than 4.0%, 3.0%, 2.5%, 2.0%, particularly preferably not more than 1.5%, 1.0% or 0.5%. It is particularly desirable for the L-lysine-containing fermentation broth to have a total by-product concentration of less than 0.5%.
  • the L-lysine-containing product after dewatering and subsequent granulation the L-lysine-containing product has an average particle size of > 0.1 to 1.0 mm, preferably in an amount of more than 97%, in particular more than 98%.
  • the L-lysine- containing product has a bulk density of at least 600 kg/m 3 , preferably 650 kg/m 3 , in particular 700 kg/m 3 , but preferably greater than 750 kg/m 3 .
  • an additive can be added to the L-lysine-containing product, in order to improve the properties of the product.
  • the content of additive added, in particular oil, here should be 0.02 - 2.0 wt.%, based on the total amount of L-lysine-containing product, on the surface.
  • the L-lysine-containing product comprises as an. additive one or more of. the oils, chosen— from the group consisting of mineral oil, vegetable oils, soya oil, olive oil, soya/lecithin mixtures, edible oils, mixtures of vegetable oils, on the surface.
  • the L-lysine-containing product has a lactate content of ⁇ 3 wt.%, ⁇ 2 wt.%, ⁇ 1 wt.%, ⁇ 0.5 wt.%, ⁇ 0.1 wt . % .
  • L-lysine and other amino acids can be carried out by anion exchange chromatography with subsequent ninhydrin derivatization, 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) ) .

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Abstract

The invention relates to an improved process for the fermentative preparation of L-lysine using coryneform bacteria which produce L-lysine.

Description

Process for the preparation of L-lysine
The invention relates to an improved process for the fermentative preparation of L-lysine using coryneform bacteria.
Prior art
L-Lysine is used in human medicine, in the pharmaceuticals industry, in the foodstuffs industry and very particularly in animal nutrition.
It is known that L-lysine can be prepared by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. 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 bacterium itself.
Object of the invention
The inventors had the object of providing new measures for improved fermentative preparation of L-lysine.
Description of the invention
The invention provides a fermentation process, which comprises a procedure in which a) a coryneform bacterium which produces L-lysine 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 or several feed streams, the futher 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-lysine, 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, wherein c) over the entire period of time of step (b) a concentration of the source (s) of carbon of not more than 10 g/1. is established, and . d) over the entire period of time of step (b) the formation of L-lysine, based on lysine HC1, with an output index (01) = space/time yield [g/(l*h)] x yield [w/w] x concentration of lysine HC1 [g/1] of at least 130 g2/ (l2*h) is established.
According to the invention, the plant output of a fermentation unit which produces L-lysine can be increased by culturing by the batch process (batch) or feed process (fed batch) in the first culturing step (a) described above, at least one additional nutrient medium being employed if the feed 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 or several feed streams and at the same time culture broth is removed from the culture with at least one 5 or several removal streams, which substantially corresponds/correspond to the feed stream or the total of the feed streams .
During the culturing step (a) , the bacterium is inoculated 10 in at least a first nutrient medium and cultured by the batch process (batch) or feed process (fed batch) . If the feed 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, preferably after 2 to 10 hours and 15 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
20 sugar, fructose> glucose, starch hydrolysate, lactose,., galactose, maltose, xylose, acetic acid, ethanol and methanol, in concentrations of 1 to 50 g/kg, preferably 5 to 40 g/kg, particularly preferably 10 to 30 g/kg. Starch hydrolysate is understood according to. the invention as the
2.5 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 30 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 50 g/kg, preferably 3 to 40 g/kg, particularly preferably 5 to 30 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 2,0 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 0.0003 to 15 g/kg. Finally, essential growth substances, such as amino acids (e.g. homoserine) and vitamins (e.g. thi,.amine) , ,. can be employed in addition to the , abovementioned 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 feed process (fed batch) 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, 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. 5 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 10 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 .
15 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
20..and .methanol, in concentrations of 20 to.700 g/kg, . .. ,r. „ preferably 50 to 650 g/kg.
The further nutrient medium or the further nutrient media furthermore comprises or comprise a source of nitrogen
25 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
30 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 500 g/kg, preferably 25 to 400 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 0.0003 to 15 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) , can be employed in addition to the abovementioned 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.
The feed stream or the sum of the feed streams in the process according to the invention are added at a rate corresponding to an average residence time of less than 60 hours, less than 55 hours, less than 50 hours, less than 45 hours, less than 40 hours, preferably less than 35 hours, very particularly preferably less than 30 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 [Biotechnology] ; H. Weide, J. Paca and . 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 rate of the removal stream or the removal streams corresponds to 80% - 120%, 90% - 110% of the feed stream or of the sum 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 over the entire period of time of step (b) and/or in the case of a prior culturing in the feed process (fed batch) , also during the feeding of the additional .nutrient medium, is adjusted to not more than 10 g/1, not more than 5 g/1* preferably not more than 3 g/1, particularly preferably not more than 1 g/1. 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 until the concentration of the source of carbon falls below 2 g/1; below 1 g/1; or below 0.5 g/1.
According to the invention, the yield (Yp/s) is at least. 43 wt.%; at least 45 wt.%; at least 48 wt.%; at least 50 wt.%; at least 52 wt.%. The yield YP/S is defined here as the ratio of the total amount of L-lysine formed in a culturing to the total amount of the source of carbon employed or• consumed. • • • • • •
According to the invention, L-lysine is formed with a space/time yield (STY) of at least 2.5 g/1, preferably at least 3.5 g/1, in particular at least 2.5 to 3.0 g/1 per h, at least 3.0 to more than 4.0 g/1, at least 4.0 to
5.0 g/1 per h, or at least 5.0 to 8.0 g/1 or more per h. The space/time yield is defined here as the ratio of the total amount of L-lysine formed in a culturing to the actively producing volume of the culture over the total period of time of culturing. The space/time yield is also called the volumetric productivity. According to the invention, the L-lysine concentration (c) , based on lysine HCl, in the fermentation broth led off is at least 100 g/1, at least 110 g/1, at least 120 g/1, preferably more than 130 g/1, particularly preferably more than 140 g/1.
In the fermentative preparation of L-lysine, an attempt is made to achieve and maintain an optimum as a function of the three output features of yield, productivity and product concentration. Such an optimum can be described by an output index (01), which is composed, of the product of the space/time yield (STY) , yield (Yp/s) and L-lysine concentration (c) , based on lysine HCl, (01 [g2/(l2*h)] = STY [g/l*h] * Yp/s[ /w] * c [g/1]). It should be remembered that the output index according to the invention relates to the total period of time of step (b) of the process according to the invention. According to the invention, the output index, based on the process according to the invention, reaches at least 130 g2/(l2*h), at least 140 g2,/(l2,*h), at least 150 g2./(l2*h), at least ... 160 ga/(l2*h), at least 170 g2/(l2*h), at least 190 g2/(l2*h), at least 210 g2/(l2*h), at least 230 g2/(l2*h), at least 250 g2/(l2*h).
During the culturing the temperature is adjusted in a range from 28°C to 40°C, preferably 30 to 35°C. The fermentation can be carried out under normal pressure or optionally under increased pressure, preferably under an increased pressure of 0 to 2.5 bar, particularly preferably under 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 ammonia gas or 25% aqueous ammonia. The culturing conditions can remain constant during the culturing or can be changed. Likewise, the culturing conditions in step (a) and (b) can be identical or can differ.
To meet the requirement of the output index, during the fermentation not only is an adequate oxygen partial pressure to be ensured, but also an adequate biological activity of the cells. To ensure the biological activity, the process as claimed in claim 1 comprises a procedure in which the oxygen uptake rate (OUR) established in step b) is not more than 350 mmol/ (l*h) , not more than 325 mmol/(l*hj, not more than 300 mmol/ (l*h) , not more than 275 mmol/ (l*h) , not more than 250 mmol/ (l*h) , not more than 225 mmol/ (l*h) , not more than 200 mmol/ (l*h) , not more than 175 mmol/ (l*h) , not more than 150 mmol/ (l*h) . The oxygen uptake rate OUR here describes the specific oxygen absorption rate by the microorganisms in mmol 02 per litre of fermentation broth and hour (Biotechnologie [Biotechnol.ogy]; D. Schlee and H.-P. Kleber, Gustav. Fischer Verlag Jena; 1991) .
The process according to the invention is operated for at least 100 hours, preferably more than 150 hours, in particular more than 200 hours, preferably more than 250 hours, particularly preferably more than 300 hours. During this procedure the volume of the culture is exchanged at least 1 time, at least 2 times, at least 4 times, at least 6 times, at least 8 times, at least 10 times, at least 12 times, at least 20 times.
The streams described for the first nutrient media and further nutrient media or the sum of the streams of first nutrient media and further nutrient media comprise, complex constituents. Sources of carbon or nitrogen which have a purity of less than 95% in the form employed are described as complex constituents. Such a complex constituent is one or more compounds from the group consisting of peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor and soya bean flour. According to the invention, the content of complex constituents in the nutrient media employed is less than 10 wt.%, less than 5 wt.%, less than 2.5 wt.%, less than 1.0 wt.%, less than 0.5 wt.%.
According to the invention, the osmolarity of the L-lysine- containing fermentation broth led off is less than 2,100 mosm/1, preferably less than 1,800 mosm/1, in particular less than 1,500 mosm/1, preferably less than 1,200 mosm/1. The osmolarity is defined as the concentration of osmotically active particles in 1 litre of liquid volume. For example, a 1 molar glucose solution corresponds to 1,000 mosm/1 (Biotechnologie [Biotechnology] ;. H. Wei.de, J. Paca and W. A. Knorre; Gustav
Fischer Verlag Jena; 1991) .
Representatives of coryneform bacteria, in particular of the genus Corynebacterium, are suitable for carrying out the process according to the invention. The coryneform bacteria are, in particular, the genus Corynebacterium. Of the genus Corynebacterium, the species Corynebacterium glutamicum and furthermore the species Brevibacterium flavum and Corynebacterium thermoaminogenes are to be mentioned in particular. These are known among experts for their ability to produce L-lysine. Information on the taxonomic classification of strains of this group of bacteria is to be found, inter alia, in Kampfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)) and in US-A-5, 250 , 434.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild-type strains
Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium thermoaminogenes FERM BP-1539 Corynebacterium efficiens DSM44547 Corynebacterium efficiens DSM44548 . Corynebacterium efficiens DSM44549 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869, and Brevibacterium divaricatum ATCC14020
and .L-lysin -producing .strains prepared therefrom ..._:, .
The coryneform bacteria contain at least one copy of an lysC gene or allele, which codes for an aspartate kinase, which is insensitive towards the inhibition of lysine or mixtures of lysine and threonine (lysCfbr) . Such bacteria are typically resistant to the lysine analogue S-(2- aminoethyl) -cysteine . (AEC) .
L-Lysine-producing coryneform bacteria which one or more of the features chosen from the group consisting of lysC allele (lysCfbr) , horn allele (homleaky) , zwf allele, coding for an NADPH-insensitive glucose 6-phosphate dehydrogenase, and the pyc allele which codes for pyruvate carboxylase are furthermore suitable. The pyc allele is described in EP 1 108 790.
L-Lysine-producing coryneform bacteria which have one or more resistances chosen from the group consisting of azauracilr (Azar) , rifamycinr (Rifr) , streptomycinr (Strepr) are also suitable.
L-Lysine-producing coryneform bacteria which include. at least the following properties are furthermore suitable: two (2) copies of an lysC allele, which codes for a lysine- resistant aspartate kinase (lysCfbr) , a hom allele, which codes for an attenuated homoserine dehydrogenase (homleay) and two (2) copies of a zwf allele, which codes for an NADPH-insensitive glucose 6-phosphate dehydrogenase.
L-Lysine-producing coryneform bacteria which contain one or more of the properties chosen from the group consisting of three (3) , four (4) or five (5) copies of an lysC allele (lysCfbr) , two (2).. copies of an lysE gene, two (2) copiea ιof a zwal gene are furthermore suitable.
L-Lysine-producing coryneform bacteria which are sensitive towards diaminopimelic acid analogues are moreover suitable. According to the present invention, the term diaminopimelic acid analogues includes compounds such as 4-fluoro-diaminopimelic acid, 4-hydroxy-diaminopimelic acid, 4-oxo-diaminopimelic acid or 2,4, 6-triaminopimelic acid.
Mutagenesis methods are used to produce the coryneform bacteria according to the invention which are sensitive towards 4-hydroxy-diaminopimelic acid. Conventional in vivo mutagenesis methods using mutagenic substances, such as, for example, N-methyl-N' -nitro-N-nitrosoguanidine, or ultraviolet light (Miller, J. H. : A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992) can be used for the mutagenesis.
The coryneform bacteria which are sensitive towards 4-hydroxy-diaminopimelic acid can be identified by plating out on nutrient medium plates containing 4-hydroxy- diaminopimelic acid. Final concentrations of approx. 5 to 15 g/1, for example approx. 10 g/1 4-hydroxy-diaminopimelic acid in the nutrient medium are particularly suitable for this. At this concentration, mutants which are sensitive towards 4-hydroxy-diaminopimelic acid can be distinguished from the unchanged parent strains by a slowed-down growth. After selection has taken place, the mutants which are sensitive towards 4-hydroxy-diaminopimelic acid show an improved L-lysine production. A . , ,■■■«-
Appropriately stable strains which do not lose their production properties in the course of the process are particularly suitable for the process described.
The targeted changing of strain properties of coryneform bacteria, the composition of the nutrient media and the type of process procedure all have the aim of converting the source of carbon employed into L-lysine as effectively as possible. For this, it is necessary for the carbon to flow through the cell metabolism in the direction of L- lysine synthesis as far as possible without consuming side reactions and very largely unimpeded. In addition to the precise knowledge of the cell metabolism, a' substance flow analysis in which, by using, for example, 13C-labelled substrate, the carbon flow through the metabolism can be reconstructed and the flow circumstances at branchings can be determined in the form of relative flows is necessary for a model-based improvement in the strain and the production process (Bioreaktionstechnik: Bioprozesse mit Mikroorganismen und Zellen: Prozessύberwachung [Bioreactions: Bioprocesses with Microorganisms and Cells: Process Monitoring]; K. Schύgerl; Birkhauser Verlag Basle - Bosten - Berlin; 1997) . The carbon flow is defined as the. ratio of the molar rates, based on carbon, of an individual reaction or reaction sequences taking place in the metabolism to the carbon uptake. If a 13C. compound is used for the labelling, determination of the distribution of the labelled atoms in the various metabolites is possible, for example, by means of nuclear magnetic resonance spectroscopy ( MR) . -, . _ ., ..,
According to the invention, the L-lysine-producing coryneform bacteria have the ability to divert the carbon flow through the oxidative pentose phosphate pathway with a percentage content of more than 75%, more than 85%, more than 95%, more than 105%, more than 115%, more, than 125%, more than 135%, more than 145%.
The L-lysine-producing coryneform bacteria furthermore have the ability to divert the carbon flow through the tricarboxylic acid cycle, based on the acetyl radicals which are transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction, with a percentage content of at least 1% but not more than 20%, at least 2% but. not more than 18%, at least 3% but not more than 16%..
In addition to providing reduction equivalents, the tricarboxylic acid cycle also serves for synthesis, of compounds which are essential precursors of the amino acid synthesis pathway. Oxaloacetate serves, for example, as a precursor of lysine. synthesis. The withdrawal of these precursors from the tricarboxylic acid cycle is compensated by replenishing reactions, so-called anaplerotic reactions- Depending on the nature of the source of carbon, growth rate and product formation of the coryneform bacteria, these reactions can proceed forwards or backwards. Forwards in this connection means that the carbon flow takes place from glycolysis in the direction of the tricarboxylic acid cycle (e.g. from pyruvate to oxaloacetate and/or from phosphoenol pyruvate to oxaloacetate) . Backwards in this connection means that the carbon flow proceeds from the tricarboxylic acid cycle in the direction of glycolysis (e.g. from oxaloacetate to pyruvate) . The flows can proceed here simultaneously in both directions. The sum of all the forwards- and backwards-directed flows is called the net flow. If the net flow is directed forwards (that is to say from glycolysis to the tricarboxylic acid cycle) it is given a positive sign, if it is directed backwards it is given a negative sign. Lysine-producing coryneform bacteria in general have the ability to divert the carbon through the anaplerotic reactions from glycolysis in the direction of the tricarboxylic acid cycle.
L-Lysine-producing coryneform bacteria which are particularly suitable according to the invention are those which have the ability to divert the carbon flow through the anaplerotic reactions, based on the sum of pyruvate and phosphoenol pyruvate (PEP) , which are converted into oxaloacetate by PEP carboxylase and pyruvate carboxylase respectively, coded by ppc and pyc respectively, with a percentage content of more than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, more than 37%. According to the definition, this corresponds to a net flow through the anaplerotic reactions of more than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, more than 37%.
L-Lysine-producing coryneform bacteria which are particularly suitable according to the invention are those which have the ability to divert the carbon flow through aspartate kinase, coded by lysC, with a percentage content of at least 28% but not more than 60%, at least 30% .but not more than 57%, at least 32% but not more than 53%, at least 33% but not more than 50%. .> . . .. - • .
L-Lysine-producing coryneform bacteria which are furthermore suitable according to the invention are those which have the ability to divert the carbon flow through diaminopimelate dehydrogenase, coded by ddh, with a percentage content of at least 49% but not more than 98%, at least 53% but not more than 95%, at least 56% but not more than 91%, at least 58% but not more than 87%.
According to the invention, the L-lysine-producing coryneform bacteria have the ability to establish a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow through the anaplerotic reactions (oxidative pentose phosphate pathway [%] / anaplerotic reactions [%] = PPP/Ana [-] ) of at least 3.4 but not more than 4.6, at least 3.5 but not more than 4.5, at least 3.6 but not more than 4.4, at least 3.7 but not more than 4.3.
Coryneform bacteria which produce, according to the invention, L-lysine and which are furthermore suitable are those which have the ability to establish a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow into the tricarboxylic acid cycle (oxidative pentose phosphate pathway [%] / tricarboxylic acid cycle [%] = PPP/TCA [-] ) of at least 7 but not more than 150, at least 10 but not more than 125, at least 13 but not more than 100, or at least.16 but not more than 75.
The L-lysine containing fermentation broth from the process according to the invention which is led off has a solids content of at least 10 wt.%, at least 12.5 wt.%, at least 15 wt.%, at least 17.5 wt.%.
The L-lysine produced can then be purified from the fermentation broth. 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 L-lysine- containing broth is then purified by known methods, such as, for example, by ion exchange chromatography, ion exclusion chromatography, extraction, crystallization, precipitation or a combination thereof.
As an alternative to this, the L-lysine-containing fermentation broth led off can also be dewatered. For this, the fermentation broth is 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. The water is removed by this means to the extent of 10% to 90%.
For improved preserving of the L-lysine-containing liquid product, the pH can be changed into the acidic (pH 2 to 5) or alkaline (pH 9 to 12) range by addition of acid or alkali.
It is also possible to prepare a product from the fermentation broth removed 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. The water is removed by this means to the extent of 10% to 90%. Concentrated broth furthermore can then be 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 by converted by suitable compacting or granulating processes into a coarse-grained, readily free-flowing, storable and largely dust-free product. The water is then removed to the extent in total 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 process according to the invention is distinguished with respect to the conventional fed batch process above all by an increased space/time yield.
An L-lysine-containing product of the following composition is preferably obtained from the fermentation broth by removal of water: Lysine 35 - 80 wt Protein max. 7 wt . Carboxylic aci .ds ma . 7 wt . Total sugars ma . 9 wt . Fats and oils max. 5 wt . Minerals 3 -30 wt.%
According to the invention, after dewatering this L-lysine- containing product has a water content of at least 0.5 wt . %, but. not more than 5.0 wt . % . For preparation of the lysine-containing product, the fermentation broth led off should furthermore adhere to the following by-product concentrations. A trehalose concentration of less than or equal to (<) 10 g/1,
< 5.0 g/1, ≤ 2.0 g/1, < 0.5 g/1. An L-alanine concentration of < 5.0 g/1, < 2.5 g/1, < 1.0 g/1, < 0.25 g/1. An L-valine concentration of < 5.0 g/1, < 2.5 g/1, < 1.0 g/1,
< 0.25 g/1. An L-glutamate concentration of < 7.5 g/1, < 5.0 g/1, < 2.0 g/1, < 0.5 g/1. An ethanol concentration of < 8.0 g/1, < 4.0 g/1, < 2.0 g/1, < 0.5 g/1. A lactate concentration of < 8.0 g/1, < 4.0 g/1, < 2.0 g/1, < 0.5 g/1. A ketoglutarate concentration of < 10 g/1, < 5.0 g/1, < 2.0 g/1, < 0.5 g/1. A succinate concentration of < 10 g/1, < 5.0 g/1, < 2.0 g/1, < 0.5 g/1. A malate concentration of < 10 g/1, < 5.0 g/1, < 2.0 g/1, < 0.5 g/1. An oxaloacetate concentration of < 10 g/1, < 5.0 g/1, < 2.0 g/1, < 0.5 g/1. An acetate concentration of < 10 g/1, < 5.0 g/1, < 2.0 g/1,
< 0.5 g/1. A pyruvate concentration of < 10 g/1, < 5.0 g/1, < 2.0 g/1, < 0.5 g/1.
According to the invention, for the preparation of an L-lysine-containing product it is preferable for the L-lysine-containing fermentation broth to have a total by-product concentration of not more than 5.0%, preferably not more than 4.0%, 3.0%, 2.5%, 2.0%, particularly preferably not more than 1.5%, 1.0% or 0.5%. It is particularly desirable for the L-lysine-containing fermentation broth to have a total by-product concentration of less than 0.5%. According to the invention, after dewatering and subsequent granulation the L-lysine-containing product has an average particle size of > 0.1 to 1.0 mm, preferably in an amount of more than 97%, in particular more than 98%.
Furthermore, after dewatering and granulation the L-lysine- containing product has a bulk density of at least 600 kg/m3, preferably 650 kg/m3, in particular 700 kg/m3, but preferably greater than 750 kg/m3.
As described in US Patent Application Serial No. 10/319,843, after dewatering and granulation an additive can be added to the L-lysine-containing product, in order to improve the properties of the product. The content of additive added, in particular oil, here should be 0.02 - 2.0 wt.%, based on the total amount of L-lysine-containing product, on the surface.
According to the invention, the L-lysine-containing product comprises as an. additive one or more of. the oils, chosen— from the group consisting of mineral oil, vegetable oils, soya oil, olive oil, soya/lecithin mixtures, edible oils, mixtures of vegetable oils, on the surface.
It. is recommended in all cases that after dewatering and granulation the L-lysine-containing product has a lactate content of ≤ 3 wt.%, < 2 wt.%, < 1 wt.%, < 0.5 wt.%, < 0.1 wt . % .
The analysis of L-lysine and other amino acids can be carried out by anion exchange chromatography with subsequent ninhydrin derivatization, 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) ) .

Claims

Patent claims
1. A process for the fermentative preparation of an L-lysine-containing product using coryneform bacteria which produce L-lysine, which comprises a procedure in which a) the bacterium is inoculated and cultured in at least a first nutrient medium, b) at least a further nutrient medium or further nutrient media is/are then fed continuously to the culture in one or several feed streams, 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-lysine, 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, wherein
. c) over the entire period of time of step (b) a concentration of the source (s) of carbon of not more than 10 g/1 is established, and d) over the entire period of time of step (b) the formation of L-lysine, based on lysine HCl, takes place with an output index (01) = space/time yield [g/ (l*h) ] x yield [w/w] x concentration of Lys-HCl [g/1]) of at least 130 g2/ (l2*h) .
2. A process as claimed in claim 1, wherein the culturing step (a) is carried out by the batch process (batch) .
3. A process as claimed in claim 1, wherein the culturing step (a) is carried out by the feed process (fed batch) , at least one additional nutrient medium being employed.
4. A process as claimed in claim 1, 2 or 3, 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, maltose, xylose, acetic acid, ethanol and methanol .
5. A process as claimed in claim 1, 2 or 3, 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.
6. A process as claimed in claim 5, 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.
7. A process as claimed in claim 1, 2 or 3 , wherein the source of phosphorus is alkali metal or alkaline earth metal salts of phosphoric acid or polymers thereof or of phytic acid.
8. A process as claimed in claim 7, wherein the alkali metal salts of phosphoric acid are potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.
9. A process as claimed, in claim 1, 2 or 3, wherein the feed stream established in step b) or the sum of the feed streams is fed in at a rate corresponding to an . average residence time of less than or equal to (<) 60 h, < 55 h, < 50 h, < 45 h, < 35 h, or < 30 h.
10. A process as claimed in claim 9, wherein the average residence time is less than or equal to 35 h.
11. A process as claimed in claim 1, 2 or 3, wherein the rate of the removal, stream or the removal streams ' " corresponds to "80% - 120%, preferably 90%'- 110% of the feed stream or of the sum of the feed streams.
12 A A process as claimed in claim 1 or 3, wherein the concentration of the source of carbon over the entire period of time of step (b) and/or in the case of a prior culturing in the feed process (fed batch) , also during the feeding. of the additional nutrient medium, is not more than 10 g/1.
13. A process as claimed in claim 1 or 3, wherein the concentration of the source of carbon over the entire period of time of step (b) and/or in the case of a prior culturing in the feed process (fed batch) , also during the feeding of the additional nutrient medium, is not more than 5 g/1.
14. A process as claimed in claim 1 or 3 , wherein the concentration of the source of carbon over the entire period of time of step (b) and/or in the case of a prior culturing in the feed process (fed batch) , also during the feeding of the additional nutrient medium, is not more than 3 g/1.
15. A process as claimed in claim 1, 2 or 3, wherein the culture broth removed can be provided with oxygen or an oxygen-containing gas until the concentration of the source of carbon is not more than 2 g/1, not more than 1 g/1, or not more than 0.5 g/1.
16. A process as claimed in claim 1, 2 or 3 , wherein the yield is at least 43 wt.%, based on the sugar employed. -
17. A process as claimed in claim 16, wherein the yield is at least 48 wt.%, based on the sugar employed.
18. A process as claimed in claim 1, 2 or.3, wherein the space/time yield is at least 2.5 g/ (l*h) .
19. A process as claimed in claim 18, wherein the space/time yield is at least 3.5 g/ (l*h) or more.
20. A process as claimed in claim 1, 2 or 3, wherein the concentration of the lysine HCl in the fermentation broth led off is > 100 g/1, > 110 g/1, > 120 g/1, > 130 g/1, or > 140 g/1.
21. A process as claimed in claim 1, 2 or 3, wherein the formation of the L-lysine, based on lysine HCl, takes place with an output index (01) of at least 170 g/(l *h) .
22. A process as claimed in claim 21, wherein the formation of the L-lysine, based on lysine HCl, takes place with an output index (01) of at least 200 g2/ (l2*h) .
23. A process as claimed in claim 1, 2 or 3, wherein step b) is carried out for more than 100 hours.
24. A process as claimed in claim 23, wherein step b) is carried out for more than 200 hours.
25. A process as claimed in claim 1, 2 or 3, wherein the nutrient media employed in step a) or b) comprise complex constituents from the group consisting of" peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor and soya bean flour.
26. A process as claimed in claim 25, wherein the content of complex constituents in the nutrient media employed is less than 5 wt.%.
27. A process as claimed in claim 1, 2 or 3, wherein the L-lysine-containing fermentation broth led off has an osmolarity of not more than 2,100 mosm/1, not more than 1,800 mosm/1, not more than 1,500 mosm/1, or not more than 1,200 mosm/1.
28. A process as claimed in claim 1, 2 or 3 , wherein the coryneform bacteria are the genus Corynebacterium.
29. A process as claimed in claim 1, 2, 3 or 28, wherein 5 the L-lysine-containing fermentation broth produced and led off is prepared as a liquid product with 0% - 100% of the biomass formed in the fermentation after a 10% - 90% dewatering.
10 30. A process as claimed in claim 1, 2, 3 or 28, wherein the coryneform bacterium contains at least one lysC gene or allele, which codes for an aspartate kinase, which is insensitive towards inhibition by lysine or mixtures of lysine and threonine .
15 31. A process as claimed in claim 1, 2, 3 or 28, wherein the L-Lysine-producing coryneform bacteria has one or more of the features chosen from the group consisting of. lysC allele (lysCfbr) , horn allele (homleaky) , zwf
■20- allele, coding for an NADPH-insensitive .glucose £-- phosphate dehydrogenase, and pyc allele, coding for a pyruvate carboxylase.
32. A process as claimed in claim 1, 2, 3 or 28, wherein 25 the L-Lysine-producing coryneform bacterium has one or more resistances chosen from the group consisting of azauracilr (Azar) , rifamycinr (Rifr) , streptomycin1 (Strepr) .
30 33. A process as claimed in claim 1, 2, 3 or 28, wherein the L-Lysine-producing coryneform bacterium has at least the following properties: two (2) copies of an lysC allele (lysCfbr) , a horn allele (homleay) and two (2) copies of a zwf allele, which codes for an NADPH-insensitive glucose 6-phosphate dehydrogenase.
34. A process as claimed in claim 1, 2, 3 or 28, wherein the L-Lysine-producing coryneform bacterium has one or more of the properties chosen from the group consisting of three (3) , four (4) or five (5) copies of an lysC allele (lysCfbr) , two (2) copies of an lysE gene, two (2) copies of a zwal gene.
35. A process as claimed in claim 1, 2, 3 or 28,. wherein the L-Lysine-producing coryneform bacterium is sensitive toward diaminopimelic acid analogues chosen from the group consisting of 4-hydroxy-diaminopimelic acid, 4-fluoro-diaminopimelic acid, 4-oxo- diaminopimelic acid and 2 , 4, 6-triaminopimelic acid, preferably 4-hydroxy-diaminopimelic acid.
36. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the L-lysine-producing coryneform. acterium •■ . has the ability to divert the carbon flow through the oxidative pentose phosphate pathway with a percentage content of more than 75%, more than 85%, more than 95%, more than 105%, more than 115%, more than 125%, more than 135% or more than 145%.
37. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the L-Lysine-producing coryneform bacterium has the ability to divert the carbon flow through the anaplerotic reactions, based on the sum of pyruvate and phosphoenol pyruvate (PEP) , which are converted into oxaloacetate by PEP carboxylase and pyruvate carboxylase respectively, coded by ppc and pyc respectively, with a percentage content of more than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, or more than 37%.
38. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the L-lysine-producing coryneform bacterium has the ability to divert the carbon flow into the tricarboxylic acid cycle, based on the acetyl radicals which are transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction, with a percentage content of at least 1% but not more than 20%, at least 2% but not more than 18%, or at least 3% but not more than 16%.
39. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the L-lysine-producing coryneform bacterium has. the ability to divert the carbon flow through the . lysine-resistant aspartate kinase, coded by lysC, with a percentage content of at least 28% but not more than 56%, at least 30% but not more than 54%, at least 32% but not more than 52%, or at least 33% but not more than 50%.
40. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the. L-lysine-producing coryneform bacterium has the ability to divert the carbon flow through diaminopimelate dehydrogenas, coded by ddh, with a percentage content of at least 49% but not more than . 98%, at least 53% but not more than 95%, at least 56% but not more than 91%, or at least 58% but not more than 87%.
41. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the L-lysine-producing coryneform bacterium, has the ability to establish a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow through the anaplerotic reactions (oxidative pentose phosphate pathway [%] / anaplerotic reactions [%] = PPP/Ana [-] ) of at least 3.4 but not more than 4.6, at least 3.5 but not more than 4.5, at least 3.6 but not more than 4.4, or at least 3.7 but not more than 4.3.
42. A process as claimed in claim 1, 2, 3, 28 or 29, wherein the L-lysine-producing coryneform bacterium has . the ability to establish a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow into the tricarboxylic acid cycle (oxidative pentose phosphate pathway [%] / tricarboxylic acid cycle [%] = PPP/TCA [-] ) of at least 7 but not more than 150, at least 10 but not - more than 1-2,5, at • least 13 but not more than lO0.,,?,or at least 16 but not more than 75.
43. A process as claimed in claim 1, 2 or 3, wherein the L-lysine contained in the fermentation broth removed. is purified.
44. A process as claimed in claim 1, 2, 3 or 29, wherein the fermentation broth led off has a solids content of at least 10 wt.%, at least 12.5 wt.%, at least 15 wt.%, or at least 17.5 wt.%.
45. A process as claimed in claim 1, 2 or 3, wherein the fermentation broth led off is concentrated by the removal of water and an L-lysine-containing product comprising Lysine 35 - 80 wt.% Protein ma . 7 wt . % Carboxylic acids max. 7 wt . % Total sugars max. 9 wt . % Fats and oils max. 5 wt . % Minerals 3 - 30 wt.% is obtained.
46. A iorocess as claimed in claim 45, wherein after dewatering the L-lysine-containing product has a water content of at least 0.5 wt.%, but not more than 5.0 wt.%.
47. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product trehalose in the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not • more -than.0.5 g/1. . ,. . ,,.■«•, .. „
48. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product lactate in the . fermentation broth led off is not more than 8.0 g/1, not more than 4.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
49. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product ethanol in the fermentation broth led off is not more than 8.0 g/1, not more than 4.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
50. A process as claimed in claim 1, 2 or 3 , wherein the concentration of the by-product L-alanine in the fermentation broth led off is not more than 5.0. g/1, not more than 2.5 g/1, not more than 1.0 g/1, or not more than 0.25 g/1.
51. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product L-valine in the fermentation broth led off is not more than 5.0 g/1, not more than 2.5 g/1, not more than 1.0 g/1, or not more than 0.25 g/1.
52. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product L-glutamate in the fermentation broth led off is not more than 7.5 g/1, not more than '5.0 g/1/ not more than 2.0 g/1, or not more than 0.5 g/1.
53. A process as claimed in claim 1, 2 or, 3, wherein the - concentration of. the by-product ketoglutarate-.vin the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
54. A process as claimed in claim 1, 2 or 3 , wherein the concentration of the by-product succinate in the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
55.. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product malate in the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
56. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product oxaloacetate in the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
57. A process as claimed in claim 1, 2 or 3 , wherein the concentration of the by-product acetate in the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1.
58. A process as claimed in claim 1, 2 or 3, wherein the concentration of the by-product pyruvate in the fermentation broth led off is not more than 10 g/1, not more than 5.0 g/1, not more than 2.0 g/1, or not more than 0.5 g/1. -:, ,- ..
59. A process as claimed in claim 1, 2, 3, 29 or 45, wherein the L-lysine-containing fermentation broth led off has a total by-product concentration of not more than 2,5%.
60. A process as claimed in claim 1, 2, 3, 29 or 45, wherein the L-lysine-containing fermentation broth led off has a total by-product concentration of not more than 1,0%.
61. A process as claimed in claim 1, 2, 3, 29 or 45, wherein the L-lysine-containing fermentation broth led off has a total by-product concentration of not more than 0.5%.
62. A process as claimed in claim 45, wherein after dewatering and granulation the L-lysine-containing product has an average particle size of at least 0.1 to 1.0 mm, at least 97% or at least 98% [sic] .
63. A process as claimed. in claim 45, wherein the L-lysine-containing product has a lactate content of not more than 3 wt.%, not more than 2 wt.%, not more than 1 wt . %, not more than 0.5 wt . %, or not more than 0.1 wt . % .
64. A process as claimed in claim 45, wherein after dewatering and granulation the L-lysine-containing product has bulk density of at least 600 kg/m3, at least 650 kg/m2, at least 700 kg/m3, or at least 750 kg/m3. . .. . .
65. A process as claimed in claim 45, wherein after dewatering and granulation the L-lysine-containing product contains a content of additive added, in particular oil, at a level of 0.02 -2 wt.%, based on the total amount of L-lysine-containing product, on the surface .
66. A process as claimed in claim 65, wherein the L-lysine-containing product contains as an additive one or more of the oils chosen from the group consisting of: mineral oil, vegetable oils, soya oil, olive oil, soya/lecithin mixtures, edible oils, mixtures of vegetable oils, on the surface.
PCT/EP2004/008882 2003-08-29 2004-08-07 Process for the preparation of l-lysine WO2005021772A1 (en)

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