DE102004035347A1 - Fermentative preparation of L-lysine-containing product, involves inoculating and culturing bacterium in nutrient medium using coryneform bacteria through oxidative pentose phosphate pathway or tricarboxylic acid cycle - Google Patents

Abstract

Fermentative preparation of an L-lysine-containing product using coryneform bacteria which produces L-lysine, involves inoculating and culturing the bacterium in a first nutrient medium using a coryneform bacteria having one or more carbon flows chosen from coryneform bacteria which direct the carbon flow through the oxidative pentose phosphate pathway, tricarboxylic acid cycle, citrate synthase reaction or aspartate kinase. Fermentative preparation (M1) of an L-lysine-containing product using coryneform bacteria which produces L-lysine, involves inoculating and culturing the bacterium in a first nutrient medium, where a concentration of the carbon source of not more than 10 g/l is established in the feed-in phase during the culturing, and using a coryneform bacteria having one or more, preferably two, or three or more carbon flows chosen from coryneform bacteria which direct the carbon flow through the oxidative pentose phosphate pathway with a percentage of more than 75%, coryneform bacteria which direct the carbon flow through the tricarboxylic acid cycle, referred to the acetyl radicals which are transferred by acetyl-CoA to oxaloacetate by the citrate synthase reaction, with a percentage of 1% but not more than 20%, coryneform bacteria which direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage of 28% but not more than 60%, coryneform bacteria which direct the carbon flow through diaminopimelate dehydrogenase, coded for by ddh, with a percentage of 49% but not more than 98%, coryneform bacteria which direct the carbon flow through anaplerotic reactions, based on the sum of pyruvate and phosphoenol pyruvate (PEP), which are converted by PEP and pyruvate carboxylase, respectively, coded for by ppc and pyc, respectively, into oxaloacetate, with a percentage of more than 19%, coryneform bacteria which 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 3.4 but not more than 4.6, and coryneform bacteria 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 7.

Description

object The invention is an improved process for fermentative production of L-lysine using coryneform bacteria.

L-lysine is used in human medicine, in the pharmaceutical industry, in the food industry and especially in animal nutrition application.

It It is known that L-lysine is coryneformer by fermentation of strains Bacteria in particular Corynebacterium glutamicum are produced can. Because of the big one Meaning of this amino acid is constantly worked on improving the manufacturing process. process improvements can fermentation measures, such as. emotion and supply of oxygen, or the composition of the nutrient media, such as. the sugar concentration during fermentation, or the workup to the product form, by e.g. Ion Exchange or the intrinsic, i. the genetic performance characteristics of the bacterium itself.

In closely related to the performance of a bacterium in a given process are the rates at which metabolic processes expire. Advantageous for the fermentative production of amino acids such as L-lysine are, for example one possible high lysine production rate with the highest possible ratio of Lysine production to sugar uptake rate (yield) as well as possible low rates (ideally zero) of by-product formation.

The Measuring rates like the ones mentioned above, either recording of nutrients from the culture fluid or the delivery of products into the culture fluid by the cells (im following extracellular Called rates) is state of the art. It is too Prior art, extracellular rates within the fermented by the intrinsic properties of the fermented Organism set limits through process control measures Cheap influence, for example, the sugar intake and affect the lysine production rate through a feeding strategy as reported by Ensari and Lim (Applied Microbiology and Biotechnology 62: 35-40 (2003)). Shimizu et al. (Metabolic Engineering 1: 299-308 (1999)) describe a procedural method, the lysine productivity in the context the boundaries of a given Corynebacterium glutamicum strain by controlling the growth rate to maximize.

In contrast to the extracellular rates, the rates of metabolic intermediate metabolism within the cell are intertwined, the so-called intracellular rates or flows. For example, intracellular fluxes can be determined using isotopic labeling techniques such as 13C labeling in conjunction with nuclear magnetic resonance (NMR) or mass spectrometry (Wiechert, Metabolic Engineering 3: 195-206 (2001)). Since intracellular reactions are, with a few exceptions, enzyme-catalyzed reactions, intracellular flows can be influenced by changing the amounts of the enzymes involved or by altering the amounts of enzyme in competing metabolic pathways (eg WO 01/07626; US 6,586,214 ; Koffas et al. (Metabolic Engineering 5: 32-41 (2003)). As a measure of the amount of a particular enzyme usually its so-called specific activity is used, which can be measured, for example, in cell extracts. Neither the amount nor the specific activity of an enzyme are a measure of the flow actually catalyzed in a fermentation process.

While the Determination of intracellular rivers not in different due to their intrinsic properties optimally lysine-producing bacterial strains, in particular strains of Corynebacterium glutamicum, numerous described in the literature (e.g., Marx et al., Biotechnology and Bioengineering 49: 111-129 (1996); Wittmann and Heinzle, European Journal of Biochemistry 268: 2441-2455 (2001); Drysch et al., Biotechnology and Bioengineering 5: 497-505 (2004)), is a specification of for optimal production conditions necessary rivers so far only on the basis of non-real, because much simplified, metabolic models he follows. Stephanopoulos and Vallino (Science 252: 1675-1681 (1991)) for example, those calculated for a theoretically maximum lysine yield of necessary intracellular fluxes a model that exclusively uses the central metabolism and lysine biosynthesis, i. neither the residual growth nor considers the maintenance metabolism of a real organism.

From Varma et al. (Biotechnology and Bioengineering 42: 59-73 (1993)) was shown using the example of the bacterium Escherichia coli, such as the theoretically maximum yields of various amino acids ter also lysine, as well as the yields underlying these rivers can be calculated based on the existing biochemical reactions. Burgard and Maranas (Biotechnology and Bioengineering 74: 364-375 (2001)) extended this work to a universal, species-independent model. However, in both cases, residual growth and maintenance metabolism are also disregarded. Petersen et al. (in: Metabolic Engineering in the Post Genomic Era, Kholodenko and Westerhoff (eds.), Horizon Bioscience, Wymondham, UK (2004), pp. 237-275) describe in one example an approach to be expected in the event of a change in specific enzyme activities To calculate changes in the rivers and thus partly improvements of the lysine productivity without, however, responding to the necessary for optimal lysine productivity rivers themselves.

It It was an object of this invention to provide new measures for improved fermentative To provide production of L-lysine.

Summary the invention

• a) ein L-Lysin produzierendes coryneformes Bakterium in mindestens einem ersten Nährmedium inokuliert und kultiviert, wobei
• b) die Konzentration der Kohlenstoffquelle(n) während der Kultivierung in der Zufütterungsphase von maximal 10 g/l eingestellt wird, und
• c) coryneforme Bakterien einsetzt werden, die mindestens einen oder mehrere Kohlenstoffflüsse aufweisen, ausgewählt aus der Gruppe von i) coryneformen Bakterien die den Kohlenstofffluss durch den oxidativen Pentose-Phosphat-Weg mit einem prozentualen Anteil von mehr als 75% lenken; ii) coryneformen Bakterien die den Kohlenstofffluss durch den Tricarbonsäurezyklus, bezogen auf die Acetyl-Reste, die von Acetyl-CoA auf Oxalacetat durch die Citratsynthase Reaktion übertragen werden, mit einem prozentualen Anteil von mindestens 1% höchstens jedoch 20% lenken; iii) coryneforme Bakterien die den Kohlenstofffluss durch die Aspartatkinase, kodiert durch lysC, mit einem prozentualen Anteil von mindestens 28% höchstens jedoch 60% lenken; iv) coryneforme Bakterien die den Kohlenstofffluss durch die Diaminopimelatdehydrogenase, kodiert durch ddh, mit einem prozentualen Anteil von mindestens 49% höchstens jedoch 98% lenken; v) coryneforme Bakterien die den Kohlenstofffluss durch die anaplerotischen Reaktionen, bezogen auf die Summe an Pyruvat und Phosphoenolpyruvat (PEP), die durch die PEP- bzw. Pyruvatcarboxylase, kodiert durch ppc bzw. pyc, in Oxalacetat umgewandelt werden, mit einem prozentualen Anteil von mehr als 19% lenken; vi) coryneformen Bakterien, die die Fähigkeit besitzen, ein Verhältnis des Kohlenstoffflusses durch den oxidativen Pentose-Phosphat-Weg zu dem Kohlenstofffluss durch die anaplerotischen Reaktionen (oxidativer Pentose-Phosphat-Weg [%]/anaplerotische Reaktionen [%] = PPP/Ana [-]) von mindestens 3,4 höchstens jedoch 4,6 einzustellen; und vii) coryneforme Bakterien die die Fähigkeit besitzen, ein Verhältnis des Kohlenstoffflusses durch den oxidativen Pentose-Phosphat-Weg zu dem Kohlenstofffluss in den Tricarbonsäurezyklus (oxidativer Pentose-Phosphat-Weg [%]/ Tricarbonsäurezyklus [%] = PPP/TCA [-]) von mindestens 7 einzustellen.

The invention relates to a fermentation process, which is characterized in that

• a) inoculating and culturing an L-lysine-producing coryneform bacterium in at least one first nutrient medium, wherein
• b) the concentration of the carbon source (s) during cultivation is set at a maximum of 10 g / l in the feeding phase, and
• c) using coryneform bacteria having at least one or more carbon fluxes selected from the group of i) coryneform bacteria which direct carbon flux through the oxidative pentose-phosphate pathway at a percentage greater than 75%; ii) coryneform bacteria which direct the carbon flux through the tricarboxylic acid cycle, based on the acetyl residues transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction, at a percentage of at least 1% but at most 20%; iii) coryneform bacteria that direct carbon flux through the aspartate kinase encoded by lysC at a percentage of at least 28% but not more than 60%; iv) coryneform bacteria which direct carbon flux through the diaminopimelate dehydrogenase encoded by ddh at a percentage of at least 49% but at most 98%; v) coryneform bacteria which convert the carbon flux into an oxoacetate by the anaplerotic reactions, based on the sum of pyruvate and phosphoenolpyruvate (PEP) converted by the PEP or pyruvate carboxylase encoded by ppc and pyc respectively, into a percentage of steer more than 19%; vi) coryneform bacteria having the ability to control a ratio of carbon flux through the oxidative pentose-phosphate pathway to carbon flux through the anaplerotic reactions (oxidative pentose-phosphate pathway [%] / anaplerotic reactions [%] = PPP / Ana [ -]) of at least 3.4 but not more than 4.6; and vii) coryneform bacteria that have the ability to convert a ratio of carbon flux through the oxidative pentose-phosphate pathway to carbon flux into the tricarboxylic acid cycle (oxidative pentose-phosphate pathway [%] / tricarboxylic acid cycle [%] = PPP / TCA [-] ) of at least 7.

Of the Carbon flux is defined as the ratio of molar rates of a metabolic metabolite Single reaction or reaction sequence to the carbon uptake rate.

In In one aspect of this invention, the method is characterized that in step (c) coryneform bacteria are used which are at least the carbon flux through the oxidative pentose-phosphate pathway with a percentage of more than 75%. It is still possible, that in step (c) coryneform bacteria are used in addition to the flow of material through the oxidative pentose-phosphate pathway, a selected further material flow from group (ii) to (vii).

In Another aspect of this invention is the method step (c) characterized in that coryneform bacteria are used, the at least two of the material flows selected from group (i) to (vii).

description the invention

In addition to the exact knowledge of the cellular metabolism, a material flow analysis is necessary for a model-based improvement of the strain and the production process in which the carbon flux can be understood through the metabolism by using, for example ¹³ C-labeled substrate and the flow conditions at branches in the form of relative flows can be determined (bioreactor technique: bioprocesses with microorganisms and cells: process monitoring, K. Schügerl, Birkhäuser Verlag Basel - Boston - Berlin, 1997). Carbon flux is defined as the ratio of carbon-related molar rates of a metabolic single reaction or reaction sequence to carbon uptake. If a 13C compound is used for the labeling, it is possible to determine the distribution of the labeled atoms in the various metabolites, for example by means of nuclear resonance spectroscopy (NMR).

Own according to the invention the coryneform bacteria the ability the carbon flux through the oxidative pentose-phosphate pathway with a percentage of more than 75%, more than 85%, more than 95%, more than 105%, more than 115%, more than 125%, more than 135%, more as 145%, to steer.

Farther L-lysine producing coryneform bacteria have the ability to the carbon flux through the tricarboxylic acid cycle, based on the Acetyl residues ranging from acetyl-CoA to oxaloacetate by citrate synthase Transmit reaction with a percentage of at least 1%, however, at most 20%, at least 2% at most however 18%, at least 3% at most however 16% to steer.

Of the tricarboxylic serves in addition to the provision of reduction equivalents for synthesis of compounds that are essential precursors of amino acid synthesis pathways are. Oxaloacetate, for example, serves as a precursor to lysine synthesis. The removal of these precursors from the tricarboxylic acid cycle is facilitated by replenishment reactions, compensated for so-called anaplerotic reactions. Depending on the type of Carbon source, growth rate and product formation of coryneforms Bacteria can these reactions forward or backwards. forward in this context means that the carbon flow from the Glycolysis towards tricarboxylic acid cycle for example, from pyruvate to oxaloacetate and / or phosphoenolpyruvate to oxaloacetate). Backwards means in this context, that the carbon flux from the tricarboxylic acid cycle in the direction of glycolysis (e.g., from oxaloacetate to pyruvate). The rivers can be simultaneously in both directions. The sum of all forward minus the sum all backward rivers is called the net flow. Is the net flow forward (ie from glycolysis to the tricarboxylic acid cycle), receives he has a positive sign, he is directed backwards, he gets one negative sign. Lysine producing coryneform bacteria generally have the ability the carbon by the anaplerotic reactions of the glycolysis towards the tricarboxylic acid cycle to steer.

Especially According to the invention, L-lysine is suitable producing coryneform bacteria that have the ability to control the carbon flux by the anaplerotic reactions, based on the sum of pyruvate and phosphoenolpyruvate (PEP) produced by the PEP or pyruvate carboxylase, encoded by ppc or pyc, converted into oxaloacetate, with a percentage of more than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, more to steer as 37%. This corresponds by definition to one 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%.

Particularly according to the invention suitable are L-lysine-producing coryneform bacteria that the ability , the carbon flux through the aspartate kinase encoded by lysC, with a percentage of at least 28% at most however 60%, at least 30% at most however 57%, at least 32% at most however 53%, at least 33% at most however, to steer 50%.

Farther are according to the invention L-lysine producing coryneform bacteria suitable for the ability the carbon flux through diaminopimelate dehydrogenase, encoded by ddh, with a percentage of at least 49% at most however 98%, at least 53% at most however 95%, at least 56% at most however 91%, at least 58% at most However, to steer 87%.

According to the invention, the L-lysine producing coryneform bacteria possessing the ability to have a ratio of the carbon flux through the oxidative pentose-phosphate pathway to the carbon flux through the anaplerotic reactions (oxidative pentose-phosphate pathway [%] / anaplerotic reactions [%] logo CNRS logo INIST PPP / Ana [-]) of at least 3.4 but not more than 4.6, at least 3.5 but not more than 4.5 however, at most 3.6, but not more than 4.4, but not less than 3.7, but not more than 4.3.

Farther are L-lysine according to the invention producing suitable coryneform bacteria that have the ability to maintain a relationship of Carbon flux through the oxidative pentose-phosphate pathway too the carbon flux into the tricarboxylic acid cycle (oxidative pentose-phosphate pathway [%] / Tricarboxylic acid cycle [%] = PPP / TCA [-]) of at least 7, at least 7 but not more than 150, at least 10 at most however 125, at least 13 at most however 100, or at least 16 at most however, to set 75.

The Microorganisms which are the subject of the present invention can amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be representative Coryneformer bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art Corynebacterium glutamicum, which in the professional world for their ability is known, L-amino acids to produce.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are especially the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium molassecola ATCC17965
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-amino acid producing mutants or strains thereof, such as the L-lysine producing strains
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Corynebacterium glutamicum FERM-P 6463
Corynebacterium glutamicum FERM-P 6464 and
Corynebacterium glutamicum DSM 5715
or such as the L-methionine producing strain
Corynebacterium glutamicum ATCC21608.

With tribes the designation "ATCC" can of of the American Type Culture Collection (Manassas, Va, USA) become. strains labeled "FERM" can be dated from National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) be obtained. The mentioned strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in US-A-5,250,434.

One Another aspect of this invention is as described above Method in which the coryneform bacteria described in (c) at least three carbon flows, selected from (i) to (vii).

One Another aspect of this invention is as described above in which the coryneform bacteria described in (c) at least four or more carbon flows, selected from (i) to (vii).

Yet Another aspect of this invention is as described above Method in which the coryneform bacteria described in (c) all Carbon fluxes steer (i) to (vii).

According to the invention, the Plant performance of a L-lysine-producing fermentation unit be increased by that in the first described above Cultivation step (a) according to the batch process or feed process (fed batch) cultivated, wherein when using the feed process at least one additional nutrient medium is used. I

During the Cultivation step (a) becomes the bacterium in at least one inoculated first nutrient medium and after the batch process or fed batch process cultured. When using the feed process, after more than 0 to a maximum of 10 hours, preferably after 1 to 10 hours, preferably after 2 to 10 hours and more preferably after 3 to 7 hours an additional nutrient medium fed.

The first nutrient medium contains as carbon source one or more of the compounds selected from the group sucrose, molasses from sugar beet or cane, fructose, Glucose, starch hydrolyzate, lactose, Galactose, maltose, xylose, acetic acid, ethanol and methanol in the concentrations of 1 to 50 g / kg, preferably of 5 to 40 g / kg, more preferably from 10 to 30 g / kg. By starch hydrolyzate is meant According to the invention, the hydrolyzate of strength from corn, cereals, potatoes or tapioca.

When Nitrogen source in the first nutrient medium can organic, nitrogen-containing compounds such as peptones, yeast extract, Meat extract, malt extract, corn steep liquor, soybean meal and Urea or inorganic compounds such as ammonia, ammonium sulfate, Ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate Potassium nitrate, potassium sodium nitrate can be used. The nitrogen sources can be individually or as a mixture in the concentrations of 1 to 50 g / kg, preferred from 3 to 40 g / kg, more preferably from 5 to 30 g / kg become.

When Phosphorus source in the first nutrient medium can Phosphoric acid, Alkali or alkaline earth 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 phytic acid called in the concentrations of 0.1 to 5 g / kg, preferably from 0.3 to 3 g / kg, more preferably from 0.5 to 2.0 g / kg become. The culture medium must continue to contain salts of metals such as. Magnesium sulfate or iron sulfate necessary for growth. These substances are in the concentrations of 0.0003 to 15 g / kg before. After all can essential growth factors such as amino acids (e.g., homoserine) and vitamins (e.g., thiamine) in addition used for the above-mentioned substances. To control the Foam development can Antifoam agents such as e.g. fatty acid be used.

The Additional nutrient medium, which is used in a fed batch process generally contains merely as carbon source one or more of the compounds selected from the group sucrose, molasses from sugar beets or sugarcane, fructose, Glucose, starch hydrolyzate, Lactose, galactose, maltose, xylose, acetic acid, ethanol and methanol in the concentrations of 300 to 700 g / kg, preferably from 400 to 650 g / kg, and optionally an inorganic nitrogen source such as. Ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium carbonate, ammonium nitrate, potassium nitrate or potassium sodium nitrate. Alternatively you can These and other components can also be fed separately.

strong Growth at the beginning of cultivation is usually a logarithmic one Growth phase. The logarithmic growth phase generally follows a phase of lesser cell growth than in the logarithmic Phase.

According to the invention Concentration of carbon source during cultivation in the feed phase of not more than 10 g / l, not more than 5 g / l, preferably not more than 3 g / l, more preferably set at a maximum of 1 g / l. This is the concentration Determining the carbon source by methods, the state of the Technology are. β-D-glucose is e.g. in a glucose analyzer YSI 02700 Select of the company Yellow Springs Instruments (Yellow Springs, Ohio, USA). The term feeding phase refers to the phase of fermentation in the at least one Carbon source continuous or discontinuous to the medium supplied becomes. For fermentations in the feeding process This is usually done after full or almost complete consumption the initially submitted carbon source.

To the feeding the nutrient media, is the fermentation of the invention done so long until the concentration of the carbon source is a maximum of 2 g / l, maximum 1 g / l, or at most 0.5 g / l.

According to the invention, the yield (Y _{P / S} ) is at least 43% by weight; at least 45% by weight; at least 48% by weight; at least 50% by weight; at least 52 wt .-% amount. The yield Y _{P / S is} defined as the ratio of the total amount of L-lysine formed in a cultivation to the total amount of carbon source used or consumed.

According to the invention, L-lysine with a space-time yield (RZA) of at least 2.5 to 3.0 g / l per hour, from at least 3.0 to more than 4.0 g / l per hour, from at least 4.0 to 5.0 g / l per hour, or from at least 5.0 to 8.0, g / l or more per hour. Here is the space-time yield defined as the ratio from the total amount of L-lysine formed in a culture the actively producing volume of culture over the entire period of Cultivation seen. The space-time yield is also volumetric productivity called.

According to the invention, the L-lysine Concentration (c), based on lysine HCl, in the fermentation broth removed at least 100 g / l, at least 110 g / l, at least 120 g / l, preferably more than 130 g / l, more preferably more than 140 g / l.

In the fermentative production of L-lysine, an attempt is made to achieve and maintain an optimum function of the three performance characteristics yield, productivity and product concentration. Such an optimum can be obtained by a performance index (LI), which is composed of the product of space-time yield (RZA), yield (Y _{P / S} ) and L-lysine concentration (c) (LI [g ² / (1 ² · h) J = RZA [g / l · h] · Y _{P / S} [w / w] · c [g / l]). According to the invention, the performance index relative to the process according to the invention reaches at least 110 g ² / (1 ² .h), at least 120 g ² / (1 ² .h), at least 130 g ² / (1 ² .h), at least 150 g ² / (1 ² .h), at least 170 g ² / (1 ² .h), at least 190 g ² / (1 ² .h), at least 210 g ² / (1 ² .h), at least 230 g ² / (1 ² · h), at least 250 g ² / (1 ² · h).

During the Cultivation is the temperature in a range of 28 ° C to 40 ° C, preferably of 30 ° C up to 35 ° C, set. The fermentation may be at atmospheric pressure or optionally at overpressure, preferably at 0 to 2.5 bar overpressure, particularly preferably be carried out at 0 to 1.5 bar. The oxygen partial pressure is controlled to 5 to 50%, preferably about 20%, air saturation. The regulation the pH to pH about 6 to 8, preferably 6.5 to 7.5 with ammonia gas or 25% ammonia water. The conditions of cultivation while of cultivation remain constant or changed.

In order to meet the requirement of the performance index, during the fermentation not only a sufficient partial pressure of oxygen is to be ensured, but also a sufficient biological activity of the cells. To ensure the biological activity, the method according to claim 1, characterized in that the set in step b) oxygen uptake rate (OUR), a maximum of 350 mmol / (1 · h), a maximum of 325 mmol / (1 · H), maximum 300 mmol / (1 · h), maximum 275 mmol / (1 · h), maximum 250 mmol / (1 · h), maximum 225 mmol / (1 · h), maximum 200 mmol / (1 · H), at most 175 mmol / (1 · h), at most 150 mmol / (1 · h). The oxygen uptake rate OUR refers to the specific oxygen absorption rate by the microorganisms in mmol O ₂ per liter of fermentation broth and hour (Biotechnology, D. Schlee and H.-P. Kleber, Gustav Fischer Verlag Jena, 1991).

The described currents at first nutrient media and other nutrient media or the sum of the currents at first nutrient media and other nutrient media contain complex ingredients. As complex components become Carbon or nitrogen sources referred to in the used Form have a purity of less than 95%. In such a complex component is one or more compounds from the group Peptone, yeast extracts, meat extracts, malt extracts, Corn steep liquor and soybean meal. According to the invention, the proportion less than 10% by weight of complex constituents in the nutrient media used, less than 5 wt%, less than 2.5 wt%, less than 1.0 wt%, less as 0.5% by weight.

According to the invention, the osmolarity of the discharged L-lysine-containing fermentation broth is less as 2100 mosm / l, better less 1800 mosm / l, especially less 1500 mosm / l, preferably less 1200 mosm / l. Osmolarity is the Concentration of osmotically active particles in 1 liter of liquid volume called. For example, a 1 molar glucose solution corresponds to 1000 mosm / l (biotechnology; H. Weide, J. Páca and W.A. Knorre; Gustav Fischer Verlag Jena; 1991).

to execution the method according to the invention are representatives of coryneform bacteria, in particular of the genus Corynebacterium suitable. The coryneform bacteria are in particular around the genus Corynebacterium. In the genus Corynebacterium are in particular the species Corynebacterium glutamicum and continue the species Brevibacterium flavum and Corynebacterium thermoaminogenes to call. These are known in the art for their ability to L-Lysine too to produce. Information on the taxonomic classification of strains of these Group of bacteria can be found among others at fighters 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 especially the known wild-type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium molassecola 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-lysine-producing strains produced therefrom.

The coryneform bacteria contain at least one copy of a lysC gene or allele encoding an aspartate kinase insensitive to the inhibition of lysine or mixtures of lysine and threonine (lysC ^fbr ). Such bacteria are typically resistant to the lysine analog S- (2-aminoethyl) -cysteine (AEC).

Also suitable are L-lysine producing coryneform bacteria having one or more of the features selected from the group lysC allele (lysC ^fbr ), hom allele (hom ^leaky ), zwf allele encoding a NADPH insensitive glucose-6 Phosphate dehydrogenase, and the pyc allele encoding pyruvate carboxylase. The pyc allele is in EP 1 108 790 described.

Also suitable are L-lysine-producing coryneform bacteria which possess one or more resistances selected from the group Azauracil ^r (Aza ^r ), Rifamycin ^r (Rif ^r ), Streptomycin ^r (Strep ^r ).

Also suitable are L-lysine-producing coryneform bacteria having at least the following characteristics: two (2) copies of a lysC allele encoding a lysine-resistant aspartate kinase (lysC ^fbr ), a hom-allele encoding an attenuated homoserine dehydrogenase encodes (hom ^leaky ) and two (2) copies of a zwf allele encoding a NADPH-insensitive glucose-6-phosphate dehydrogenase.

Also suitable are L-lysine producing coryneform bacteria having one or more of the properties selected from the group of three (3), four (4), or five (5) copies of a lysC allele (lysC ^fbr ), two (2) Copies of a lysE gene, two (2) copies of a zwal gene.

Furthermore are L-lysine producing coryneform bacteria that are sensitive towards diaminopimelic acid analogs are. The term diaminopimelic acid analogs are used according to the present invention compounds such as 4-fluoro-diaminopimelic acid, 4-hydroxy-diaminopimelic acid, 4-oxo-diaminopimelic or 2,4,6-Triaminopimelinsäure summarized.

to Generation of the invention against 4-hydroxy-diaminopimelic acid sensitive coryneform bacteria, mutagenesis methods are used. For mutagenesis can classical in vivo mutagenesis methods using mutagenic Substances such as 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).

The coryneform bacteria that are sensitive to 4-hydroxy-diaminopimelic acid, can through Plating on 4-hydroxy-diaminopimelinsäurehaltigen Nährmediumsplatten be identified. For this purpose, final concentrations are particularly suitable from about 5 to 15 g / l. for example, about 10 g / l of 4-hydroxy-diaminopimelic acid in the nutrient medium. At this concentration can 4-hydroxy-diaminopimelic acid sensitive Mutants of the unchanged parent strains be differentiated by a slower growth. After successful Selection show the 4-hydroxy-diaminopimelic acid sensitive mutants an improved L-lysine production.

For the described Processes are correspondingly stable strains that have their production characteristics Do not lose in the course of the procedure, especially suitable.

The targeted change of strain characteristics coryneformer bacteria, the composition of the Culture Media and the nature of the litigation all have the goal, the carbon source used as possible to convert effectively into L-lysine. For this it is necessary that the Carbon as possible without consuming side reactions and largely unhindered flows through the cell metabolism towards L-lysine synthesis.

The L-lysine-containing fermentation broth from the process according to the invention has a solids content of at least 10% by weight, at least 12.5 wt .-%, at least 15 wt .-%, at least 17.5 wt .-%.

The produced L-lysine can then be purified from the fermentation broth become. Separation methods are used to remove or separate the biomass such as centrifugation, filtration, decantation, flocculation or a combination thereof. The L-lysine-containing broth is then with known methods such as ion exchange chromatography, Ion exclusion chromatography, extraction, crystallization, precipitation or a combination of this on cleaned.

alternative For this purpose, the L-lysine-containing fermentation broth can also be dehydrated. For this purpose, fermentation broth with known methods such as with the aid of a rotary evaporator, Thin film evaporator, Falling film evaporator, by reverse osmosis, by nanofiltration or a combination of this thickened or concentrated. The water is hereby removed to 10% to 90%.

to improved preservation of the L-lysine-containing liquid product can by adding acid or Lye the pH in the acidic (pH 2 to 5) or alkaline (pH 9 to 12) area changed become.

It is also possible from the fermentation broth to produce a product by containing those contained in the culture broth Biomass of bacteria completely (1000 or almost complete i.e. more than or greater than (>) 90%,> 95%,> 97%,> 99% removed and the remaining ingredients the fermentation broth largely i. to 30% -100%, 40% -100%, 50% -100%, 60% -100%, 70% -100%, 80% -100%, or 90% - 100, preferably greater or equal (≥) 50%, ≥ 60%, ≥ 70%, ≥ 80%, ≥ 90% or ≥ 95% or also completely (100%) in the product leaves.

to Removal or separation of the biomass are separation methods such as centrifugation, filtration, decantation, flocculation or a combination thereof.

The preserved broth will follow with known methods such as with the aid of a rotary evaporator, Thin film evaporator, Falling film evaporator, by reverse osmosis, by nanofiltration or a combination of this thickened or concentrated. The water is hereby removed to 10% to 90%.

Farther can concentrated broth subsequently by freeze drying, spray drying, spray granulation or by other methods to a preferably free-flowing, finely divided Be worked up powder. This free-flowing, finely divided powder can then again by suitable compacting or granulation process into a coarse-grained, good flowable, be stored storable and largely dust-free product. The water will in this case, a total of more than 90% removed, so that the water content in the product is less than 10%, less than 5%.

The specified method steps must not necessarily performed in the order listed here but can possibly combined in a technically meaningful way.

The inventive method stands opposite the usual fed batch process, mainly characterized by an increased space-time yield.

Preferably, an L-lysine-containing product of the following composition is obtained from the fermentation broth by removing water: lysine 35-80% by weight protein Max. 7% by weight carboxylic acids Max. 7% by weight total sugar Max. 9% by weight fats and oils Max. 5% by weight minerals 3-30% by weight

According to the invention this has L-lysine containing product after dehydration a water content of at least 0.5% by weight, but at most 5.0% by weight.

Furthermore, for the preparation of the lysine-containing product, the fermentation broth should be as follows Adhere to byproduct concentrations. A trehalose concentration of less than or equal to (≤) 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. An L-alanine concentration of ≤ 5.0 g / l, ≤ 2.5 g / l, ≤ 1.0 g / l, ≤ 0.25 g / l. An L-valine concentration of ≤ 5.0 g / l, ≤ 2.5 g / l, ≤ 1.0 g / l, ≤ 0.25 g / l. An L-glutamate concentration of ≤ 7.5 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. An ethanol concentration of ≤ 8.0 g / l, ≤ 4.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. A lactate concentration of ≤ 8.0 g / l, ≤ 4.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. A ketoglutarate concentration of ≤ 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. A succinate concentration of ≤ 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. A malate concentration of ≤ 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. An oxaloacetate concentration of ≤ 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. An acetate concentration of ≤ 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l. A pyruvate concentration of ≤ 10 g / l, ≤ 5.0 g / l, ≤ 2.0 g / l, ≤ 0.5 g / l.

According to the invention for the production a product containing L-lysine, that the L-lysine containing fermentation broth a total by-product concentration of not more than 5.0%, preferably maximum 4.0%, 3.0%, 2.5%, 2.0%, more preferably at most 1.5%, 1.0% or 0.5%. It is especially desirable that the L-lysine-containing fermentation broth has a total by-product concentration of less than 0.5%.

In accordance with the invention the L-lysine-containing product after dehydration and subsequent granulation 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, the product containing L-lysine after dehydration and granulation 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 .

As in US Patent Application Ser. 10 / 319,843 can the product containing L-lysine after dehydration and granulation Add additive to the properties of the product too improve. The proportion of added additive, especially oil, should be included 0.02 - 2.0 Wt .-%, based on the total amount of L-lysine product, at the surface be.

According to the invention, this contains L-lysine containing product as an additive one or more of the oils selected from the group of mineral oil, vegetable oils, Soybean oil, Olive oil, Soy / lecithin mixtures, edible oils, mixtures vegetable oils on the surface.

recommendable is in any case that the L-lysine-containing product after the drainage and granulation 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 achieved by anion exchange chromatography followed by Ninhydrin derivatization, as in Spackman et al. (Analytical Chemistry 30: 1190-1206 (1958)), or it can be done by reversed phase HPLC, as in Lindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)) described.

Claims (68)

A process for the fermentative production of a lysine-containing product using L-lysine-producing coryneform bacteria, characterized in that a) inoculating and cultivating the bacterium in at least one first nutrient medium, b) a maximum concentration of the carbon source during the cultivation in the feeding phase 10 g / l, and c) using coryneform bacteria having at least one or more carbon fluxes selected from the group of (i) coryneform bacteria that regulate carbon flux through the oxidative pentose-phosphate pathway in a percentage of more than 75% steer; (ii) coryneform bacteria which direct the carbon flux through the tricarboxylic acid cycle, relative to the acetyl residues transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction, at a percentage of at least 1% but at most 20%; (iii) coryneform bacteria which direct carbon flux through the aspartate kinase encoded by lysC at a percentage of at least 28% but not more than 60%; (iv) coryneform bacteria which direct carbon flux through the diaminopimelate dehydrogenase encoded by ddh at a percentage of at least 49% but at most 98%; (c) coryneform bacteria that control carbon flux through anaplerotic reactions, based on the The sum of pyruvate and phosphoenolpyruvate (PEP) converted by the PEP or pyruvate carboxylase, encoded by ppc and pyc respectively, into oxaloacetate at a percentage of greater than 19%; (vi) coryneform bacteria having the ability to control a ratio of carbon flux through the oxidative pentose-phosphate pathway to carbon flux through the anaplerotic reactions (oxidative pentose-phosphate pathway [%] / anaplerotic reactions [%] = PPP / Ana [-]) of at least 3.4 but not more than 4.6; and (vii) coryneform bacteria having the ability to convert a ratio of carbon flux through the oxidative pentose-phosphate pathway to carbon flux into the tricarboxylic acid cycle (oxidative pentose-phosphate pathway [%] / tricarboxylic acid cycle [%] = PPP / TCA [-] ]) of at least 7. Method according to claim 1, characterized in that coryneform bacteria are used in step (c), the at least the oxidative pentose-phosphate pathway with a percentage Steer more than 75% share Method according to claim 2, characterized in that coryneform bacteria are used in step (c), the additional at least one material flow selected from the group (ii) to (vii) directs. Process for the fermentative production of a lysine containing product using L-lysine producing coryneforms Bacteria, characterized in that a) the bacterium in at least a first nutrient medium inoculated and cultured, wherein b) a concentration of Carbon source during Cultivation in the feeding phase of a maximum of 10 g / l, and c) coryneform bacteria are used, the at least two carbon flows, selected from the group of (i) coryneform bacteria the carbon flux by the oxidative pentose-phosphate pathway with a percentage Steer more than 75% share; (ii) coryneform bacteria the carbon flux through the tricarboxylic acid cycle, based on the Acetyl residues transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction with a percentage of at least 1% at most however, steer 20%; (iii) coryneform bacteria control the carbon flux by the aspartate kinase encoded by lysC, with a percentage Share of at least 28% at most but steer 60%; (iv) coryneform bacteria control the carbon flux by diaminopimelate dehydrogenase, encoded by ddh, with one percentage of at least 49% but not more than 98%; (V) coryneform bacteria that regulate the carbon flux through the anaplerotic Reactions based on the sum of pyruvate and phosphoenolpyruvate (PEP), which by the PEP or Pyruvatcarboxylase, coded by ppc and pyc, respectively, are converted to oxaloacetate with a percentage Steer more than 19% share; (vi) coryneform bacteria the ability own a relationship the carbon flux through the oxidative pentose-phosphate pathway to the carbon flux through the anaplerotic reactions (oxidative Pentose phosphate pathway [%] / anaplerotic reactions [%] = PPP / Ana [-]) of at least 3.4 at most however, set 4.6; and (vii) coryneform bacteria the the ability own a relationship the carbon flux through the oxidative pentose-phosphate pathway to the carbon flux into the tricarboxylic acid cycle (oxidative pentose-phosphate pathway [%] / tricarboxylic acid cycle [%] = PPP / TCA [-]) of at least 7. Process for the fermentative production of a lysine-containing product using L-lysine-producing coryneform bacteria, characterized in that a) inoculating and cultivating the bacterium in at least one first nutrient medium, b) a maximum concentration of the carbon source during the cultivation in the feeding phase 10 g / l is set, and c) coryneform bacteria are used which have at least three or more carbon fluxes selected from the group of (i) coryneform bacteria that control the carbon flux through the oxidative pentose-phosphate pathway in a percentage greater than Steer 75%; (ii) coryneform bacteria which direct the carbon flux through the tricarboxylic acid cycle, relative to the acetyl residues transferred from acetyl-CoA to oxaloacetate by the citrate synthase reaction, at a percentage of at least 1% but at most 20%; (iii) coryneform bacteria which direct carbon flux through the aspartate kinase encoded by lysC at a percentage of at least 28% but not more than 60%; (iv) coryneform bacteria which direct carbon flux through the diaminopimelate dehydrogenase encoded by ddh at a percentage of at least 49% but at most 98%; (c) coryneform bacteria which convert the carbon flux by the anaplerotic reactions, in terms of the percentage of pyruvate and phosphoenolpyruvate (PEP) converted by the PEP or pyruvate carboxylase encoded by ppc and pyc into oxaloacetate, respectively steer more than 19%; (vi) coryneform bacteria having the ability to control a ratio of carbon flux through the oxidative pentose-phosphate pathway to carbon flux through the anaplerotic reactions (oxidative pentose-phosphate pathway [%] / anaplerotic reactions [%] = PPP / Ana [ -]) of at least 3.4 but not more than 4.6; and (vii) coryneform bacteria having the ability to convert a ratio of carbon flux through the oxidative pentose-phosphate pathway to carbon flux into the tricarboxylic acid cycle (oxidative pentose-phosphate pathway [%] / tricarboxylic acid cycle [%] = PPP / TCA [-] ]) of at least 7. Method according to claim 4 or 5, characterized in that the coryneform bacteria used all carbon flows steer (i) to (vii). Method according to claim 1, 4 or 5, characterized in that the culturing step (a) carried out according to the batch procedure. Method according to claim 1, 4 or 5, characterized in that the culturing step (a) is carried out according to the fed batch process, wherein at least one additional nutrient medium is used. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through the oxidative pentose-phosphate pathway with a percentage Share of more than 105%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through the oxidative pentose-phosphate pathway with a percentage Proportion of more than 125%. Method according to claim 1, 2 4, or 5, characterized in that the coryneform bacteria the Carbon flux through the anaplerotic reactions, based on the sum of pyruvate and phosphoenolpyruvate (PEP) produced by the PEP or Pyruvatcarboxylase, encoded by ppc or pyc, converted into oxaloacetate 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%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through the tricarboxylic acid cycle, based on the Acetyl residues ranging from acetyl-CoA to oxaloacetate by citrate synthase Transmit reaction with a percentage of at least 2% at most but steer 18%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through the tricarboxylic acid cycle, based on the Acetyl residues ranging from acetyl-CoA to oxaloacetate by citrate synthase Transmit reaction with a percentage of at least 3% at most but steer 16%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through aspartate kinase encoded by lysC a percentage of at least 30% but not more than 57%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through aspartate kinase encoded by lysC a percentage of at least 32% but not more than 53%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the Carbon flux through the diaminopimelate dehydrogenase encoded by ddh, with a percentage of at least 49% at most however 98%, at least 53% at most however 95%, at least 56% at most 91%, or at least 58%, but not more than 87%. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the the ability own a relationship the carbon flux through the oxidative pentose-phosphate pathway to the Carbon flux through the anaplerotic reactions (oxidative Pentose phosphate route [%] / anaplerotic reactions [%] = PPP / Ana [-]) of at least 3.4 at most however 4.6, but at most 3.5 at most 4.5, at least 3.6 at most however, set 4.4, or at least 3.7, but not more than 4.3. Method according to claim 1, 2, 4 or 5, characterized in that the coryneform bacteria the the ability own a relationship the carbon flux through the oxidative pentose-phosphate pathway to the Carbon flux into the tricarboxylic acid cycle (oxidative pentose-phosphate pathway [%] / Tricarboxylic [%] = PPP / TCA [-]) of at least 7 but not more than 150, at least 10 at most however 125, at least 13 at most however 100, or at least 16 at most however, to set 75. Method according to claim 1, 2, 3, 4, 5, 6, 7 or 8, characterized in that it is at the carbon source around one or more of the compounds selected from the group sucrose, molasses from sugar beets or sugarcane, fructose, Glucose, starch hydrolyzate, Maltose, xylose, acetic acid, Ethanol and methanol. Method according to claim 1, 2, 3, 4, 5, 6, 7 or 8, characterized in that it is at the nitrogen source to one or more organic, nitrogen-containing Substances or mixtures selected from the group Peptone, yeast extracts, meat extracts, malt extracts, Corn steep liquor, soybean meal and urea and / or one or more several of the inorganic compounds selected from the group ammonia, Ammonium-containing salts and salts of nitric acid is. Method according to claim 20, characterized in that it is in ammonium-containing salts and Salts of nitric acid ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, Ammonium nitrate, potassium nitrate and potassium sodium nitrate. Method according to claim 1, 2, 3, 4, 5, 6, 7 or 8, characterized in that it is in the phosphorus source to phosphoric acid or its polymers or phytic acid or alkali or alkaline earth salts of phosphoric acid. Method according to claim 22, characterized in that it is the alkali metal salts of phosphoric acid potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. Method according to claim 1, 2, 3, 4 or 5, characterized in that a concentration the carbon source during Cultivation in the feeding phase of a maximum of 10 g / l, 5 g / l or 3 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that a concentration the carbon source during Cultivation in the feeding phase of a maximum of 3 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that, after feeding the nutrient media, the fermentation is carried out until the concentration is reached the carbon source maximum 2 g / l, maximum 1 g / l, or maximum 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the yield is at least 43 Wt .-%, based on the sugar used. Method according to claim 1, 2, 3, 4 or 5, characterized in that the yield is at least 52 Wt .-%, based on the sugar used. Method according to claim 1, 2, 3, 4 or 5, characterized in that the space-time yield at least 2.5 g / (lh) is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the space-time yield at least 4.0 g / (lh) to 5.0 g / (lh) is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the lysine HCl in the discharged Fermentation broth ≥ 100 g / l, ≥ 110 g / l, ≥ 120 g / l, ≥ 130 g / l, or ≥ 140 g / l. A method according to claim 1, 2, 3, 4 or 5, characterized in that the formation of L-lysine, based on lysine HCl, with a power index (LI) of at least 110 g ² / (1 ² · h) he follows. A method according to claim 32, characterized in that the formation of the L-lysine, based on lysine HCl, with a power index (LI) of at least 130 g ² / (1 ² · h) takes place. Method according to claim 1, 2, 3, 4 or 5, characterized in that in step a) used nutrient media complex constituents from the group of peptones, yeast extracts, meat extracts, Malt extracts, corn steep liquor and soybean meal. Method according to claim 28, characterized in that the proportion of complex components in the nutrient media used less than 5 wt .-% is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the discharged L-lysine containing fermentation broth an osmolarity of a maximum of 2100 mosm / l, a maximum of 1800 mosm / l, a maximum of 1500 mosm / l, or at most 1200 mosm / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that it is in the Coryneform bacteria is the genus Corynebacterium. Method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the L-lysine containing fermentation broth with 0% -100% the biomass produced in the fermentation after a 10% -90% dehydration as a liquid product will be produced. Method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the coryneforme Bacterium contains at least one lysC gene or allele for one Aspartate kinase, which is opposite to the inhibition by lysine or mixtures of lysine and threonine is insensitive. A method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the L-lysine producing coryneform bacterium has one or more of the features selected from the group lysC allele (lysC ^fbr ), hom allele (hom ^leaky ) , zwf allele encoding an NADPH-sensitive glucose-6-phosphate dehydrogenase, and pyc allele encoding a pyruvate carboxylase. A method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the L-lysine-producing coryneform bacterium one or more resistances selected from the group azauracil ^r (Aza ^r ), rifamycin ^r (Rif ^r ), streptomycin ^r (Strep ^r ), owns. A method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the L-lysine producing coryneform bacterium comprises at least the following properties: two (2) copies of a lysC allele (lysC ^fbr ), a hom allele ( hom ^leaky ) and two (2) copies of a zwf allele encoding a NADPH-sensitive glucose-6-phosphate dehydrogenase. A method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the L-lysine producing coryneform bacterium has one or more of the properties selected from the group of three (3), four (4), or five (5) Copies of a lysC allele (lysC ^fbr ), two (2) copies of a lysE gene, two (2) copies of a zwal gene. Method according to claim 1, 2, 3, 4, 5 or 37, characterized in that the L-lysine producing coryneform bacterium sensitive to diaminopimelic acid analogues selected from the group 4-hydroxy-diaminopimelic acid, 4-fluoro-diaminopimelic acid, 4-oxo-diaminopimelic and 2,4,6-Triaminopimelinsäure, preferably 4-hydroxy-diaminopimelic acid. Method according to claim 1, 2, 3, 4 or 5, characterized in that contained in the fermentation broth L-lysine is purified. Method according to claim 1, 2, 3, 4, 5 or 38, characterized in that the fermentation broth a Solids content of at least 10% by weight, at least 12.5% by weight, at least 15% by weight, or at least 17.5% by weight. A method according to claim 1, 2, 3, 4 or 5, characterized in that the fermentation broth is concentrated by the removal of water and a L-lysine-containing product consisting of lysine 35-80% by weight protein Max. 7% by weight carboxylic acids Max. 7% by weight total sugar Max. 9% by weight fats and oils Max. 5% by weight minerals 3-30% by weight is won. Method according to claim 47, characterized in that the L-lysine-containing product after drainage has a water content of at least 0.5, but at most 5.0 wt .-%. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product trehalose in the fermentation broth maximum 10 g / l, a maximum of 5.0 g / l, a maximum of 2.0 g / l, or a maximum of 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product lactate in the fermentation broth maximum 8.0 g / l, maximum 4.0 g / l, a maximum of 2.0 g / l, or a maximum of 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product ethanol in the fermentation broth maximum 8.0 g / l, maximum 4.0 g / l, maximum 2.0 g / l, or maximum 0.5 g / l is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product L-alanine in the fermentation broth maximum 5.0 g / l, maximum 2.5 g / l, maximum 1.0 g / l, or maximum 0.25 g / l is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product L-valine in the fermentation broth maximum 5.0 g / l, maximum 2.5 g / l, maximum 1.0 g / l, or maximum 0.25 g / l is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product L-glutamate in the fermentation broth maximum 7.5 g / l, maximum 5.0 g / l, maximum 2.0 g / l, or maximum 0.5 g / l is. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product ketoglutarate in the fermentation broth maximum 10 g / l, a maximum of 5.0 g / l, a maximum of 2.0 g / l, or a maximum of 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product succinate in the fermentation broth maximum 10 g / l, a maximum of 5.0 g / l, a maximum of 2.0 g / l, or a maximum of 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product malate in the fermentation broth maximum 10 g / l, maximum 5.0 g / l, maximum 2.0 g / l, or at most 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product oxaloacetate in the fermentation broth maximum 10 g / l, a maximum of 5.0 g / l, a maximum of 2.0 g / l, or a maximum of 0.5 g / l. Method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of the by-product acetate in the fermentation broth maximum 10 g / l, maximum 5.0 g / l, maximum 2.0 g / l, or at most 0.5 g / l. A method according to claim 1, 2, 3, 4 or 5, characterized in that the concentration of Ne benproduktes pyruvate in the fermentation broth at a maximum of 10 g / l, a maximum of 5.0 g / l, a maximum of 2.0 g / l, or a maximum of 0.5 g / l. Method according to claim 1, 2, 3, 32 or 41, characterized in that the discharged L-lysine containing fermentation broth a total by-product concentration has a maximum of 2.5%. Method according to claim 1, 2, 3, 32 or 41, characterized in that the discharged L-lysine containing fermentation broth a total by-product concentration of not more than 1.0%. Method according to claim 1, 2, 3, 32 or 41, characterized in that the discharged L-lysine containing fermentation broth a total by-product concentration of a maximum of 0.5%. Method according to claim 47, characterized in that the L-lysine-containing product after drainage and granulation has an average particle size of at least 0.1 to 1.0 mm, at least 97%, or at least 98%. Method according to claim 47, characterized in that the L-lysine-containing product a lactate content of at most 3 wt .-%, at most 2 wt .-%, maximum 1 wt .-%, at most 0.5 wt .-%, or at most 0.1 wt .-% has. A method according to claim 47, characterized in that the L-lysine-containing product after dehydration and granulation a bulk density of at least 600 kg / m ³ , at least 650 kg / m ³ , at least 700 kg / m ³ , or at least 750 kg / m ³ has. Method according to claim 47, characterized in that the L-lysine-containing product after drainage and granulation a proportion of added additive, in particular oil in the amount of 0.02-2 wt .-% based on the total amount of the product containing L-lysine the surface contains. Method according to claim 67, characterized in that the L-lysine-containing product as an additive one or more of the oils selected from the group: mineral oil, vegetable oils, soybean oil, olive oil, soya / lecithin mixtures, Edible oils, Mixtures of vegetable oils on the surface contains.