WO2005021771A2

WO2005021771A2 - Process for the production of l-lysine

Info

Publication number: WO2005021771A2
Application number: PCT/EP2004/009385
Authority: WO
Inventors: Sören PETERSEN; Marc Gerigk; Thomas Hermann; Brigitte Bathe
Original assignee: Degussa Ag
Priority date: 2003-08-29
Filing date: 2004-08-23
Publication date: 2005-03-10
Also published as: WO2005021771A3

Abstract

The invention relates to an improved process for the production of L-lysine by fermentation using L-lysine-producing coryneform bacteria.

Description

Process for the Production of L-Lysine

Field of the Invention

The invention provides an improved process for the production of -lysine by fermentation using coryneform bacteria.

Background of the Invention

L-Lysine is used in human medicine, in the pharmaceuticals industry, in the foodstuffs industry and, very especially, in animal feeds .

It is known that L-lysine can be produced by fermentation of strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of the great importance of this amino acid, attempts are continuously being made to improve the production processes . Improvements to the processes may concern measures relating to the fermentation, such as, for example, stirring and oxygen supply, or the composition of the nutrient media, such as, for example, the sugar concentration during the fermentation, or working up to the product form by, for example, ion-exchange chromatography, or the intrinsic, i.e. the genetically determined, performance properties of the bacterium itself.

The rates at which metabolic processes occur are closely related to the efficiency of a bacterium in a given process. For example, as high a lysine production rate as possible with at the same time the highest possible ratio of lysine production rate to sugar uptake rate (yield) as well as the lowest possible (ideally zero) formation of byproducts are advantageous for the fermentative production of amino acids such as L-lysine.

The measurement of rates such as the aforementioned rates, that characterize either the uptake of nutrients from the culture liquid or the release of products to the culture liquid by the cells (hereinafter termed extra-cellular rates) , is known in the prior art. It is also known in the prior art to influence favorably extra-cellular rates within the limits imposed by the intrinsic properties of the fermented organism, by process control or process regulation measures, so that for example the sugar uptake rate and the lysine production rate can be influenced by a feed strategy, as is described by Ensari and Li (Applied Microbiology and Biotechnology 62: 35-40 (2003)). Shimizu et al . (Metabolic Engineering 1: 299-308 (1999)) describe a process technology method for maximizing lysine productivity within the framework of the limits of a given Corynebacterium glutamicum strain by controlling the growth rate.

In contrast to the extra-cellular rates, the reaction rates of metabolic intermediates within the cell, the so-called intra-cellular rates or flows, are related to one another. Intra-cellular flows can be determined for example by means of isotopic labeling techniques such as the 13C labeling technique in combination with nuclear magnetic resonance spectroscopy (NMR) or mass spectrometry (Wiechert, Metabolic Engineering 3: 195-206 (2001)). Since intra- cellular conversions are, with a few exceptions, enzyme- catalyzed reactions, intra-cellular flows can be influenced by altering the amounts of the participating enzymes or also by altering the enzyme amounts of competing metabolic pathways (e.g. WO 01/07626; US 6,586,214; Koffas et al . (Metabolic Engineering 5: 32-41 (2003)). The so-called specific activity of an enzyme is generally employed as a measure of the amount of this enzyme, and can be measured for example in cell extracts. Neither the amount nor the specific activity of an enzyme are in this connection a measure of the actually catalyzed flow in a fermentation process. Whereas the determination of the intra-cellular flows in various bacterial strains, in particular strains of Corynebacterium glutamicum, that do not optimally produce lysine on account of their intrinsic properties, has been described in numerous literature references (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)), the flows necessary for optimal production conditions have hitherto been specified only on the basis of greatly simplified and therefore unreal metabolic models . Stephanopoulos and Vallino (Science 252: 1675-1681 (1991)) calculated for example the intra-cellular flows necessary for a theoretically maximum lysine yield, on the basis of a model that involves exclusively the central metabolism and the lysine biosynthesis, i.e. that takes into account neither the residual growth nor the conservation metabolism of a real organism.

It was shown by Varma et al . (Biotechnology and

Bioengineering 42: 59-73 (1993)) with the example of the bacterium Escherichia coli how the theoretically maximum yields of different amino acids, including also lysine, as well as the flows on which these yields are based, can be calculated by means of the occurring biochemical reactions. Burgard and Maranas (Biotechnology and Bioengineering 74: 364-375 (2001)) expanded this work to a universal, species- independent model. However, in both cases residual growth and conservation metabolism are likewise not taken into account. 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 for calculating the changes in the flows and thus also to some extent improvements in lysine productivity to be expected on altering specific enzyme activities, without however discussing the flows necessary for an optimal lysine productivity.

Object of the Invention

The object of the present invention was to provide new measures for the improved fermentative production of L-lysine.

Summary of the Invention

The invention provides a fermentation process which is characterized in that a) an L-lysine-producing coryneform bacterium is inoculated and cultured in at least one first nutrient medium, wherein b) the concentration of the carbon source (s) during the culturing is set at not more than 10 g/1 in the feed-in phase, and c) coryneform bacteria are used that have at least one or more carbon flows and that are selected from the group of

(i) coryneform bacteria that direct the carbon flow through the oxidative pentose phosphate pathway with a percentage fraction of more than 75%;

(ii) coryneform bacteria that direct the carbon flow through the tricarboxylic acid cycle, referred to the acetyl radicals that are transferred by acetyl-CoA to oxaloacetate by the citrate synthase reaction with a percentage fraction of at least 1% but at most 20%; (iii) coryneform bacteria that direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage fraction of at least 28% but at most 60%;

(iv) coryneform bacteria that direct the carbon flow through diaminopimelate dehydrogenase, coded for by ddh, with a percentage fraction of at least 49% but at most 98%;

(v) coryneform bacteria that direct the carbon flow through the anaplerotic reactions, referred to the sum total of pyruvate and phosphoenol pyruvate (PEP) that are converted into oxaloacetate by PEP carboxylase and pyruvate carboxylase, coded for by ppc and pyc, with a percentage fraction of more than 19%;

(vi) coryneform bacteria that have the ability to adjust 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 at most 4.6; and

(vii) coryneform bacteria that have the ability to adjust a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow in the tricarboxylic acid cycle (oxidative pentose phosphate pathway [%] /tricarboxylic acid cycle [%] = PPP/TCA [-] ) of at least 7. The carbon flow is in this connection defined as the ratio of the molar rates referred to carbon of an individual reaction or reaction sequence occurring during metabolism, to the carbon uptake rate.

According to one aspect of the present invention the process is characterized in that in step c) coryneform bacteria are used that at least direct the carbon flow through the oxidative pentose phosphate pathway with a percentage fraction of more than 75%. It is also possible to use in step c) coryneform bacteria that, in addition to the substance flow through the oxidative pentose phosphate pathway, direct a further material flow selected from the group (ii) to (vii) .

According to a further aspect of the present invention the process step c) is characterized in that coryneform bacteria are used that direct at least two of the substance flows selected from the group (i) to (vii) .

Detailed Description of the Invention

In addition to precise knowledge of the cell metabolism, a flow analysis is necessary for a model-based improvement of the strain and of the production process, in which analysis, by the use of, for example, ¹³C-labelled substrate, the carbon flow through the metabolism can be understood and the flow ratios at branches can be determined in the form of relative flows (Bioreaktions- technik: Bioprozesse it Mikroorganismen und Zellen: Prozessϋberwachung; K. Schύgerl; Birkhauser Verlag Basel - Bosten - Berlin; 1997) . The carbon flow is defined as the ratio of the molar rates, based on carbon, of an individual reaction or reaction sequence taking place in the metabolism to the carbon uptake. If a ¹³C compound is used for labeling, it is possible to determine the distribution of the labeled atoms in the various metabolites by means of nuclear resonance spectroscopy (NMR) , for example. According to the invention, the coryneform bacteria have the ability to direct the carbon flow 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 than 145%.

Furthermore, the L-lysine-producing coryneform bacteria have the ability to direct 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 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%.

As well as providing reduction equivalents, the tricarboxylic acid cycle also serves to synthesize compounds that are important precursors of the amino acid synthesis pathways. Oxaloacetate, for example, serves as a precursor of lysine synthesis . The removal of those precursors from the tricarboxylic acid cycle is compensated for by filling-up reactions, so-called anaplerotic reactions. Depending on the nature of the carbon source, the growth rate and product formation of the coryneform bacteria, such reactions can proceed forwards or backwards. Forwards in this context means that the carbon flow takes place from glycolysis in the direction towards the tricarboxylic acid cycle (e.g. from the pyruvate to the oxaloacetate and/or from the phosphoenol pyruvate to the oxaloacetate) . Backwards in this context means that the carbon flow proceeds from the tricarboxylic acid cycle in the direction towards glycolysis (e.g. from the oxaloacetate to the pyruvate) . It is possible for the flows to run in both directions simultaneously. The sum of all forwardly-directed flows minus backwardly-directed flows is referred to as the net flow. If the net flow is directed forwards (i.e. from glycolysis to the tricarboxylic acid cycle) , it is given a positive symbol; if it is directed backwards, it receives a negative symbol. Lysine-producing coryneform bacteria generally have the ability to direct the carbon through the anaplerotic reactions from glycolysis in the direction towards the tricarboxylic acid cycle.

Particularly suitable according to the invention are L- lysine-producing coryneform bacteria that have the ability to direct the carbon flow through the anaplerotic reactions, referred to the sum total of pyruvate and phosphoenol pyruvate (PEP) that are converted by PEP carboxylase and pyruvate carboxylase, coded for by ppc and pyc, into oxaloacetate, with a percentage fraction of more . than 19%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35% and more than 37%. This corresponds according to the definition to a net flow via 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% and more than 37%.

L-Lysine-producing coryneform bacteria that are particularly suitable according to the invention have the ability to direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage 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%.

Furthermore, according to the invention L-lysine-producing coryneform bacteria are suitable that have the ability to direct the carbon flow through diaminopimelate dehydrogenase, coded for by ddh, with a percentage 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.

Furthermore, according to the invention also suitable are L-lysine-producing coryneform bacteria that 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, 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 microorganisms that are the subject of the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol . The microorganisms may be coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium there should be mentioned in particular the species Corynebacterium glutamicum, which is known to persons skilled in the art for its ability to produce L-amino acids.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the following known wild type strains

Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidophilum ATCC13870 Corynebacterium melassecola ATCC17965 O 2005/021771 10

Corynebacterium thermoaminogenes FERM BP-153 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020

and mutants and strains obtained therefrom that produce L-amino acids, such as for example 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 for example the L-methionine-producing strain Corynebacterium glutamicum ATCC21608.

Strains identified by "ATCC" can be obtained from the American Type Culture Collection (Manassas, VA, USA) . Strains identified by "FERM" can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan) . The aforementioned strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in US-A-5, 250, 434.

A further aspect of the present invention is a process as described above in which the coryneforme bacteria described in c) direct at least three carbon flows selected from (i) to (vii) .

Yet a further aspect of the present invention is a process as described above in which the coryneforme bacteria described in c) direct at least four or more carbon flows selected from (i) to (vii) . Yet another aspect of the present invention is a process as described above in which the coryneform bacteria described in c) direct all carbon flows (i) to (vii) .

According to the invention, the performance of an L-lysine- producing fermentation unit can be increased by carrying out the culturing in the above-described first culturing step (a) by the batch or fed batch process, at least one additional nutrient medium being used when the fed batch process is employed.

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

The first nutrient medium contains as the carbon source one or more compounds selected from the group saccharose, molasses from sugar beet or sugar cane, fructose, glucose, starch hydrolysate, lactose, galactose, maltose, xylose, acetic acid, ethanol and methanol in concentrations of from 1 to 50 g/kg, preferably from 5 to 40 g/kg, particularly preferably from 10 to 30 g/kg. Starch hydrolysate is understood according to the invention to be the hydrolysate of starch from corn, cereals, potatoes or tapioca.

There may be used as the nitrogen source in the first nutrient medium organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate potassium nitrate, potassium sodium nitrate. The nitrogen sources can be used individually or in the form of a mixture in concentrations of from 1 to 50 g/kg, preferably from 3 to 40 g/kg, particularly preferably from 5 to 30 g/kg.

There may be used as the phosphorus source in the first nutrient medium phosphoric acid, alkali salts or alkaline earth salts of phosphoric acid, especially 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 known as phytinic acid, in concentrations of from 0.1 to 5 g/kg, preferably from 0.3 to 3 g/kg, particularly preferably from 0.5 to 2.0 g/kg. The culture medium must also contain salts of metals, such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. These substances are present in concentrations of from 0.0003 to 15 g/kg. Finally, essential growth substances such as amino acids (e.g. homoserine) and vitamins (e.g. thiamine) can be used in addition to the above-mentioned substances. In order to control the formation of foam, antifoams, such as, for example, fatty acid polyglycol esters, can be used.

The additional nutrient medium, which is used in a fed batch process, generally contains only as the carbon source one or more compounds selected from the group saccharose, molasses from sugar beet or sugar cane, fructose, glucose, starch hydrolysate, lactose, galactose, maltose, xylose, acetic acid, ethanol and methanol in concentrations of from 300 to 700 g/kg, preferably from 400 to 650 g/kg, and optionally an inorganic nitrogen source such as, for example, 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. Pronounced growth at the beginning of the culturing is normally a logarithmic growth phase. The logarithmic growth phase generally follows a phase of lesser cell growth than in the logarithmic phase.

According to the invention, the concentration of the carbon source during the culturing is set at 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, in the feed-in phase. The concentration of the carbon source is determined by prior art methods. β-D-Glucose is determined, for example, in a YSI 02700 Select glucose analyzer from Yellow Springs Instruments (Yellow Springs, Ohio, USA) . The expression feed-in phase refers to the phase of the fermentation in which at least one carbon source is fed to the medium continuously or discontinuously. In the case of fermentations by the feed-in process, this is generally carried out after the carbon source introduced initially has been consumed completely or almost completely.

After the feed-in to the nutrient media, the fermentation according to the invention is carried out until the concentration of the carbon source is not more than 2 g/1, not more than 1 g/1 or not more than 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 Y_P/S is here defined as the ratio of the total amount of L-lysine formed in a culturing to the total amount of the carbon source used or consumed.

According to the invention, L-lysine is formed with a space/time yield (STY) of at least 2.5 to 3.0 g/1 per hour, of at least 3.0 to more than 4.0 g/1 per hour, of at least 4.0 to 5.0 g/1 per hour, or of at least 5.0 to 8.0 g/1 or more per hour. The space/time yield is here defined as the ratio of the total amount of L-lysine formed in a culturing to the actively producing volume of the culture as seen over the entire period of culturing. The space/time yield is also referred to as volumetric productivity.

According to the invention, the L-lysine concentration (c) , based on lysine-HCl, in the fermentation liquor that is drawn 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 production of L-lysine by fermentation, an attempt is made to achieve and maintain an optimum in dependence on the three performance features yield, productivity and product concentration. Such an optimum can be described by a performance index (PI) , which is composed of the product of space/time yield (STY) , yield (Yp_/s) and L-lysine concentration (c) (PI [g²/(l²*h)] = STY[g/l*h] * Y_P/s[w/w] * c [g/1] ) . According to the invention, the performance index, based on the process according to the invention, reaches at least 110 g²/(l *h), at least 120 g²/(l²*h), at least 130 g²/(l²*h), at least 150 g²/(l²*h), at least 170 g²/(l²*h), at least 190 g²/(l²*h), at least 210 g /(l *h), at least 230 g²/(l²*h), at least 250 g²/(l²*h) .

During the culturing, the temperature is set in a range from 28°C to 40°C, preferably from 30°C to 35°C. The fermentation can be carried out at normal pressure or optionally at excess pressure, preferably at an excess pressure of from 0 to 2.5 bar, particularly preferably at from 0 to 1.5 bar. The oxygen partial pressure is adjusted to from 5 to 50%, preferably about 20%, air saturation.

Adjustment of the pH value to a pH of approximately from 6 to 8, preferably from 6.5 to 7.5, can be carried out using ammonia gas or 25% ammonia water. The conditions of the culturing can remain constant during the culturing or can be changed. In order to meet the performance index requirement, it is necessary during the fermentation to ensure not only that the oxygen partial pressure is sufficient but also that the biological activity of the cells is adequate. In order to ensure biological activity, the oxygen uptake rate (OUR) established in step b) in the process according to claim 1 is not more than 350 mmol./(l*h), not more than 325 mmol./(l*h), 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 refers to the specific rate of oxygen absorption by the microorganisms in mmol . of 0₂ per liter of fermentation liquor and per hour (Biotechnologie; D. Schlee and H.-P. Kleber, Gustav Fischer Verlag Jena; 1991) .

The described streams of first nutrient media and further nutrient media, or the sum of the streams of first nutrient media and further nutrient media, contain complex constituents. The term complex constituents refers to carbon sources or nitrogen sources which have a purity of less than 95% in the form in which they are used. Such a complex constituent is one or more compounds from the group peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor and soybean flour. According to the invention, the proportion of complex constituents in the nutrient media used 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 liquor that is drawn off is less than 2100 mosm/1, better less than 1800 mosm/1, especially less than 1500 mosm/1, preferably less than 1200 mosm/1. Osmolarity refers to the concentration of particles having osmotic activity in a 1 liter volume of liquid. For example, a 1 molar glucose solution corresponds to 1000 mosm/1 (Biotechnologie; H. Weide, J. Paca and W. A. Knorre; Gustav Fischer Verlag Jena; 1991) .

Representatives of the coryneform bacteria suitable for carrying out the process according to the invention are especially those of the Genus Coryneformbacterium. The coryneform bacteria are especially those of the Genus Coryneformbacterium. In the genus Corynebacterium, special mention is to be made of the species Corynebacterium glutamicum and also the species Brevibacterium flavum and Corynebacterium thermoaminogenes. These are known in the art for their ability to produce L-lysine. Information regarding the taxonomic classification of strains of this group of bacteria will 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, especially 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-lysine-producing strains produced therefrom.

The coryneform bacteria contain at least one copy of a lysC gene or allele which codes for an aspartate kinase which are 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 which one or more features selected from the group lysC allele (lysC^fbr) , horn allele (hom^leay) , zwf allele, coding for an NADPH-insensitive glucose-6-phosphate dehydrogenase, and the pyc allele coding for pyruvate carboxylase. The pyc allele is described in EP 1 108 790.

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) are likewise suitable.

Also suitable are L-lysine-producing coryneform bacteria which include at least the following properties: two (2) copies of a lysC allele which codes for a lysine-resistant aspartate kinase (lysC^fbr), a horn allele which codes for an attenuated homoserine dehydrogenase (hom^leakγ) and two (2) copies of a zwf allele which codes for an NADPH-insensitive glucose-6-phosphate dehydrogenase.

Also suitable are L-lysine-producing coryneform bacteria which contain one or more properties selected from the group 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.

Also suitable are L-lysine-producing coryneform bacteria which are sensitive towards diaminopimelic acid analogs . According to the present invention, the expression diaminopimelic acid analogs includes compounds such as 4-fluoro-diaminopimelic acid, 4-hydroxy-diaminopimelic acid, 4-oxo-diaminopimelic acid or 2, 4, 6-triaminopimelic acid. To produce the coryneform bacteria according to the invention which are sensitive to 4-hydroxy-diaminopimelic acid, methods of mutagenesis are used. It is possible to use for the mutagenesis conventional in vivo mutagenesis processes 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) .

The coryneform bacteria that are sensitive towards 4- hydroxy-diaminopimelic acid can be identified by plating out on nutrient medium plates containing 4-hydroxy- diaminopimelic acid. To that end there are especially suitable final concentrations of approximately from 5 to 15 g/1. for example approximately 10 g/1 of 4-hydroxy- diaminopimelic acid in the nutrient medium. At that concentration, mutants that are sensitive to 4-hydroxy- diaminopimelic acid can be distinguished from the unchanged parent strains by retarded growth. Once selection has been carried out, the mutants sensitive to 4-hydroxy- diaminopimelic acid exhibit improved L-lysine production.

Correspondingly stable strains, which do not lose their production properties during the process, are particularly suitable for the described process.

The targeted modification of original properties of coryneform bacteria, the composition of the nutrient media and the manner in which the process is carried out all have the aim of converting the carbon source used into L-lysine as effectively as possible. To that end it is necessary for the carbon to flow through the cell metabolism in the direction towards L-lysine synthesis as far as possible without entering into secondary reactions in which it is consumed, and largely unhindered. The L-lysine-containing fermentation liquor from the process according to the invention 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 that is produced can subsequently be purified from the fermentation liquor. In order to remove or separate off the biomass, separation methods such as, for example, centrifugation, filtration, decantation, flocculation or a combination thereof are used. The L-lysine-containing liquor is then purified by known methods, such as, for example, by ion-exchange chromatography, ion-exclusion chromatography, extraction, crystallisation, precipitation or a combination thereof.

Alternatively, it is also possible to remove water from the L-lysine-containing fermentation liquor. To that end, the fermentation liquor is thickened, or concentrated, by known methods such as, for example, by means of a rotary evaporator, a thin-layer evaporator, a falling film evaporator, by reverse osmosis, by nanofiltration or by a combination thereof. From 10% to 90% of the water is remove .

For better preservation of the L-lysine-containing liquid product, the pH value can be changed to the acid (pH 2 to 5) or alkaline (pH 9 to 12) range by addition of acid or alkaline solution.

It is also possible to prepare a product from the fermentation liquor, by removing the biomass of the bacterium contained in the culture liquor completely (100%) or almost completely, i.e. more than or greater than (>) 90%, >95%, >97%, >99% and leaving the remaining constituents of the fermentation liquor 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 alternatively completely (100%) in the product.

Separation methods such as, for example, centrifugation, filtration, decantation, flocculation or a combination thereof are used for removing or separating off the biomass .

The resulting liquor is then thickened, or concentrated, by known methods, such as, for example, by means of a rotary evaporator, a thin-layer evaporator, a falling film evaporator, by reverse osmosis, by nanofiltration or by a combination thereof. From 10% to 90% of the water is removed.

Furthermore, concentrated liquor can subsequently be worked up by methods of lyophilisation, spray drying, spray granulation or by other methods to form a finely divided, preferably pourable powder. That pourable, finely divided powder can then in turn be converted into a coarse-grained, readily pourable, storable and largely dust-free product by suitable compacting or granulating methods. More than 90% of the water is removed in total, so that the water content in the product is less than 10%, less than 5%.

The mentioned process steps do not necessarily have to be carried out in the order indicated here but may, where appropriate, be combined in a technically expedient manner.

The process according to the invention is distinguished, as compared with the conventional fed batch process, especially by an increased space/time yield.

There is preferably obtained from the fermentation liquor, by removal of water, an L-lysine-containing product having the following composition: lysine 35 - 80 wt.% protein max. 7 wt.% carboxylic acids max. 7 wt.% total sugars max. 9 wt.% fats and oils max. 5 wt.% minerals 3 - 30 wt.%

According to the invention, that L-lysine containing product, after the removal of water, has a water content of at least 0.5 wt.% but not more than 5.0 wt.%.

For the production of the lysine-containing product, the fermentation liquor should also contain the following concentrations of secondary products. 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.

For the production of an L-lysine-containing product it is preferred, according to the invention, for the L-lysine- containing fermentation liquor to have a total secondary 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 liquor to have a total secondary product concentration of less than 0.5%. According to the invention, the L-lysine-containing product, after the removal of water and subsequent granulation, has a mean particle size of from > 0.1 to 1.0 mm, preferably in an amount of more than 97%, especially more than 98%.

Furthermore, the L-lysine-containing product, after the removal of water and granulation, has an apparent density of at least 600 kg/m³, preferably 650 kg/m³, especially 700 kg/m³, but preferably greater than 750 kg/m³.

As described in US Patent Application Serial No.

10/319,843, it is possible to add an additive to the L-lysine-containing product, after the removal of water and granulation, in order to improve the properties of the product. The proportion of added additive, especially oil, at the surface should be from 0.02 to 2.0 wt.%, based on the total amount of the L-lysine-containing product.

According to the invention, the L-lysine-containing product contains as additive at the surface one or more oils selected from the group mineral oil, vegetable oils, soybean oil, olive oil, soya/lecithin mixtures, edible oils, mixtures of vegetable oils.

It is recommended in every case that the L-lysine- containing product, after the removal of water and granulation, should have a lactate content of 3 wt.%, ≤ 2 wt.%, < 1 wt.%, < 0.5 wt.%, ≤ 0.1 wt.%.

Analysis of L-lysine and other amino acids can be carried out by anion-exchange chromatography with subsequent ninhydrin derivation, as described in Spackman et al . (Analytical Chemistry 30: 1190-1206 (1958)), or it can be effected by reversed phase HPLC, as described in Lindroth et al . (Analytical Chemistry 51: 1167-1174 (1979)).

Claims

What is claimed is:

1. Process for the production of a lysine-containing product by fermentation using L-lysine-producing coryneform bacteria, wherein a) the bacterium is inoculated and cultured in at least one first nutrient medium, wherein b) a concentration of the carbon source of not more than 10 g/1 is established in the feed-in phase during the culturing, and c) coryneform bacteria are used that have at least one or more carbon flows selected from the group of

(i) coryneform bacteria which direct the carbon flow through the oxidative pentose phosphate pathway with a percentage of more than 75%;

(ii) 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 at least 1% but not more than 20%;

(iii) coryneform bacteria which direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage of at least 28% but not more than 60%;

(iv) coryneform bacteria which direct the carbon flow through diaminopimelate dehydrogenase, coded for by ddh, with a O 2005/021 24 percentage of at least 49% but not more than 98%;

(v) coryneform bacteria which direct the carbon flow through the 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%;

(vi) 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 at least 3.4 but not more than 4.6; and

(vii) 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 at least 7.

2. Process according to claim 1, wherein in step (c) coryneform bacteria are used that direct at least the oxidative pentose phosphate pathway with a percentage fraction of more than 75%.

3. Process according to claim 2, wherein in step c) coryneform bacteria are used that in addition direct at least one substance flow selected from the group (ii) to (vii) .

4. Process for the fermentative production of a lysine- containing product using L-lysine-producing coryneform 5 bacteria, wherein a) the bacterium is inoculated and cultured in at least one first nutrient medium, wherein b) a concentration of the carbon source of at most 10 g/1 is established in the feed-in phase during

L0 the culturing, and c) coryneform bacteria are used that have at least two carbon flows selected from the group of

(i) coryneform bacteria that direct the carbon flow through the oxidative pentose 5 phosphate pathway with a percentage fraction of more than 75%;

(ii) coryneform bacteria that direct the carbon flow through the tricarboxylic acid cycle, referred to the acetyl radicals that are 0 transferred by acetyl-CoA to oxaloacetate by the citrate synthase reaction with a percentage fraction of at least 1% but at the most 20%;

(iii) coryneform bacteria that direct the carbon 5 flow through aspartate kinase, coded for by lysC, with a percentage fraction of at least 28% but at most 60%;

(iv) coryneform bacteria that direct the carbon flow through diaminopimelate 0 dehydrogenase, coded for by ddh, with a percentage fraction of at least 49% but at most 98%;

(v) coryneform bacteria that direct the carbon flow through the anaplerotic reactions, referred to the sum total of pyruvate and phosphoenole pyruvate (PEP) that are converted into oxaloacetate by PEP carboxylase and pyruvate carboxylase, coded by ppc and pyc, with a percentage fraction of more than 19%;

(vi) coryneform bacteria that have the ability to adjust a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow by the anaplerotic reactions (oxidative pentose phosphate pathway [%] /anaplerotic reactions [%] = PPP/Ana [-]) of at least 3.4 but at most 4.6; and

(vii) coryneform bacteria that have the ability to adjust a ratio of the carbon flow through the oxidative pentose phosphate pathway to the carbon flow in the tricarboxylic acid cycle (oxidative pentose phosphate pathway [%] /tricarboxylic acid cycle [%] = PPP/TCA [-] ) of at least 7.

5. Process for the fermentative production of a lysine- containing product using L-lysine-producing coryneform bacteria, wherein a) the bacterium is inoculated and cultured in at least one first nutrient medium, wherein b) a concentration of the carbon source of at most 10 g/1 is established in the feed-in phase during the culturing, and

c) coryneform bacteria are used that have at least three or more carbon flows selected from the group of

(ii) coryneform bacteria that direct the carbon flow through the tricarboxylic acid cycle, referred to the acetyl radicals that are transferred by acetyl-CoA to oxaloacetate by the citrate synthase reaction with a percentage fraction of at least 1% but at the most 20%;

(iii) coryneform bacteria that direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage fraction of at least 28% but at most 60%;

6. Process according to claim 4 or 5 , wherein the coryneform bacteria that are used direct all carbon flows (i) to (vii) .

7. Process according to claim 1, 4 or 5, wherein the culturing step (a) is carried out by the batch process .

8. Process according to claim 1, 4 or 5, wherein the culturing step (a) is carried out by the fed batch process, with the use of at least one additional nutrient medium.

9. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria direct the carbon flow through the oxidative pentose phosphate pathway with a percentage of more than 105%.

10. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria direct the carbon flow through the oxidative pentose phosphate pathway with a percentage of more than 125%.

11. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria direct the carbon flow through the anaplerotic reactions, referred to 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%, more than 23%, more than 26%, more than 28%, more than 30%, more than 33%, more than 35%, or more than 37%.

12. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria 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 at least 2% but not more than 18%.

13. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria 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 at least 3% but not more than 16%.

14. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage of at least 30% but not more than 57%.

15. Process according to claim 1, 2, 4 or 5, wherein the coryne orm bacteria direct the carbon flow through aspartate kinase, coded for by lysC, with a percentage of at least 32% but not more than 53%.

16. Process according to claim 1, 2, 4 or 5, wherein the coryneform bacteria direct the carbon flow through diaminopimelate dehydrogenase, coded for by ddh, with a percentage 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%.

17. Process according to claim 1, 2, 4 or 5, wherein the 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 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.

18. Process according to claim 1, 2, 4 or 5, wherein the 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 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.

19. Process according to claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein the carbon source is one or more compounds selected from the group sucrose, molasses from sugar beet or sugar cane, fructose, glucose, starch hydrolysate, maltose, xylose, acetic acid, ethanol and methanol.

20. Process according to claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein the nitrogen source is one or more organic, nitrogen-containing substances or substance mixtures selected from the group peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor, soybean flour and urea, and/or one or more inorganic compounds selected from the group ammonia, ammonium-containing salts and salts of nitric acid.

21. Process according to claim 20, 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.

22. Process according to claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein the phosphorous source is phosphoric acid or its polymers or phytic acid or alkali metal or alkaline earth metal salts of phosphoric acid.

23. Process according to claim 22, wherein the alkali salts of phosphoric acid are potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts .

24. Process according to claim 1, 2, 3, 4 or .5, wherein a concentration of the carbon source during the culturing of not more than 10 g/1, 5 g/1 or 3 g/1 is established in the feed-in phase.

25. Process according to claim 1, 2, 3, 4 or 5, wherein a concentration of the carbon source during the culturing of not more than 3 g/1 is established in the feed-in phase.

26. Process according to claim 1, 2, 3, 4 or 5, wherein after the feed-in to the nutrient media, the fermentation is carried out until the concentration of the carbon source is not more than 2 g/1, not more than 1 g/1, or not more than 0.5 g/1.

27. Process according to claim 1, 2, 3, 4 or 5, wherein the yield is at least 43 wt.%, referred to the sugar used.

28. Process according to claim 1, 2, 3, 4 or 5, wherein the yield is at least 52 wt.%, referred to the sugar used.

29. Process according to claim 1, 2, 3, 4 or 5, wherein the space/time yield is at least 2.5 g/(l*h).

30. Process according to claim 1, 2, 3, 4 or 5, wherein the space/time yield is at least 4.0 g/(l*h) to 5.0 g/(l*h) .

31. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of lysine-HCl in the fermentation liquor that is drawn off is ≥ 100 g/1, > 110 g/1, > 120 g/1, > 130 g/1, or > 140 g/1.

32. Process according to claim 1, 2, 3, 4 or 5, wherein the formation of L-lysine, referred to lysine-HCl, takes place with a performance index (PI) of at least 110 g²/(l²*h) .

33. Process according to claim 32, wherein the formation of L-lysine, referred to lysine-HCl, takes place with a performance index (PI) of at least 130 g²/(l²*h).

34. Process according to claim 1, 2, 3, 4 or 5, wherein the nutrient media used in step a) contain complex constituents from the group peptones, yeast extracts, meat extracts, malt extracts, corn steep liquor and soybean flour.

35. Process according to claim 28, wherein the proportion of complex constituents in the nutrient media used is less than 5 wt.%.

36. Process according to claim 1, 2, 3, 4 or 5, wherein the L-lysine-containing fermentation liquor that is drawn off has an osmolarity of not more than 2100 mosm/1, not more than 1800 mosm/1, not more than 1500 mosm/1, or not more than 1200 mosm/1.

37. Process according to claim 1, 2, 3, 4 or 5, wherein the coryneform bacteria are the genus Corynebacterium.

38. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the L-lysine-containing fermentation liquor is produced in the form of a liquid product containing from 0% to 100% of the biomass formed in the fermentation after the removal of from 10% to 90% of the water.

39. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the coryneform bacterium contains at least one lysC gene or allele which codes for an aspartate kinase which is insensitive to inhibition by lysine or mixtures of lysine and threonine.

40. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the L-lysine-producing coryneform bacterium has one or more features selected from the group lysC allele (lysCfbr) , horn allele (homleaky) , zwf allele, coding for an NADPH-insensitive glucose-6-phosphate dehydrogenase, and pyc allele, coding for a pyruvate carboxylase.

41. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the L-lysine-producing coryneform bacterium has one or more resistances selected from the group azauracil^r (Aza^r) , rifamycin^r (Rif^r) , streptomycin^r

42. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the L-lysine-producing coryneform bacterium includes at least the following properties: two (2) copies of a lysC allele (lysC^fbr) , a horn allele (hom^leay) and two (2) copies of a zwf allele which codes for an NADPH-insensitive glucose-6-phosphate dehydrogenase.

43. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the L-lysine-producing coryneform bacterium has one or more properties selected from the group 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.

44. Process according to claim 1, 2, 3, 4, 5 or 37, wherein the L-lysine-producing coryneform bacterium is sensitive towards diaminopimelic acid analogs selected from the group 4-hydroxy-diaminopimelic acid, 4- fluoro-diaminopimelic acid, 4-oxo-diaminopimelic acid and 2 , 4, 6-triaminopimelic acid, preferably 4-hydroxy- diaminopimelic acid.

45. Process according to claim 1, 2, 3, 4 or 5, wherein the L-lysine contained in the fermentation liquor is purified.

46. Process according to claim 1, 2, 3, 4, 5 or 38, wherein the fermentation liquor has a solids content of at least 10 wt.%, at least 12.5 wt.%, at least 15 wt.%, or at least 17.5 wt.%.

47. Process according to claim 1, 2, 3, 4 or 5, wherein the fermentation liquor is concentrated by the removal of water, and an L-lysine-containing product consisting of lysine 35 - 80 wt.% protein max. 7 wt.% carboxylic acids max. 7 wt.% total sugars max. 9 wt . % fats and oils max. 5 wt.% minerals 3 - 30 wt.% is obtained.

48. Process according to claim 47, wherein the L-lysine- containing product, after the removal of water, has a water content of at least 0.5 wt.% but not more than 5.0 wt.%.

49. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product trehalose in the fermentation liquor 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.

50. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product lactate in the fermentation liquor 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.

51. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product ethanol in the fermentation liquor 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.

52. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product L-alanine in the fermentation liquor 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.

53. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product L-valine in the fermentation liquor 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.

54. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product L-glutamate in the fermentation liquor 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.

55. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product ketoglutarate in the fermentation liquor 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. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product succinate in the fermentation liquor 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. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product malate in the fermentation liquor 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. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product oxaloacetate in the fermentation liquor 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. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product acetate in the fermentation liquor 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.

60. Process according to claim 1, 2, 3, 4 or 5, wherein the concentration of the secondary product pyruvate in the fermentation liquor 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.

61. Process according to claim 1, 2, 3, 32 or 41, wherein the L-lysine-containing fermentation liquor that is drawn off has a total secondary product concentration of not more than 2.5%.

62. Process according to claim 1, 2, 3, 32 or 41, wherein the L-lysine-containing fermentation liquor that is drawn off has a total secondary product concentration of not more than 1.0%.

63. Process according to claim 1, 2, 3, 32 or 41, wherein the L-lysine-containing fermentation liquor that is drawn off has a total secondary product concentration of not more than 0.5%.

64. Process according to claim 47, wherein the L-lysine- containing product, after the removal of water and granulation, has a mean particle size of at least 0.1 to 1.0 mm, at least 97%, or at least 98%.

65. Process according to claim 47, 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.%.

66. Process according to claim 47, wherein the L-lysine- containing product, after the removal of water and granulation, has an apparent density of at least 600 kg/m³, at least 650 kg/m³, at least 700 kg/m³, or at least 750 kg/m³.

67. Process according to claim 47, wherein the L-lysine- containing product, after the removal of water and granulation, contains an amount of added additive, especially oil, at the surface of from 0.02 to 2 wt.%, referred to the total amount of the L-lysine- containing product.

68. Process according to claim 67, wherein the L-lysine- containing product contains as additive at the surface one or more oils selected from the group: mineral oil, vegetable oils, soybean oil, olive oil, soya/leciuhin mixtures, edible oils, mixtures of vegetable oils.