Biotechnological Manufacture of Lysine

Walter Pfefferle1 · Bettina Möckel · Brigitte Bathe · Achim Marx 2

Degussa AG, Feed Additives Division, R&D Feed Additives/Biotechnology, Kantstrasse 2,33790 Halle-Kuensebeck, Germany1 E-mail: walter.pfefferle@degussa.com2 E-mail: achim.marx@degussa.com

l-Lysine has been manufactured using Corynebacterium glutamicum for more than 40 years.Nowadays production exceeds 600,000 tons per year. Based on conventionally bred strains, fur-ther improvement of lysine productivity has been achieved by genetic engineering. Pyruvatecarboxylase, aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase andthe specific lysine exporter were shown to be key enzymes for lysine production and were char-acterized in detail. Their combined engineering led to a striking increase in lysine formation.Pathway modeling with data emerging from 13C-isotope experiments revealed a coordinatedflux through pentose phosphate cycle and tricarboxylic acid cycle and intensive futile cyclingbetween C3 compounds of glycolysis and C4 compounds of tricarboxylic acid cycle. Process eco-nomics have been optimized by developing repeated fed-batch techniques and technical con-tinuous fermentations. In addition, on-line metabolic pathway analysis or flow cytometry mayhelp to improve the fermentation performance. Finally, the availability of the Corynebacteriumglutamicum genome sequence has a major impact on the improvement of the biotechnologi-cal manufacture of lysine. In this context, all genes of the carbon flow from sugar uptake to ly-sine secretion have been identified and are accessible to manipulation. The whole sequence in-formation gives access to post genome technologies such as transcriptome analysis,investigation of the proteome and the active metabolic network. These multi-parallel workingtechnologies will accelerate the generation of knowledge. For the first time there is a chance ofunderstanding the overall picture of the physiological state of lysine overproduction in a tech-nical environment.

Keywords. l-Lysine, Fermentation, Strain development, Corynebacterium glutamicum, Scaledown, Metabolic engineering

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

2 Strain Development . . . . . . . . . . . . . . . . . . . . . . . . . 62

2.1 Historical Routes . . . . . . . . . . . . . . . . . . . . . . . . . . . 622.2 Genetic Engineering of the Key Enzymes Aspartate Kinase

and Dihydrodipicolinate Synthase . . . . . . . . . . . . . . . . . 632.3 Availability of Oxaloacetate as a Precursor for the Aspartic Acid

Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682.4 Availability of NADPH for Biosynthetic Reductive Power . . . . . 702.5 Enhancement of Secretion: Influence on Activity of Amino Acid

Exporters by the Lipid Environment . . . . . . . . . . . . . . . . . 73

CHAPTER 1

Advances in Biochemical Engineering/Biotechnology, Vol. 79Managing Editor: T. Scheper© Springer-Verlag Berlin Heidelberg 2003

2.6 Combination of Enhancement of Enzymes for the Lysine Biosynthetic Pathway . . . . . . . . . . . . . . . . . 77

2.7 Enhancement of Stress Resistance . . . . . . . . . . . . . . . . . 79

3 Generation of Basic Knowledge for the Understanding of Lysine Production with Corynebacterium glutamicum for Further Strain and Process Development . . . . . . . . . . . . . . . . . . . . . . 81

3.1 Corynebacterium glutamicum Genome Analysis – Identification of All Genes Leading from Sugar Uptake to Lysine Secretion . . . . 81

3.2 Expression Profiling as a Tool for Strain and Process Optimization 863.3 Pathway Modeling with Data Emerging from 13C-Isotope

Experiments and Applications for Biochemical and Metabolic Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

3.3.1 Genotype and Metabolic Phenotype . . . . . . . . . . . . . . . . 873.3.2 Short Review on Isotope Analysis . . . . . . . . . . . . . . . . . . 883.3.3 Homologous and Heterologous Glutamate Dehydrogenase Mutant 893.3.4 Amphibolic Function of the Glyoxylate Cycle – Anaplerosis and

Link to C4-Compound Decarboxylation . . . . . . . . . . . . . . 923.3.5 Comparison of in-Vivo and in-Vitro Characterization of

Enzyme Activities . . . . . . . . . . . . . . . . . . . . . . . . . . 943.3.6 Application of Fluxomics – Exploitation of Metabolic Flux

Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

4 Optimization of the Lysine Fermentation Process . . . . . . . . . 95

4.1 Improvement of Process Economics by Process Intensification . . 954.2 Scale up and Scale down Techniques . . . . . . . . . . . . . . . . 974.3 Process Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994.3.1 Physiological State Estimation and Optimization of Nutrient

Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994.3.2 Single Cell Analysis by Flow Cytometry . . . . . . . . . . . . . . . 103

5 Large Scale Lysine Manufacture . . . . . . . . . . . . . . . . . . . 103

6 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

1Introduction

l-Lysine is an essential amino acid that has to be available in sufficient amountsin feed-stuffs to meet the nutritional requirements of the animals. Especially feedwhich is based on corn, wheat or barley is poor in lysine. Therefore, supplemen-tation of a lysine-rich source is necessary to increase the efficacy of the feed(Fig. 1).

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This supplementation can be realized by adding feed ingredients which exhibitsignificant higher lysine content like soybean meal or by directly adding lysine.The advantage of supplementing lysine is that intake of other essential aminoacids is not increased concomitantly. For example, addition of 0.5% lysine in-creases the protein quality of the feed as effectively as 20% soy meal. Since the ni-trogen of any superfluously added non-limiting amino acids of a high proteindiet is degraded to ammonia which is excreted by the animals, the supplemen-tation with l-lysine makes it possible to reduce the amount of protein supple-ment thereby decreasing environmental pollution by manure.

For the last ten years a tremendous growth in the market has taken place(Fig. 2).

In 2000, the world-wide production of l-lysine used as a feed additive was ap-prox. 550,000 tons and the market still shows a growth potential of 7–10% peryear. Since only l-lysine is effective as a feed additive, all today’s manufacturingprocesses use the fermentation route [1].

The sole production organism is Corynebacterium glutamicum (including subspecies, Table 1), whose capability of secreting amino acids was discovered by Kinoshita et al. [2, 3] and Udaka [4].

Corynebacterium glutamicum is a gram-positive, irregularly shaped, non-motile, non-spore forming aerobic rod-like bacterium having a peptido-glycancrosslinked via meso-diaminopimelic acid, a cell wall with an arabino-galactanand mycolic acids showing 26 to 36 carbons [6]. Corynebacterium glutamicumshows a GC content of 54.1%.

Large scale production with Corynebacterium glutamicum started as early as1958 at Kyowa Hakko’s plant in Japan. Other companies joined the business andsubsequently, during 4 decades of production with Corynebacterium glutamicum,

Biotechnological Manufacture of Lysine 61

Fig. 1. Demand of lysine, threonine and methionine in piglet feed and natural content of theseamino acids in plant biomass (digestible amino acids)

the biotechnological manufacture of l-lysine has been improved constantly bystrain improvement and process engineering. These optimizations made possi-ble the availability of the large amounts of lysine required nowadays.

2Strain Development

2.1Historical Routes

Soon after discovery of the ability of Corynebacterium glutamicum to secreteamino acids, large scale production of lysine was started using mutants aux-otrophic for amino acids. Even these early mutants displayed a remarkable lysineproductivity. Within 70 hours, one of the first generation strains such as the ho-

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Fig. 2. Fermentative lysine production in tons per year during the last 3 decades

0

100

200

300

400

500

600

1970 1975 1980 1985 1990 1995 2000 2005

Year

Ann

ual L

ysin

e P

rod

uctio

n [1

0 t

ons

]3

Table 1. Subspecies belonging to Corynebacterium glutamicum ATCC13032 according to [5]

Corynebacterium glutamicum ATCC13032

Brevibacterium flavum ATCC14067Brevibacterium lactofermentum ATCC13869Corynebacterium lilium ATCC15990Brevibacterium divaricatum ATCC14020

moserine auxotrophic strain ATCC13287, patented in 1961 as US 2,979,439 [7],yielded 44 g L–1 with a conversion yield of approx. 26% g lysine · HCl (g sugar)–1

[8].Further strain development was carried out by introducing additional amino

acid auxotrophies, vitamin auxotrophies and resistances to antimetabolites.These strains produce lysine up to a conversion yield of more than 50% based onsugar. A summary is given by Leuchtenberger [1].

As early as1970 [8], there were efforts to overcome the disadvantage of the aux-otrophic strains that fermentation broths need substantial and defined growthfactor supplementation. Looking for strains with lower requirements for the de-manded compounds led to so-called leaky strains; a leaky biosynthetic pathwayis still functional but can only provide very limited amounts of the requiredmetabolite. So this metabolite will become growth limiting without showing thetypical metabolic pattern of starvation. In consequence, the intracellular level willbe very low thus avoiding feedback inhibition and repression of a key enzyme ofthe desired production pathway, respectively. Examples are strains with reducedactivity of citrate synthase [9] or reduced homoserine dehydrogenase [10, 11].Sugimoto et al. [12] revealed that leaky mutations may be caused by single basechanges leading to relevant amino acid replacements in the catalytic domain ofthe enzyme.

2.2Genetic Engineering of the Key Enzymes Aspartate Kinase and Dihydrodipicolinate Synthase

The first reaction initiating l-lysine synthesis is catalyzed by the aspartate kinasewhich has been shown to be a key enzyme in l-lysine biosynthesis (Fig. 3).

In Escherichia coli, three isoenzymes with activity and expression controlledby different regulatory mechanisms are present. In contrast to the situation ofE. coli, only one aspartate kinase has been detected in C. glutamicum. In the early days of lysine production with C. glutamicum, it became apparent that the aspartate kinase of C. glutamicum is inhibited by the two end products ofthe emerging pathways, l-lysine and l-threonine and that the main control ofthe carbon flow into the pathway is mediated by this concerted feedback inhibi-tion.

In the screening process to obtain feedback resistant strains, mutants resistantto the mixtures of the l-lysine analogue AEC (amino-ethyl-cysteine) and l-thre-onine were isolated [13]. The reduction of the susceptibility of aspartate kinaseto feedback inhibition is the most important step in the development of l-lysineproducing strains.

In 1990, the corresponding gene of an AEC resistant mutant, the feedback re-sistant aspartate kinase of strain DM 58–1 with a LysC activity completely in-sensitive to feedback inhibition was isolated [14]. The mutation leading to AECresistance of the lysC gene was elucidated [15]. These studies showed that the lysClocus encoding aspartate kinase is composed of two overlapping genes, lysCa andlysCb with lysCb being responsible for the feedback inhibition of the enzyme [15,16]. Also the enzyme has an interesting structure as shown in Fig. 4.

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Fig. 3. The split pathway of l-lysine biosynthesis in C. glutamicum

Two a-subunits and two b-subunits of 421 and 171 amino acid residues re-spectively together form the active enzyme with the sequence of the b-subunitsbeing identical to the carboxy terminal part of the a-subunits.

Sequencing of a number of lysC genes from strains carrying a feedback re-leased aspartate kinase provided further evidence that lysCb encodes the regu-latory feature of the enzyme [15–19].

With such an insensitive LysC protein, C. glutamicum secretes some l-lysine.Cloning a lysC(fbr) gene on a high copy plasmid in the Corynebacterium glutam-icum wild type strain ATCC 13032 results in the accumulation of about 38 mMl-lysine compared to no lysine secretion of the parent strain [17].

In a recent study using site-directed mutagenesis, a set of mutant lysC geneswas constructed. In these mutant genes, nucleotide substitutions of codon 279(threonine) of the a-subunit (corresponding to amino acid residue 30 of the b-subunit) revealed nine mutants with different amino acid mutations at the mu-tation point. For the mutant with a proline residue introduced at position 279, thelargest reduction of susceptibility to l-lysine inhibition was observed [19]. Thismutation is very likely to have the strongest effect on the structure of the enzymeof all amino acid residues chosen in this study. Since, in proline residues, the sidechain is fixed covalently to the main chain of the protein, there is less conforma-tional freedom in the backbone of the protein chain [20].

Jetten et al. [21] analyzed the effect of the copy number of a feedback releasedaspartate kinase gene on l-lysine production and growth in Brevibacteriumlactofermentum ATCC 21799. The strain B. lactofermentum ATCC21799 was cho-sen because of its high resistance to AEC and high lysine productivity [22] re-sulting from a feedback released aspartate kinase [21]. In this study, the parentalstrain was compared to a strain carrying two copies of the mutant lysC gene,showing that the growth rates of the two strains were indistinguishable, but thestrain with the second lysC copy produced significantly more l-lysine. With the

Biotechnological Manufacture of Lysine 65

Fig. 4. Model of the native aspartate kinase with the four identical polypeptide sequences oftwo lysCb subunits and the C-terminal part of lysCa arranged as the allosteric center

nativeaspartate kinase

lysCbsubunit

lysC asubunit

catalyticdomain

allostericcenter

a

lysC (fbr) gene on a high copy number plasmid, the resulting strain did not growas well as the parent strain, but produced larger amounts of lysine. However, thelysine production rate was lower. These results made it clear that, in some cases,a feedback released aspartate kinase is not sufficient to overcome the first limit-ing step in l-lysine production, but the optimal enzyme activity has to be ad-justed to maximize the production rate.

The third step in the l-lysine biosynthetic pathway starting from aspartate isthe condensation of l-aspartate semialdehyde with pyruvate to l-2,3-dihy-drodipicolinate. This reaction is catalyzed by the dihydrodipicolinate synthase.The synthase is located at the branch point of metabolic distribution to either ly-sine or threonine and competes with homoserine dehydrogenase for their com-mon substrate l-aspartate semialdehyde (Fig. 3).

Cloning and overexpression of the dapA gene encoding dihydrodipicol-inate synthase revealed that DapA can provide a powerful target in the develop-ment of l-lysine producers [17, 23–25]. Overexpression of only dapA in the wild type strain ATCC13032 resulted in the production of already about 11 mMl-lysine [17] which is a similar effect like the enhancement of the aspartate kinase.

To analyze the function of DapA in metabolic flux toward l-lysine, severalstrains were constructed with different levels of dapA overexpression [26]. It wasshown that the degree of dapA expression is linked to a decreased metabolic fluxtowards the homoserine dehydrogenase. Strains with a second copy of the dapAgene inserted into the genome, with the dapA gene cloned onto a low copy num-ber plasmid and on a multi-copy plasmid were elucidated. An increased copynumber of the dapA gene leads to increased DapA activity and results in a step-wise increase of l-lysine formation. However, interestingly, the growth rate of theresulting strains is reduced with increased DapA activity. This is similar to the ef-fect of lysC overexpression in C. lactofermentum observed by Jetten et al. [18]. Aglobal response of the metabolism to the DapA activity became apparent(Table 2).

Besides increasing the copy number of a gene there are several more ways tomodulate gene expression in prokaryotes. One of these is to modify transcriptioninitiation signals. However, there is still limited knowledge available about pro-moter structure in C. glutamicum and the way how to optimize the transcriptionsignal to enhance gene expression. Studying promoter structure and function in

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Table 2. The effect of various dapA copy numbers on growth rate, internal alanine concentra-tions and l-lysine excretion rates. Dihydrodipicolinate synthase activity (mmol min–1 mg–1 pro-tein). Excretion rates (nmol min–1 (mg dry weight)–1)

Strain dapA Synthase Growth Intracellular ExcretionCopies activity rate (h–1) alanine (mM) rates

13032 1 0.051 0.43 3 0.013032::dapA 2 0.072 0.37 6 0.2513032/pKW3::dapA 6 0.250 0.36 8 2.7013032/pJC23 20 0.630 0.22 9 3.80

C. glutamicum showed that the activity of the dapA promoter is moderately highcompared to other promoters of the bacterium [27].

In comparison to E. coli or B. subtilis, there is a low conservation of the –10 inC. glutamicum promoters and in some cases the –35 region is missing [28].

A mutational analysis of the dapA promoter was performed to identify regionsand the particular nucleotides important for its function. It was found that astretch of six Ts at position –55 to –50 and an extended –10 region were the mostimportant elements in the promoter function. For the dapA promoter the ex-tended –10 motive was defined (AGGTAACCTTG) and no –35 region. This situ-ation is similar to E. coli sigma –70 depending promoters with an extended –10region.Vasicova et al., [28] made it clear, that the dapA promoter activity can beincreased 3.9 times by introducing single nucleotide exchanges in the extended–10 region (Fig. 5).

Subsequent studies with C. glutamicum MH20–22B mutants carrying a sec-ond copy of the dapA gene with the wild type promoter or the MA16 or MC20mutation were performed (Fig. 6). As expected, the DapA activity of the strainswas significantly enhanced compared to the parent strain MH20–22B resultingin enhanced l-lysine production [29].

Biotechnological Manufacture of Lysine 67

Fig. 5. DNA sequence of the dapA promoter region as described by Vasicova et al. (1999). Thearrows mark an inverted repeat in the –35 region. Replacement bases are indicated in bold faceand underlined. Sequences of wild type P-dapA, mutant MC20 and MA16 are described. TSmarks the transcription start (underlined A)

Fig. 6. The effect of a second dapA copy with different dapA promoter mutations on dihy-drodipicolinate synthase activity and l-lysine production. Dihydrodipicolinate synthase ac-tivity (mmol min–1 mg–1). l-lysine production (g L–1). Black columns represent the dihy-drodipicolinate synthase activity. Grey columns indicate the amount of l-lysine HCl produced

2.3 Availability of Oxaloacetate as a Precursor for the Aspartic Acid Family

Genetic and biochemical investigations on the complex network of anapleroticcarboxylation (Fig. 7) and opposed decarboxylation have been reviewed recently[21, 30].

Oxaloacetate is a direct precursor for aspartic acid, therefore its availability isof central importance for the production of lysine.

As early as1969, Ozaki and Shiio [32] investigated the regulation of phospho-enolpyruvate carboxylase and pyruvate kinase in Brevibacterium lactofermentumto understand this branching point of glucose metabolism.

Enhancement of phosphoenolpyruvate carboxylase was shown to be positivefor the formation of aspartic acid derived amino acids in Brevibacterium lactofer-mentum [33, 34].

Later on, it was shown that in Corynebacterium glutamicum, phospho-enolpyruvate carboxylase is dispensable for growth and lysine production [35].Therefore, it was obvious that another anaplerotic reaction has to be responsiblefor replenishing the oxaloacetate pool.

The most prominent candidate was pyruvate carboxylase, an enzyme whichcatalyzes the conversion of pyruvate and carbon dioxide to oxaloacetate underhydrolysis of ATP.

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Fig. 7. Complex network of anaplerotic carboxylation of C3-compounds and decarboxylationof C4-compounds. In C. glutamicum the anaplerotic carboxylation and opposed decarboxyla-tion is potentially catalyzed by five enzymes. In addition, pyruvate kinase and malate dehy-drogenase might catalyze the conversion of phosphoenolpyruvate (PEP) into pyruvate and ox-aloacetate into malate, respectively. This way the operation of futile cycles might becomepossible. (From [31]. Kindly provided by Dr. L. Eggeling, Research Center Juelich, Germany)

Initially, pyruvate carboxylase activity could only be demonstrated by 13C-iso-tope investigations [36, 37] but recently, significant in-vitro activities were mea-sured and it was shown that growth is decreased in pyruvate carboxylase dele-tion mutants [38].

Overexpression of this enzyme confirmed that this anaplerotic reaction playsa key role in lysine biosynthesis. Enhancement of pyc in Corynebacterium glu-tamicum MH20–22B increased lysine production by 50% [39].

The computer-aided 13C-isotope analysis indicated for the first time for C. glu-tamicum that not only C3-compounds of glycolysis are carboxylated to C4-com-pounds of the tricarboxylic acid cycle but at the same time a decarboxylation ofC4- to C3-compounds takes place [40, 41] (Fig. 8).

A detailed sensitivity analysis revealed a high sensitivity between the 13C-en-richment in position oxaloacetate C-2 and the bidirectional flux between C4- andC3-compounds [31]. This bidirectional interconversion represents a futile cyclewhich can result in the consumption of energy without contribution to biosyn-thesis [43, 44]. This substrate cycle or futile cycle contributes to the high flexibilityof the central metabolism of C. glutamicum and was identified as a target formetabolic engineering. To improve metabolite production the net flux has to be

Biotechnological Manufacture of Lysine 69

Fig. 8. Net supply of C4- and C3-compounds (black bar) for different physiological states whichis the result of simultaneously operating (white bar) carboxylation of C3-compounds and de-carboxylation of C4-compounds which has been described elsewhere [31, 40, 41].All values areexpressed as percentage of the molar flux of glucose uptake. The particular physiological statesfor isogenic mutant strains were characterized in chemostat culture [31, 40, 41]. The isocitratedehydrogenase mutant was cultivated in a shake flask batch culture [42]. A, lysine productionwith 0.29 g lysine·HCl (g glucose)–1; B, lysine production with 0.18 g lysine·HCl (g glucose)–1;C, growth; D, glutamate production; E, growth for an isocitrate dehydrogenase deletion mutant

increased. The strategy to improve lysine overproduction by inactivating energydissipating futile cycles has already been validated experimentally for C. glu-tamicum by inactivating the phosphoenolpyruvate carboxykinase [45]. Recently,detailed 13C-isotope analysis revealed which particular enzyme catalyzes whichportion of the total in-vivo flux of carboxylation and decarboxylation [46].

The net flux of decarboxylation of C4-compounds turned out to be maximumfor an isocitrate dehydrogenase mutant of C. glutamicum (Fig. 8). For this phys-iological state the decarboxylation of C4-compounds plays an essential role as alink between the flux through pyruvate dehydrogenase and glyoxylate cycle tomaintain the oxidative metabolism of glucose when the tricarboxylic acid cycleis blocked. In contrast to the situation of the isocitrate dehydrogenase mutant, thenet flux of decarboxylation of C4-compounds was decreased to maximize the netsupply of C4-compounds in a homologous glutamate dehydrogenase mutant witha high requirement for C4-compounds due to the high lysine yield (Fig. 17; Fig. 8;cf. Sect. 3.3). In conclusion, a significant decarboxylation of C4-compounds wasobserved although this was not expected for cells metabolizing glucose. A de-crease in the decarboxylation of C4-compounds in combination with increasinglysine yield indicates that the futile cycle between carboxylation of C3-com-pounds and decarboxylation of C4-compounds is decreased in relation to ahigher demand for C4-compounds.

The decarboxylation of C4-compounds can potentially contribute to theNADPH synthesis when the flux is catalyzed by the malic enzyme. In this case, itbecomes obvious that especially for the overproduction of lysine, a compromisebetween the optimization of C4-compound formation and NADPH supply mustbe achieved since the intracellular provision of both intermediates might repre-sent a bottleneck for lysine formation. For Aspergillus nidulans [47] and C. glu-tamicum [48] it has been proposed that the malic enzyme is of crucial impor-tance for NADPH synthesis. In contrast, 13C-isotope investigations for the lysinemodel strain MH20–22B revealed that, at least for a high lysine yield achievedwith the homologous glutamate dehydrogenase mutant, the decarboxylation ofC4-compounds was low compared to the overall NADPH synthesis by the pentosephosphate pathway and tricarboxylic acid cycle. This indicates only a minor con-tribution of the malic enzyme to NADPH synthesis in C. glutamicum for this par-ticular physiological state of lysine overproduction.A minor contribution of themalic enzyme to intracellular NADPH supply has also been observed in relationto the overproduction of riboflavin by B. subtilis [44].

2.4Availability of NADPH for Biosynthetic Reductive Power – NADPH Metabolism in C. glutamicum

One use of 13C-isotope experiments based metabolic flux analysis which is de-scribed in detail in Sect. 3.3 is that, without making assumptions about bacter-ial energetics, the carbon flux estimations can be used to establish a NADPHbalance. From the 13C-isotope analysis based flux distributions for different phys-iological states [41] the NADPH supply by glucose 6-phosphate dehydrogenase,6-phosphogluconate dehydrogenase and isocitrate dehydrogenase was calcu-

70 W. Pfefferle et al.

lated.A comparison of this supply to the requirement of the synthesis of biomassbuilding blocks and products revealed an unassigned NADPH consumptionwhich varied according to the physiological state under investigation (Fig. 9).

Since the unassigned NADPH consumption (flux of 16 to 67% relative to theglucose uptake rate) was minimal for the observed maximum lysine yieldNADPH limitation of lysine production is likely. Recently, the increase of nico-tinamide nucleotide transhydrogenase activity in microbial cells has been identified as a means for removing the bottle-neck of limited NADPH supply and thereby facilitating the formation of amino acids and microbial pro-ducts in general [49]. A similarly unusually high capacity for the reoxidation of NADPH has been revealed by 13C-isotope investigations for C. glutamicumin batch culture [50] and for riboflavin-producing Bacillus subtilis in chemostatculture [44]. This indicates that in both gram-positive bacteria, in-vivo NADPHconsumption processes must be active which are still unassigned to a metabolicfunction and are not related to the synthesis of biomass building blocks andproducts.

Since the NADPH requirement for the synthesis of biomass building blocksand products for C. glutamicum was partially derived from data for the gram-negative bacterium Escherichia coli [40] and it has been confirmed that gram-positive bacteria such as B. subtilis contain more peptidoglycan [51] the re-quirement of NADPH might be higher than indicated in Fig. 9. A correction ofthe data would result in an increase in the NADPH requirement equal to a fluxof only 4.6% relative to the glucose uptake rate which is less than the observedunassigned NADPH consumption of up to a flux of 67% relative to the glucoseuptake rate. It is likely that the unassigned NADPH consumption is required forcellular processes which depend on the activity of NADPH oxidizing enzymeslike glutathione reductase [52]. Processes such as the detoxification of reactiveoxygen intermediates have been described for Saccharomyces cerevisiae [53] andE. coli [54]. Furthermore, for C. glutamicum, an NADPH oxidase activity has beendescribed recently [55] and in particular an in-vitro activity of 16.5 mU (mg pro-tein)–1 has been observed upon incubation of crude extract with NADPH [56]which corresponds to a NADPH consumption flux of about 30% relative to theglucose uptake rate referring to the above mentioned metabolic flux data.

NADPH is consumed by the lysine biosynthetic pathway in four steps eitherdirectly in the dihydrodipicolinate dehydrogenase and diaminopimelate dehy-drogenase reaction or indirectly by the reactions aspartate aminotransferase andsuccinylaminoketopimelate aminotransferase. It turned out that the overexpres-sion of a homologous glutamate dehydrogenase as described in Sect. 3.3 led to animprovement in lysine formation most likely because a better channeling ofNADPH into the amination reactions of lysine synthesis.A scenario could be thatthe cytoplasmatic glutamate concentration was increased and thereby the cyto-plasmatic substrate availability for lysine synthesis specific aspartate amino-transferase and succinylaminoketopimelate aminotransferase has been opti-mized. Recently an attempt to make glutamate formation dependent on NADHby introduction of a heterologous glutamate dehydrogenase from P. assaccha-rolyticus has been described [31]. Initially it was expected that lysine formationwould be increased for the heterologous glutamate dehydrogenase mutant since

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72 W. Pfefferle et al.

Fig. 9. Some NADPH fluxes in Corynebacterium glutamicum indicated with the unit mole ofNADPH per 100 mol of glucose taken up by the bacterial cells. An unexpected and still unas-signed NADPH consumption flux (bar with lines) was calculated from the supply of NADPHby pentose phosphate pathway (black bar) plus tricarboxylic acid cycle (bar with rectangles)minus the requirement of NADPH which was derived from product synthesis (bar with dia-monds) and C. glutamicum biomass formation (white bar) as described elsewhere [40].All val-ues are expressed as percentage of the molar flux of glucose uptake. The particular physiolog-ical states for isogenic mutant strains were characterized in chemostat culture [31, 40, 41]:A, lysine production with 0.29 g lysine · HCl (g glucose)–1; B, lysine production with 0.18 g ly-sine · HCl (g glucose)–1; C, growth; D, glutamate production

NADH was required for glutamate synthesis by the heterologous NADH-depen-dent glutamate dehydrogenase to circumvent a NADPH limitation of lysine pro-duction. However, complex adjustments of some cytoplasmatic pools in responseto the introduction of the heterologous glutamate dehydrogenase [57] might haveled to the observed decrease in pentose phosphate cycle flux. In this way, the over-all NADPH supply for enzyme reactions, such as for diaminopimelate dehydro-genase, which are directly dependent on NADPH might be reduced and mighthave limited lysine formation. Further changes in cytoplasmatic pools of im-portant intermediates might have influenced the regulatory network as a re-sponse to the introduction of a heterologous enzyme.

The in-vivo monitoring of the cytoplasmatic concentration of inter-mediates as was published for lysine production with C. glutamicum [58] mightcontribute to an understanding of how changes in cytoplasmatic pool sizes influence metabolic flux patterns and lysine production. This kind of investiga-tion, called metabolome analysis, has recently been introduced for E. coli [59],and might be a valuable tool for understanding and engineering the centralmetabolism of industrial microorganisms, especially for lysine producing C. glutamicum.

2.5Enhancement of Secretion: Influence on Activity of Amino Acid Exporters by the Lipid Environment

Detailed knowledge of the mechanism by which l-lysine crosses the permeabil-ity barrier of the plasma membrane is also significant for considerations con-cerning process efficiency and yield.

Studying the l-lysine export of C. glutamicum, Bröer et al. [60, 61] showed thatl-lysine is secreted by a carrier mediated secondary transport process. However,the molecular basis for lysine export was unknown until 1996. One importantstep towards the cloning of the gene was the isolation of a l-lysine excretion-de-fective mutant [62]. Cloning the gene encoding the lysine carrier protein LysEfrom C. glutamicum provided the breakthrough [63]. The comparatively smallprotein of 236 amino acids in size or 25.4 kDa performs the translocation of l-lysine. In a lysE disruption mutant, l-lysine excretion is blocked, which provesthat no second function can substitute the LysE-mediated lysine export [64].Withoverexpression of lysE, the excretion rate for l-lysine is enhanced five times to arate of 3.76 nmol min–1 (mg dry weight)–1 compared to the wild type strain. Thesefindings indeed show that lysE is a potential target in strain development, avoid-ing the bottleneck of product excretion.

The LysE protein represents a new family of transport proteins consisting of5 transmembrane spanning helices.A large hydrophilic loop in the middle of thepolypeptide chain is probably involved in the export of the positively charged l-lysine (Fig. 10).

The translocation mechanism of l-lysine has been studied in detail and it has been discovered that the driving force for the entire translocation process is the membrane potential DY with the export activity being modulated by the pH and the l-lysine gradient between the cytosol and the external medium.

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A model describing the l-lysine translocation differentiated four distinct steps:

(i) The negatively charged LysE carrier protein is loaded with l-lysine and twohydroxyl ions.

(ii) l-Lysine is translocated across the membrane.(iii) The substrate l-lysine and the associated hydroxyl ions are released on the

outside of the membrane.(iv) In the last step, the carrier is reoriented to be ready to be loaded up again on

the inside of the membrane.

In this model the driving force DY is required for the reorientation of the carrier[61] (Fig. 11).

However, is lysE the only key to modulate the export rate of lysine producers?It has been shown that different lysine-producing strains show differences in ex-port capabilities. The idea was that mutations in the lysE gene should be re-sponsible for the variations in lysine export. Cloning and sequencing of the genesof several C. glutamicum strains made it clear that the lysE gene itself is not theonly source to modulate export capabilities of production strains [65]. But whatis the story behind this evidence?

It is well known that the lipid environment determines the fluidity of the cellwall and the activity of transport proteins. Detailed studies indicate that lipidsplay a supporting role in the stabilization of transport proteins in a native and ac-tive conformation [66].

Integral membrane proteins usually require specific lipids for their optimal ac-tivity and are inhibited by other lipid species. The activity of transport proteins

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Fig. 10. Model of the topology of the lysE product encoding the l-lysine exporter of C. glu-tamicum. Conserved aminoacyl residues proposed to be of major importance for translocationare given in the single-letter code

is modulated by bulk physical characteristics of the lipid bilayer, while specificrequirements rarely appear.

The influence of the lipid content of the membrane on amino acid efflux inCorynebacteria has been intensively studied for the glutamate excretion (cf.chapter “Metabolic Engineering of Glutamate Production”) and interestinglythere is also an effect on the activity of the lysine secretion system. Recently twogenes (dtsR1 and dtsR2) were cloned whose expression influence the lipid trans-porters of the cell.

Another protein encoded by the accBC gene is a two-domain protein com-posed of a N-terminal biotin carboxylase and a C-terminal biotin-carboxyl-car-rier protein. The high homology to an acyl-coenzyme A carboxylase from My-cobacterium leprae led to the conclusion that the identified gene is a subunit ofthe AccBCDA complex [67].

Thus, it has been assumed that DtsR1 forms a complex with the biotin-con-taining protein AccBC, as does DtsR2 [30]. Evaluation of sequence identities leadto the conclusion that AccBC is the alpha-chain of acetyl-coenzyme A-carboxy-lase and DtsR1 and DtsR2 function as the b-subunit of the enzyme. There is ex-perimental evidence that DtsR1/AccBC and DtsR2/AccBC complexes are formedin vivo (Fig. 12).

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Fig. 11. Putative kinetic mechanism of l-lysine efflux in C. glutamicum. Abbreviations: lys+,lysine base; C+, carrier; Dy, transmembrane potential

Besides the dtsR1 and dtsR2 genes, a third orthologous gene accDA has beencloned and identified [68]. Now there is experimental evidence that the genes en-coding the acetyl-coenzyme A-carboxylase have been identified. Enzyme mea-surements made it clear that a complex of AccBC/AccDA shows acetyl-coenzymeA-carboxylase activity (Eggeling, personal communication). Furthermore, in ad-dition to acetyl-coenzyme A, propionyl-coenzyme A can be used as a substrateof the enzyme complex, indicating that beside malonyl-coenzyme A, methyl-manoyl-coenzyme A is formed (Eggeling, personal communication).Acyl-coen-zyme A carboxylase is known to provide building blocks for fatty-acids and my-colic acids.

Interestingly, amplifying the dtsR1 gene in an l-lysine producing strain leadsto enhanced l-lysine production. The idea is that the changed lipid environmentof the carrier protein is responsible for enhanced l-lysine efflux. Similar resultswere obtained by amplifying accDA and the concerted amplification of both accBC and accDA in the l-lysine producing strain MH20–22B (DSM5715) [68,69] (Table 3).

These findings show that modulating the lipid environment of transport pro-teins can be used to enhance l-lysine secretion. Further studies will reveal themolecular mechanism of the interaction between LysE and the lipids of the cellmembrane in C. glutamicum.

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Fig. 12. Probable structure of accCBDA complex in C. glutamicum. In E. coli four sub-units are encoded by separated genes. In C. glutamicum two subunits were detected. DtsR1,DtsR2 and AccDA form homologous proteins and may function as the carboxyltran-ferase- acetyl-/propionyl binding domain of the acetyl-CoA-carboxylase or propionyl-CoA-carboxylase

The expression of the lysE gene encoding the lysine carrier protein is regu-lated by the lysG gene located immediately adjacent to the lysE gene on the opposite DNA strand [64]. LysG is homologue to a group of DNA regulatorswhich belong to the LysR type transcriptional regulators family [70] and acts as a positive control element, activating the transcription of lysE and dis-playing autoregulatory activities. One characteristic of this type of activator isthat they act in response to small molecules. The molecule controlling lysGactivity is probably l-lysine. Beside lysE and the lipid environment of lysE, lysGis indeed a powerful target for modulating the excretion capacity of l-lysine producing strains.

2.6Combination of Enhancement of Enzymes for the Lysine Biosynthetic Pathway

Several successful attempts have been made to enhance l-lysine productivity ofstrains by means of amplification of l-lysine biosynthesis genes or introductionof mutant genes in coryneform bacteria. It has been shown that aspartate kinaseand dihydrodipicolinate synthase are the most important enzymes in regulatingthe carbon flux throughout the l-lysine biosynthetic pathway. The dapA gene am-plification by genetic engineering methods has two effects on the cell, as de-scribed above, not only l-lysine production is enhanced, but also growth speedis decreased resulting in increased intracellular pyruvate availability favorable forl-lysine synthesis.

The amplification of both the feedback released aspartate kinase together withthe dihydrodipicolinate synthase is used to further enhance l-lysine formation[17] but leaving the problem of growth decrease still existing.

The amplification of single genes is a well known tool and proved to be suc-cessful not only for the amplification of l-lysine specific genes. Several genes ofthe central metabolic pathways from C. glutamicum like phosphoenolpyruvatecarboxylase or pyruvate carboxylase have been used to enhance l-lysine pro-ductivity of several strains [17, 38, 71].

The combination of several genes from the l-lysine biosynthetic pathway isalso applied to achieve an improved metabolic flux in the pathway to l-lysine.Amplification of dapB alone does not have any effect on l-lysine formation [17],but in combination with dapA and lysC(fbr) overexpression the positive effect ofdapB on lysine production became apparent [72].

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Table 3. l-Lysine secretion of recombinants of Coryne-bacterium glutamicum MH20–22B carrying different plasmids

Strain OD660 l-Lysine (g L–1)

DSM5175 31.4 7.2DSM5715/pZ1accBC 27.6 9.6DSM5715/pZ1accDA 43.1 8.0

In this study the authors showed that the concerted overexpression of 5 genesof the l-lysine biosynthetic pathway, lysC, dapA, dapB, ddh and lysA enhanced l-lysine accumulation from 29.8 g L–1 to 47.0 g L–1 after 72 h incubation with onlyslightly reduced growth from 0.450 to 0.409 (OD). They report that dapB is im-portant for restoring growth rate.Enhancing lysC(fbr) in a moderate lysine producerlead to reduced growth and increased yield as expected; lysine formation after 24 hwas slowed down by 24 h due to poor growth rate. Additional overexpression ofdapB restored growth and overcompensated productivity losses (Table 4).

Nevertheless, the effect of high dihydrodipicolinate reductase activity on themetabolic flux is not obvious and still unknown. There is no explanation whydapB amplification is able to overcome the growth reduction resulting from over-expression of aspartate kinase.

Beside the already mentioned steps in l-lysine biosynthesis there are somemore genes whose expression is capable to influence l-lysine productivity dra-matically.A new aspect in enhancing l-lysine productivity is the attempt to mod-ulate the expression of several genes from the central metabolism, the l-lysinebiosynthetic pathway and furthermore the l-lysine export carrier gene lysE. Fur-ther work showed that overexpression of the recently cloned pyc gene encodingpyruvate carboxylase in addition to the enhanced activity of dapA and dapB incombination with lysC(fbr) increases l-lysine production. Overexpression of pyru-vate carboxylase (pyc), dihydrodipicolinate synthase (dapA) and reductase(dapB) together with the gene encoding the l-lysine export carrier lysE resultsin substantial increased accumulation of l-lysine (Fig. 13).

The parent strain MH20–22B with amplified pyc gene, accumulates 11.3 g L–1

l-lysine HCl after 72 h whereas the strain with amplified pyc, dapA, dapB and lysEgene accumulates up to 17.6 g L–1 [73]. This strong increase in l-lysine formationis due to the combination of redirecting the carbon flow in the most importantrate limiting steps of C4-compound supply, specific lysine biosynthesis and prod-uct secretion.

Ohnishi et al. demonstrated a high-speed fermentation yielding 80 g L–1 l-ly-sine · HCl in 27 h combining a deregulated aspartate kinase with a mutated pyru-vate carboxylase and homoserine dehydrogenase both bearing a single aminoacid exchange [74]. These findings make clear that there are not only one or two

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Table 4. Enhancement of dapB restores growth and accelerates lysine formation in strains withamplified lysC(fbr) and dapA (according to [72]).Abbreviation: OD600, optical density at 600 nm

Bacterial strain/ Amount of produced l-Lysine Growthamplified gene (g L–1) OD600

24 h 72 h

AJ11082 22.0 29.8 0.450+ lysC fbr 16.8 34.5 0.398+ lysC fbr dapB 23.3 35.0 0.440+ lysC fbr dapA 19.7 36.5 0.360+ lysC fbr dapA dapB 23.0 45.0 0.425

rate limiting steps in l-lysine production of the cell; in consequence amplifica-tion of single genes which were supposed to be limiting will lead to new bottle-necks to be enlarged.

2.7Enhancement of Stress Resistance

Technical manufacturing of lysine is performed in fermentation media with highosmotic pressure up to 2 osmol. Therefore an essential requirement for produc-tion strains is their osmostress resistance.

As a soil bacterium Corynebacterium glutamicum is well equipped to face hyperosmotic environments. As early as 1990, Kawahara reported that supple-mentation of glycine betaine stimulated growth of Corynebacterium glutamicumin media of inhibitory osmotic strength and that this osmoprotectant had a pos-itive influence on lysine fermentation [75, 76].

Transport studies showed that Corynebacterium glutamicum can use the com-patible solutes glycine betaine, proline and ectoine for osmoprotection [77]. Inthe meantime, a complex osmoprotective network has become known in this or-ganism (Fig. 14).

For lysine production, the constitutively expressed but strongly regulatedglycine betaine transport system BetP is the most important: on the one hand it

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Fig. 13. l-Lysine secretion of recombinants of Corynebacterium glutamicum MH20–22B. Cellswere cultivated on production medium containing 50 g glucose l–1. l-Lysine · HCl concentra-tion was determined in supernatants after 72 h. Strain MH20–22B is a leucine auxotrophic andAEC-resistant mutant derived from C. glutamicum ATCC 13032. Pyc (pyruvate carboxylase),dapA (dihydrodipicolinate synthase), dapB (dihydrodipicolinate reductase), lysE (lysine car-rier), lysC(Fbr) (feedback resistant aspartate kinase)

shows a very high maximum activity and on the other hand glycine betaine ischeap and readily available in molasses which is a natural substrate or co-sub-strate for lysine fermentation.

A more general aspect of stress tolerance is covered by the approach of Kimuraet al. [78]. The idea is that enhanced expression of heat shock proteins or ofthe alternative sigma-32 factor which activates the synthesis of heat shock proteins (for a summary of the E. coli heat shock response see [79]) will elevatethe stress tolerance of the production strains towards product concentration,temperature and osmotic stress. They report that heterologue amplification of the E. coli rpoH gene in Brevibacterium lactofermentum leads to enhanced lysine formation.

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Fig. 14. Present knowledge of the osmoprotective network in Corynebacterium glutamicum.Kindly provided by Dr. R. Krämer, University Cologne, Germany

3Generation of Basic Knowledge for Understanding of Lysine Productionwith Corynebacterium glutamicum for Further Strain and Process Development

3.1Corynebacterium glutamicum Genome Analysis – Identification of All Genes Leadingfrom Sugar Uptake to Lysine Secretion

Over the past 10 years, the roots of genetic engineering enabled the molecular bi-ologists to modulate the pathways critical for l-lysine production. As describedin Sect. 2.2 the aim is to tune the metabolic flux in C. glutamicum productionstrains towards a highly efficient conversion of the substrate glucose into the endproduct l-lysine. Methods such as transposon mutagenesis, DNA transfer byelectroporation [80] conjugational transfer of plasmids [81, 82] or disruptionmutagenesis and gene replacement [83, 84] have been successfully established forcorynebacteria to make strain optimization by genetic engineering the methodof choice.

A variety of genes of C. glutamicum have been cloned by complementation ofheterologous E. coli mutants or using the PCR technique [85]. Furthermore, theimpact of most of the genes of the lysine biosynthetic pathway on amino acidproduction has been elucidated [17, 21].

However, despite the success of metabolic engineering, there are still some se-crets remaining in the central metabolism of the coryneform bacteria which arenot only of importance for the scientific world, but are of major interest for in-dustrial biotechnology. There is only a little knowledge about the regulatory net-works of C. glutamicum limiting sugar consumption and influencing growth per-formance. The most powerful tool for filling this gap in information is theanalysis of the whole genome sequence.

Nowadays, sequencing of the entire genome of an organism is possible withina limited time and with a minimum of effort. The entire genomes of 27 mi-croorganisms have been sequenced and data are accessible in the public data-bases, the annotation of 17 genomes is in progress and more than 70 genome se-quencing projects will be completed within the first year of the new millennium(http://www.tigr.org, http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/bact.html,http://geta.life.uiuc.edu/~nikos/prokaryagenomes.html).With the developmentof high throughput sequencing capabilities, genome sequencing has become af-fordable making information available about all genes defining an organism suchas C. glutamicum.

In 1999, the amino acid producing companies BASF, Degussa, and Kyowa-Hakko completed the sequencing of the Corynebacterium glutamicum genome[86–89].

In principle, there are two different ways of starting a sequencing project, thewhole genome shotgun sequencing and sequencing based on ordered cos-mid/bac libraries [90].

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Shotgun approach:

Here the whole genome is randomly sheared and DNA fragments of about1.5 kb are subcloned into plasmids which are sequenced. These small insertsoften contain only a part of a gene thus being compatible with the metabolismof the cell. Using this technology, a significantly higher amount of DNA has tobe sequenced to provide sufficient redundancy (about 10 fold) so that com-putational assembly is also possible with repetitive sequences. In recent times,this disadvantage is compensated for by lower sequencing costs and morepowerful bioinformatics.

Ordered cosmid/BAC libraries:

The other approach is based on cosmid or BAC libraries which are screenedfor minimal overlaps by hybridization techniques. In this way the necessaryredundancy is minimized.

3.1.1Sequencing the Genome of the Amino-Acid Producing C. glutamicum

For deciphering the C. glutamicum genome, Degussa AG applied the cosmidstrategy using a physical map of the genome as a starting point [91].

It is known that many genes from C. glutamicum are effectively expressed inE. coli leading to toxic effects making the subcloning of many regions of thegenome on high copy number vectors very difficult or nearly impossible. Simi-lar experiences were made sequencing the Bacillus subtilis genome [92]. By se-quencing the Mycobacterium tuberculosis genome, several repetitive DNA ele-ments (e.g., insertion elements) were identified. In addition, about 10% of thecoding capacity of the genome is represented by two different polymorphicrepetitive sequences [93]. Because Mycobacterium tuberculosis is evolutionarilyrelated to Corynebacterium glutamicum, similar facts were expected for this se-quencing project.

Therefore, the physical map provided an important framework throughout thewhole sequencing project. The strategy was divided into 6 steps [94]:

(i) The C. glutamicum genome was cut using three rare cutting restriction en-zymes SwaI, PacI and PmeI and with the PFGE technique, a physical map ofthe genome was established. About 40 genes known from the public data-bases were localized on the map by hybridization techniques (Fig. 15).

(ii) A cosmid library of about 2300 cosmids was established.(iii) The cosmid library was screened and minimal overlapping clones were se-

lected from the library by hybridization techniques using the physical mapas a guideline throughout the project.

(iv) Sequencing of the 3,282 Kbp genome of C. glutamicum ATCC 13032 by a cos-mid-by-cosmid sequencing strategy.A set of 98 cosmids covering about 90%of the entire genome were subcloned and sequenced.

(v) Assembling of the data, identification of the remaining gaps.(vi) Closing of the remaining gaps in the 3.282 Kbp sequence was mainly per-

formed by establishing a BAC library and subsequently sequencing the re-maining gaps on BAC templates.

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Computer analysis of the genome of the lysine producer Corynebacterium glu-tamicum indicates that the present knowledge concerning the genes of the mostimportant pathways has not been completed. Publicly available microbialgenomes have shown that alternative reactions often appear to be required orcertain steps are missing.

The alignment of biochemical pathways from different species is an importantstep towards the understanding of bacterial physiology [95–97]. It is of greatvalue for pathway engineering to get detailed knowledge about the genes defin-ing the central pathways of the metabolism. It has been shown that the adapt-ability of organisms is underestimated if only textbook knowledge regarding the

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Fig. 15. Overview of the physical and genetic map of the chromosome of Corynebacterium glu-tamicum ATCC 13032. Localization of the restriction fragments derived from SwaI-cut DNA areshown as segments of the circle. Selected genes are arranged around the circle due to their ge-nomic localization

fundamental pathways is considered [98–100]. These pathways rarely occur as previously defined [101–102]. Since the data on genome sequences started toaccumulate, a number of isoenzymes have been detected and identified [103] andit has been shown that some genes are lacking in various organisms.

The comparison of the genes encoding the enzymes of the glycolytic pathwayfrom 17 sequenced genomes revealed an interesting insight into the variability ofglycolysis [98, 99, 104].

The genome sequence of C. glutamicum has been analyzed to identify all genesinvolved in glycolysis and compared to the data with the genome annotation ofMycobacterium tuberculosis one of the most closely related bacteria with itsgenome sequence already published [93]. The glycolytic pathway includes somereactions that, in different species, are catalyzed by non-orthologous enzymes,namely phosphofructokinase, fructosebisphosphate aldolase and phosphoglyc-erate mutases [98, 102] and glyceraldehyde-3-phosphate dehydrogenase (Fig. 16).

As in Mycobacterium tuberculosis, one gene encoding phosphofructokinase Aand one encoding phosphofructokinase B have both been found in C. glutam-icum [105, 106].

In the Bacillus subtilis genome, only the 6-phosphofructokinase family B ispresent. The phosphofructokinase isoenzymes are regulated differently by al-losteric activators. Family A enzymes (main 6-phosphofructokinase) are acti-vated by any nucleotide diphosphate and inhibited by PEP. Whereas family B(which supplies only 10% of the activity of phosphofructokinase family A) is in-hibited by ATP. 6-Phosphofructokinase activity has been measured in Brevibac-terium flavum strains, indicating that there is a different mode of regulation ofthis enzyme activity. It has been found, that in B. flavum, the phosphofructoki-nase activity is inhibited by ADP only [107]. These findings have to be clarified,which can now easily be performed with the sequence of the two genes at hand.

Similar to Mycobacterium tuberculosis, a second glyceraldehyde-3-phosphatedehydrogenase gene (gap) has also been detected in C. glutamicum, indicatingthat the metabolisms of C. glutamicum and M. tuberculosis are closely related.The finding that two open reading frames annotated as gpmA and gpmB encod-ing phosphoglycerate mutase are present in C. glutamicum was unexpected . Bothhomologous genes have been identified in the analyzed genome of the soil bac-terium C. glutamcium [108].

An open reading frame with significant homology to the poxB gene from E.coli indicated the existence of this enzymatic activity in C. glutamicum. The poxBgene encodes the pyruvate oxidase which catalyzes the oxidation of pyruvate toacetate. It became apparent that the disruption of the poxB gene in a l-lysine pro-ducing strain C. glutamicum MH20–22B results in an increase in l-lysine for-mation from 9.5 g L–1 to 12.9 g L–1 [109]. These findings show that genome analy-sis provides further insights into metabolic pathways.

For the pentose phosphate cycle and tricarboxylic acid cycle, all the missinggenes of the pathway have been identified. Also, the missing genes in the suc-cinylase route of the lysine specific pathway, dapC and dapF are now available.Thus both branches of the lysine biosynthesis, the 4-step tetradihydrodipicolinatesuccinylase pathway and the one-step meso-diaminopimelate dehydrogenasepathway are accessible for thorough investigation. Shaw-Reid et al. [110] inacti-

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Fig. 16. Glycolytic enzymes found in organisms with completely sequenced genomes. In C. glu-tamicum a second gap gene was found and three open reading frames with homology to thegpm genes. The enzymes are listed under E. coli gene names. Black arrows (thick lines) indicatepractically irreversible reactions, thin lines indicate reversible reactions

vated the desuccinylase gene dapE and reported that in shake flasks there was noinfluence on lysine biosynthesis indicating that the succinylase pathway is dis-pensable for lysine overproduction. In addition, they state that under these con-ditions wild type meso-diaminopimelate dehydrogenase activity is not rate lim-iting for lysine biosynthesis.

Since the dehydrogenase pathway uses NADPH as reductive power and thesuccinylase pathway glutamate (synthesized from a-ketoglutarate and NADPH),it will be interesting to see how high yielding lysine producers respond to en-hancing either of them under fermentation conditions.

To sum up the whole pathway leading from substrate uptake by the PTS sys-tem to the end product, l-lysine is now ready to be studied on the molecular level.

Metabolic pathways of C. glutamicum have been studied over the years by ge-neticists and biologists. With the knowledge of the genome sequence, the pres-ence of until now undetected genes becomes apparent. It must be considered thatthe presence of a gene sequence does not necessarily ensure that the corre-sponding enzyme is functional in the organism. To answer this question and tofind out which role the enzyme might play in the metabolism, the successful ap-proach using genetic tools, biochemical characterization, and metabolic fluxanalysis will be further applied. New software tools for the analysis of metabolicpathways will give more hints coming from the computational analysis ofgenome data [111].

Furthermore, it will be an interesting task to compare the data from the C. glu-tamicum genome with the Corynebacterium diphtheriae genome sequenced atthe Sanger Center [112] to elucidate the gene structure in both organisms and tofind differences in the metabolic pathways of these two closely related bacteria.

3.2Expression Profiling as a Tool for Strain and Process Optimization

Advances in microarray technology enable massive parallel data mining from hybridization based expression profiling on the genomic scale. The availability of genome data makes possible the production of whole genome chips for tran-scriptome analysis of C. glutamicum.

DNA arrays have already provided some important insights into gene functionin a variety of microorganisms like Saccharomyces cerevisiae, Streptococcus pneu-moniae, Haemophilus influenzae, Mycobacterium tuberculosis or Escherichia coli[113–115]. A twofold change in expression is detectable and a sensitivity of oneto five transcripts per cell is achievable with this technology [114].

A variety of different systems is available ranging from DNA arrays on nylonmembranes to high density arrays on silica chips. Printing technologies are de-veloping fast and different formats including spotted PCR fragments andoligonucleotide arrays are available [117]. There are obvious advantages of DNAchips, since chips allow miniaturization of the assay, parallelism of the analysisand automation of the assay procedure, making expression profiling an appro-priate tool also in fermentation monitoring.

Analyzing fermentation with expression profiling will lead to a better under-standing of the physiological status “lysine production”. Fermentation phases of

86 W. Pfefferle et al.

optimal growth can be compared to phases of optimal lysine production. Study-ing different conditions in general will reveal clusters of coordinated expressedgenes; the pattern of clusters should be typical for a physiological state. This waygenes and regulatory networks which are relevant to, e.g., exponential growth,maximal lysine overproduction, osmotic stress, toxification, or oxygen depletion,can be identified and investigated in detail. In consequence, they become targetsfor site specific manipulation to achieve further improvements.

The combined use of different postgenomic techniques such as DNA chips,2D gel-electrophoresis and old-fashioned physiology will provide the means tounderstand the cell’s regulators networks [118].

A second application field of expression profiling is the monitoring of routineproduction.

Applying DNA chips in production will help to assess the culture quality of in-ocula and seed fermenters as well as the lysine formation capability in a pro-longed fermentation mode. In addition, the fermentation may be run at the edgeof the mass transfer capacity of the fermentation tank due to a precise monitor-ing of the physiological state of the cell.

There are some basic requirements DNA chips have to match with regard tothis technology as a tool in routine production monitoring, e.g., for the moni-toring of process related target genes in fermentation [119]:

– Costs of such a diagnostic DNA-chip system have to be low– A quantity of DNA chips of uniform quality must be available– Improved and reliable methods for RNA isolation and labeling– Easy handling of the array during hybridization (automation required)– Short hybridization time– Sensitive detection system– Data handling and data evaluation tools for detection of the crucial steps in

fermentative production and to detect changes of the expression profile whichmight influence the quality of the production process.

3.3Pathway Modeling with Data Emerging from 13C-Isotope Experiments and Applicationsfor Biochemical and Metabolic Engineering

3.3.1Genotype and Metabolic Phenotype

Very recently, bioinformatics programs for the improvement of lysine productionby C. glutamicum have been initiated ([111] cf. Sect. 3.1). Once the C. glutamicumgenome has been completely sequenced and annotated an in-silico metabolicphenotype may be defined. With the use of this approach recently forHaemophilus influenzae Rd metabolic capabilities were assessed and differentoptimal phenotypes arising from the same metabolic genotype were derived,each with different constraint features [120]. Especially, high flexibility in themetabolic system to generate redox equivalents has been demonstrated compu-tationally. This kind of metabolic flexibility has recently been revealed experi-mentally for C. glutamicum [31]. In parallel to phenotype characterization by

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transcriptome and proteome analysis, the physiological state of bacterial cells ischaracterized by an array of metabolic fluxes through all reactions that occur inan organism which is called fluxome [121] as a particular feature of the wholemetabolic phenotype. Transcriptome analysis will in future reveal to which ex-tent genes are switched on and off for a particular physiological state. The actualuse of the single reaction steps or whole metabolic pathways will be quantifiedby characterization of the fluxome by metabolic flux analysis. The combinationof both techniques will have great impact on advanced process control.

In the past, metabolic flux analysis research into lysine production by C. glu-tamicum was mainly focused on the supply of carbon precursors and NADPH forproduct formation. Without the availability of transcriptome information, thisapproach was severely dependent on assumptions of the metabolic network ar-chitecture [56]. Detailed reviews on the biochemistry of C. glutamicum centralmetabolism are available as a basis for the establishment of metabolic models [30,56, 122–124]. Stoichiometric balances were used to estimate theoretical maxi-mum lysine yield on glucose of 0.45 to 0.75 g lysine · HCL (g glucose)–1 depend-ing on the model assumptions as, e.g., for minimized biomass formation or re-strictions by the metabolic network structure [56, 125]. Assuming a fluxlimitation due to succinyl-coenzyme A supply, the theoretical maximum yieldwas estimated to be 0.56 g lysine · HCl (g glucose)–1 [126]. Some uncertainty is in-troduced into the estimation of metabolic fluxes with the use of stoichiometricmodels, since bacterial energetics are still subject to reassessment [127] and gen-eral assumptions on the substrate demand to cover bacterial energetics may notbe valid for particular physiological states. Nevertheless, with the help of pub-lished data on the efficiency of the electron transport chain for C. glutamicum[55, 128, 129] and with progress in the field of bioinformatics as has been de-scribed for the analysis of the electron transport chain in B. subtilis [130] it mightbecome possible to establish reliable metabolic flux models for particular phys-iological states changing during the course of lysine fermentation. The questionof which of the alternative cytochrome oxidases are switched on or off in a par-ticular physiological state will be answered by transcriptome analysis. Whenmetabolic flux analysis is combined with fast DNA micro-array-techniques,which have recently been applied to investigate E. coli physiology [131], changesin the structure of the metabolic network might be monitored during the courseof fermentations. Models for process monitoring and control might be adjustedproperly, especially the clustering of genes might be monitored and together withthe identification of metabolic patterns by fluxome analysis might be used as animportant input for physiological state estimation.

3.3.2Short Review on Isotope Analysis

Recently, isotope analysis has been used to determine metabolic fluxes for C. glu-tamicum in general [132–134] and for lysine production in particular [31, 36, 40,41, 50, 135, 136]. Isotope investigations were used to reduce the number of as-sumptions on the metabolic network which must be made for the flux estimationby exclusive stoichiometric balancing. For the growth on fructose it was shown

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that the pentose phosphate cycle flux was lower than for growth on glucose [134].As a compensation for the lack of NADPH supply by the pentose phosphate cycle, the NADPH generating flux through the malic enzyme seemed to be in-creased and the ratio of cytoplasmatic NADH versus NAD was increased. For lysine production and glutamate production the split ratio of the pentose phos-phate cycle versus glycolysis was 70 versus 30 and 20 versus 80, respectively [135].The split ratio of tricarboxylic acid cycle versus anaplerotic carboxylation whichleads to C4-compound synthesis was shown to be 52 versus 48 for lysine pro-duction [137]. Recently, the above mentioned split ratios for lysine productionwere roughly confirmed by extensive 13C-isotope investigations for various fer-mentation runs [31, 40, 41] which indicates that at least for a subset of the meta-bolic network some split ratios can be predetermined by 13C-isotope analysis. Incombination with transcriptome analysis, it should be possible in future to re-solve singularity problems for the establishment of stoichiometric models forprocess control.

Bidirectional metabolic fluxes are exclusively accessible by isotope analysis.The investigation of bidirectional fluxes is useful for the identification of targetsfor metabolic engineering [31, 41] and mandatory during the phase of the es-tablishment of metabolic models. For routine metabolic flux analysis, bidirec-tional fluxes cannot be monitored continuously and the use of simplified mod-els turned out to be valuable for the control of lysine fermentation [138].

Very recently, very comprehensive approaches which make use of the 13C-en-richment in biomass constituents like amino acids were introduced to access notonly some but all major fluxes of central metabolism for lysine production withC. glutamicum [40, 139] and riboflavin production with B. subtilis [44]. This com-prehensive treatment of all the metabolic branch-points, rather than a portion ofthe central metabolism, allows us to reveal metabolic patterns which might beused for the design of metabolic and biochemical engineering strategies. An ex-tensive validation of a stoichiometric metabolic model by 13C-isotope analysis forthe lysine producer strain MH20–22B has been published [31, 40, 41, 50, 140]. Insummary, the comparison of six metabolic patterns which were revealed for C.glutamicum by 13C-isotope analysis showed that in C. glutamicum some meta-bolic fluxes depend severely on the physiological state of the cells: (1) coordinatedflux through pentose phosphate cycle and tricarboxylic acid cycle, (2) still unas-signed NADPH consumption or in other words high capacity for the reoxidationof NADPH, and (3) futile cycling between C3-compounds of glycolysis and C4-compounds of tricarboxylic acid cycle.

3.3.3Homologous and Heterologous Glutamate Dehydrogenase Mutant

An extensive validation of a stoichiometric metabolic model by 13C-isotopeanalysis for the lysine producer strain MH20–22B has been published [31, 40, 41,50, 140]. The computer-aided overall analysis of the 13C-isotope data for two iso-genic glutamate dehydrogenase mutants of the lysine producer strain C. glu-tamicum MH20–22B [31] revealed that the pentose phosphate cycle flux was onlyhigh for a high demand of NADPH and a low tricarboxylic acid cycle flux. The

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Fig. 17. In-vivo flux distribution in the central metabolism for C. glutamicum MH20–22B (a)using its NADP-dependent glutamate dehydrogenase (homologous C. glutamicum mutantMH20–22DBgdh pEK1.9gdh-1) and (b) using the NAD-dependent glutamate dehydrogenaseof P. asaccharolyticus (heterologous mutant C. glutamicum MH20–22BDgdh pEKEXpgdh).Numbers in oval symbols near thick lines give the estimated net fluxes; those near the thin ar-rows give the measured fluxes required for biomass synthesis. Numbers in hexagons give theestimated additional exchange fluxes in reversible reactions. All fluxes are given as molar

a

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b

metabolite flux expressed as a percentage of the glucose uptake rate, which was (a) 26.9 nmol(mg bio-dry mass)–1 min–1 and (b) 23.0 nmol (mg bio-dry mass)–1 min–1. Abbreviations: g6p,glucose 6-phosphate; f16 bp, fructose 1,6-bisphosphate; f6p, fructose 6-phosphate; gap, glycer-aldehyde 3-phosphate; pep, phosphoenolpyruvate; pyr, pyruvate; oaa, oxaloacetate; fum,fumarate; icit, isocitrate, akg, a-ketoglutarate; p5p, pentose 5-phosphate; s7p, sedoheptulose 7-phosphate; e4p, erythrose 4-phosphate. (From [31] with permission of Academic Press)

observed metabolic patterns indicated that the pentose phosphate cycle flux wasadjusted to the tricarboxylic acid cycle flux. In the heterologous glutamate de-hydrogenase mutant NADH was required for glutamate synthesis by a heterolo-gous NADH-dependent glutamate dehydrogenase form Peptostreptococcus asac-charolyticus. When a high tricarboxylic acid cycle flux occurred (Fig. 17) re-sulting from a high demand of NADH for glutamate synthesis much NADPH wassupplied by the isocitrate dehydrogenase as a reaction step in the tricarboxylicacid cycle pathway.

In response to this the NADPH formation by the pentose phosphate cycle waslow. This indicates that the reoxidation of NADPH might limit the pentose phos-phate cycle flux in C. glutamicum as has been described for liver tissue [141]. Foran increased reoxidation of NADPH in the homologous glutamate dehydroge-nase mutant during lysine production in which much NADPH was needed forglutamate synthesis by the NADPH-dependent glutamate dehydrogenase from C.glutamicum and in which a higher lysine yield was observed than in the het-erologous mutant resulting in a high NADPH demand the pentose phosphate cy-cle flux was increased and at the same time the tricarboxylic acid cycle flux wasdecreased (Fig. 17).

3.3.4Amphibolic Function of the Glyoxylate Cycle – Anaplerosis and Link to C4-Compound Decarboxylation

An illustrative example for validation of metabolic models is that for an isocitratedehydrogenase deletion mutant of C. glutamicum ATCC 13032. It was shown thata blocked tricarboxylic acid cycle was bypassed by high glyoxylate cycle activityand conversion of dicarboxylic acids into pyruvate or phosphoenolpyruvate asintermediates of glycolysis which afterwards entered the glyoxylate cycle se-quence for a second time [42]. In this way, pyruvate was oxidized to carbon diox-ide and redox equivalents were provided for energy generation.At the same time,in response to the blocked tricarboxylic acid cycle, the pentose phosphate cycleflux was high to provide sufficient amounts of NADPH. This is in agreement withthe generally observed inverse coordination of tricarboxylic acid cycle and pen-tose phosphate cycle in C. glutamicum. In general, a problem for the establish-ment of a stoichiometric model for lysine fermentation arises when two alter-native pathways are available which can operate in parallel as is the case for thetricarboxylic acid cycle and the glyoxylate cycle. In this case, singularity problemsprevent mathematical modeling of the metabolic network.An analysis of the sen-sitivity between the 13C-enrichment in position oxaloacetate C-4 and the two al-ternative anaplerotic pathways of glyoxylate cycle and anaplerotic carboxylationfor a particular data set [40] showed that a low 13C enrichment in oxaloacetate C-4 indicates a high glyoxylate cycle flux (Fig. 18).

Such a low 13C enrichment in oxaloacetate C-4 was observed experimentallyfor the above mentioned isocitrate dehydrogenase mutant. The computer-aidedoverall analysis of the 13C-enrichment data for this mutant resulted in a metabolicflux distribution with a high glyoxylate cycle flux. The glyoxylate cycle fulfils anamphibolic function in this mutant because on the one hand it provides ox-

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aloacetate which contributes to the supply of biomass building blocks so that itcan be called anaplerotic [142]. On the other hand, the glyoxylate cycle catalyzesthe interconversion of isocitrate to oxaloacetate and malate providing two C4-compounds which are subsequently decarboxylated to acetyl-coenzyme A and can enter the glyoxylate cycle again. By this metabolic cycle, glucose can be oxidized totally to CO2 in eight steps which provide as much NADH andFADH2 as an intact tricarboxylic acid cycle would do [143].All other 13C-isotopeinvestigations so far undertaken for the strain MH20–22B during cultivation on glucose have not revealed significant glyoxylate cycle flux at all which meansthat the glyoxylate cycle pathway can be excluded from a stoichiometric modelfor process control as long as no additional metabolic engineering is carried out. As described for the isocitrate dehydrogenase mutant where the glyoxylatecycle is active even during growth on glucose metabolic engineering resulted inan overall response of the metabolic network so that the stoichiometric model forthe particular strain has to be reevaluated by isotope investigations.

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Fig. 18. Influence of the net flux through the two alternative metabolic pathways ana-plerotic carboxylation of C3-compounds and the glyoxylate cycle on the 13C-enrichment in position oxaloacetate C-4 (OAA C-4). The sensitivity was investigated for a particular data set [40]. Flux values are expressed as percentage of the molar flux of glucose uptake rate.(From [42])

3.3.5Comparison of in-Vivo and in-Vitro Characterization of Enzyme Activities

Metabolic flux analysis was used for the determination of the in-vivo activity ofenzyme reactions of the split lysine synthesis pathway (cf. Sect. 2.2).Although thein-vitro activity of the succinylase pathway enzymes and of the diaminopimelatedehydrogenase are in the range of up to 100 mU (mg protein)–1 and 1 U (mg pro-tein)–1 respectively, at the end of a batch fermentation when ammonia concen-tration is very low, the in-vivo metabolic flux of about 40 mU (mg protein)–1 isexclusively catalyzed by the succinylase pathway enzymes for which lower in-vitro activity values had been determined [140]. Here it becomes obvious that cytoplasmatic substrate availability determines flux split ratios since theMichaelis-Menten constant of diaminopimelate dehydrogenase for ammonia wasdetermined to be high with about 36 mM [144]. In a chemostat culture with anammonia concentration of 150 mM and 600 mM, the split ratio of succinylase todehydrogenase flux was about 2.8 to 1 and 1.1 to 1 respectively [31, 40], whichagain stresses the importance of substrate availability as a key factor which in-fluences metabolic flux split ratios during lysine production. As is valid for thediaminopimelate dehydrogenase, the isocitrate dehydrogenase, in-vitro and in-vivo activities of up to 1 U (mg protein)–1 [145] and up to 50 mU (mg protein)–1

[31, 40, 41] have been published for comparable physiological states, respectively,which again stresses that the in-vitro activity does not give any information onthe actual in-vivo metabolic activity of the reaction step as a part of the wholemetabolic network.

Fluxome research has been applied to analyze the in-vivo split ratio betweenlow- and high-affinity metabolic pathways which operate in parallel. Isotope in-vestigations with the use of 13C and 15N lead to the elucidation of the metabolicfluxes through the split lysine biosynthetic pathway [140] and of intracellular glutamate formation [146], respectively. It was revealed that, with progressing fermentation time and decreasing extracellular ammonia concentration, the in-vivo dehydrogenase pathway activity decreased indicating that the high-affinityroute succinylase pathway became predominant. Intracellular glutamate forma-tion might be crucial for an optimal operation of the succinylase pathway oflysine biosynthesis. It was found that glutamate formation was catalyzed to onethird by the high-affinity route glutamine synthetase. This reaction was especiallyactive to provide glutamine N-5 atoms for the synthesis of biomass components.The other two thirds of intracellular glutamate synthesis was catalyzed by the glutamate dehydrogenase reaction.

The significant in-vivo activity of high-affinity routes indicates that re-duced extracellular ammonia concentrations could be sufficient for ammonia in-put. Furthermore, by 15N-isotope data analysis, a bidirectional metabolic flux was quantified in addition to the net synthesis of glutamate from ammonia and a-ketoglutarate by the glutamate dehydrogenase reaction which was morethan half the net flux. Metabolic engineering could target a reduction of bi-directional fluxes in order to increase the net flux in the desired direction. Thesame applies to the formation of C4-compounds, as described in Sect. 2.3.Summarizing, these few examples of fluxome research by metabolic flux analy-

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sis in C. glutamicum during growth and amino acid overproduction illustratehow important it is to establish metabolic models for particular strains and phys-iological states stressing the differences between in-vitro and in-vivo enzyme activities.

3.3.6Application of Fluxomics – Exploitation of Metabolic Flux Information

One use of stoichiometric metabolic flux models is that, knowing all re-quirements for the synthesis of biomass and product, it should be possible to design proper substrate feed profiles with which neither overflow metabolismnor precursor limitation of biomass and lysine synthesis occur. Especially the demand for NADPH, oxaloacetate, pyruvate, and glutamate should not be-come limiting in lysine production process. For the strain MH20–22B it was revealed that, with increasing lysine formation, the net synthesis of oxalocatetateincreased because the reaction rate of the oxaloacetate consuming decarboxy-lations decreased (cf. Sect. 2.3; [31, 41]). In parallel, the total NADPH supply increased with enhanced lysine formation mainly by an increase in pentose phosphate cycle flux (cf. Sect. 2.4; [31, 41]). It should be stressed that conclusionsabout metabolic patterns can only be extrapolated partially and that especially during scale-up it must be considered that cell physiology in generaland especially metabolic fluxes change rapidly when the micro-environment of bacterial cells varies within the different spatial zones of a large bioreactor[147, 148].

4Optimization of the Lysine Fermentation Process

4.1Improvement of Process Economics by Process Intensification

Lysine is mainly produced in fed-batch mode and from data on the specific ratesfor sugar consumption and lysine formation which were published in a study on the use of osmoprotectants for optimization of lysine production experi-mental values on the apparent yield of up to 0.62 g lysine · HCl (g glucose)–1

were determined for resting cells [75, 76]. This result indicates that quite high experimental product yields can be achieved which are even higher than theo-retically determined values (cf. Sect. 3.3) based on particular assumptions whichmight not apply to the above mentioned physiological state. A target for furtherimprovement of process economics is to repeat batch or fed-batch fermenta-tions without the need of repeated preparation of the bioreactor and new in-ocula. In order to improve the overall productivity of lysine fermentation, a portion of about 60 to 95% of the final culture volume might be removed and replaced by fresh medium. Using this mode of repeated batch or fed-batch fermentation successive fermentation runs can be carried out without introduc-ing fresh inoculum and down time for the preparation of the sterile bioreactor.Up to 2000 only repeated batch for the production of lysine has been described

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[149–151]. Only few examples for repeated fed-batch as for phenylalanine production have been reported [152] where strain instability was revealed as amajor problem. Strain instability becomes a major problem when lysine pro-duction strains are used which are auxotrophic for amino acids [153]. With a stable model strain for which acetylcysteine-resistance and feed-back resistanceof aspartate kinase has been determined, in two successive fed-batch runs a lysine · HCl concentration of 53.4 and 55.6 g L–1, respectively was measured [154]. The lysine · HCl yield on sucrose was 0.209 and 0.228 g g–1 and the volu-metric productivity was 1.71 and 2.01 g L–1 h–1, respectively (Fig. 19).With about30 h of pure fermentation time per single fed-batch run, the preparation time of10 h could be saved for the second fed-batch run. Thus with one repetition, a 15%increase in overall productivity was achieved for this repeated fed-batch fer-mentation where the performances of the two successive fed-batch runs werecomparable.

The next step for the improvement of process economics is the establishmentof continuous culture fermentation but up to now has not yet been published forlarge-scale lysine production. Investigations on glutamate production revealedthat the volumetric productivity with 5 to 8 g L–1 h–1 for continuous cultivationswas at least twice the value for comparable fed-batch fermentations [155]. For a two-step continuous culture system for lysine production where for up to 400 hours in the “growth”bioreactor and “production”bioreactor pH is controlledat 7.2 and 6.2, respectively, the productivity through the whole process has been

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Fig. 19. Repeated fed-batch process for lysine production with a stable model strain for whichacetylcysteine-resistance and feed-back resistance of aspartate kinase was determined. Timecourse of Optical density at 535 nm (1) and concentration (g L–1) of ammonia (2), dextrose (3),and lysine · HCl (4)

shown to be about twice that of a batch type cultivation [156]. In small-scalecontinuous culture fermentations for the production of lysine, maximum ap-parent yields for lysine·HCl on glucose in basic research experiments with modelstrains were 0.25 g g–1 [157], 0.29 g g–1 [31], and 0.30 g g–1 [158] which indicatesa maximum intrinsic product yield of 0.41 g g–1.A maximum of 1.0 g lysine · HClL–1 h–1 was achieved for a strain requiring homoserine and leucine and having aresistance to an isoleucine analogue [157]. Continuous culture experiments byHirao et al. with a strain derived from ATCC13032 by selecting mutants resistantto S-(2-aminoethyl)-l-cysteine, rifampicin, streptomycin, and 6-azauracil resultedafter optimization of the culture redox potential kinetics in a maximum appar-ent yield for lysine · HCl on glucose of about 0.40 g g–1 and a maximum of 5.6 glysine · HCl L–1 h–1 [159]. This clearly indicates that there is potential for thedevelopment of more economic processes once limitations such as insufficientoxygen supply have been overcome. In a more detailed analysis of the cultureredox potential, a strategy for the addition of dithiothreitole has been used to op-timize lysine production in batch fermentations [160, 161].

By the introduction of simultaneous carbon and phosphate limitation, con-tinuous fermentation for lysine production has been optimized [162].

Compared to simple carbon limitation, with the double limitation, thevolumetric productivity increased from 3.18 to 3.75 g lysine · HCl L–1 h–1. The bio-mass specific product formation rate increased from 0.084 to 0.240 g lysine · HCl(g bio-dry mass)–1 h–1 so that for the production of the same amount of lysine,65% less biomass is produced in the double limitation process. Furthermore, itwas observed that with the use of the double limitation culture, instability as typ-ically observed for the continuous cultivation of amino acid production strainscould be avoided for up to 30 generations. In conclusion, process intensificationfor lysine production has been achieved in the past by introduction of fed-batchand continuous culture fermentation as well as by optimization of oxygen avail-ability. Modern techniques as fluxomics, transcriptomics, and single cell analy-sis by cytometry might help to extend the cultivation time of this culture modese.g. by detection of the inoculum viability and metabolic activity before succes-sive repeats are started.

4.2Scale up and Scale down Techniques

Lysine fermentations are performed in very large tanks with a working vol-ume of several hundred cubic meters. Fermentations performed in such large tanks may be severely limited by mass transport phenomena such as mixing and oxygen transfer. Einsele [163] reports that mixing time in large fermenterscan easily exceed 100 s. Due to different kinetics of mass transfer, mixing and consumption gradients of dissolved oxygen, pH and substrates are formedto which the microorganisms are exposed during their circulation in large vessels. Even in comparatively small vessels of 20 m3, strong oxygen profiles have been reported [164]. These gradients affect the overall performance negatively so that scaling up becomes a very important factor in bioengineer-ing.

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Adjusting a bioprocess to a large tank at the production site is very costly.Therefore, there is a series of reports which deal with the investigation of in-sufficient mixing in lab fermenters simulating fluctuating conditions that occur on the production scale. Especially the effects of changes in oxygen andsubstrate availability were investigated in detail in a one fermenter system[165–169].

Another quite common experimental design is to connect 2 lab tanks in seriesin which defined but different conditions are set [170–172].

The aim of scale down investigations is to mimic the large-scale microen-vironment of the bacterial cell in a small-scale bioreactor.

A special reactor design for investigation mixing time effects in one reactor isreported by Schilling et al. [147]. This scale down lab tank is equipped with cylin-drical disks and Rushton turbines leading to reduced axial mixing (Fig. 20).

Due to the reactor design, the apparent mixing time q90 was prolonged to130 scompared to 10 s of the reference reactor. Comparison studies with a leucine-aux-otrophic lysine strain were performed in fed-batch fermentations with feeding ofl-leucine. Table 5 shows that the lysine fermentation in the scale-down reactorwas significantly slowed down indicating that varying availability of the processlimiting substrate directly affects the fermentation performance.

Since the authors report that these findings are reflected in a lower specific ac-tivity of enzymes such as citrate synthase, aspartate kinase and phosphoenolpyruvate carboxylase, the cellular sensing and responding to substrate gradientsis of general interest.

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Fig. 20. Scale down lab tank. A prolonged mixing time (q90 =130 s) is achieved by cylindricaldisks which form compartments and thus reduce axial mixing. Potential pH and pO2 gradientsare monitored using probes on two levels

A scale down system which allows us to investigate these assumed rapid re-sponses of the microorganism to changing environments is a reactor designwhere a lab fermenter is connected to a plugged flow reactor bypass withoutsparging leading to oxygen depletion [148]. Sampling along the plugged flow re-actor with subsequent expression profiling and proteome analysis can follow thequick reactions which take place inside the microorganisms.

Besides working on the elucidation of the influence of gradients on microbialphysiology improved stirring systems with strong axial mixing have been devel-oped. This way spatial gradients are considerably reduced improving the fer-mentation performance in large tanks.

4.3Process Control

4.3.1Physiological State Estimation and Optimization of Nutrient Supply

For the metabolic state of overproduction Neijssel and Tempest [173, 174; forsummary 175] developed a concept of “overflow metabolism”. They report thatcarbon sufficient chemostat cultures often metabolize the substrate inefficientlyleading to excretion of energy rich compounds like acetate, pyruvate, 2-oxoglu-tarate or gluconic acid.

Characteristics of the investigated conditions are quite similar to lysine pro-duction conditions:

– Carbon source in excess– Growth limitation of the culture– No strict regulation of substrate uptake

Although lysine overproduction itself can be understood as a metabolic state of favorable overflow metabolism, the above mentioned characteristics suggestthat lysine fermentations may also suffer from concomitant by-product for-mation. This was confirmed by Hadj Sassi et al. [176] and Hua et al. [177], whofound that under fully aerobic conditions in cultures with growth limitations andcarbon excess, significant formation of organic acids like acetate and pyruvateoccurs.

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Table 5. Experimental results after 53 h cultivation of parallel exponential fed-batch cultiva-tions with the leucine auxotrophic Corynebacterium glutamicum DSM5715 in a standard anda scale down reactor with prolonged mixing time

Reference Reactor Model Reactorq90 =10 s q90 =130 s

Sugar Consumption 4.48 kg 3.85 kgLysine Production 0.82 kg 0.72 kgTotal CO2-evolution 3.22 kg 2.07 kgDry cell weight 0.33 kg 0.31 kg

The concept of monitoring and control of the physiological state of cell cul-tures has been reviewed recently [178]. For phenylalanine production with E. coliespecially exact monitoring of specific growth rate enabled one to adjust the glu-cose feed properly and changes in metabolic rate ratios as oxygen to glucose up-take and in feed ratios as ammonia to glucose feed revealed the onset of growthdecline and by-product formation, respectively [179]. Figure 21 shows that thetypical E. coli by-product acetate is secreted when substrate consumption rate ex-ceeds a so-called critical glucose consumption rate. Below this rate, acetate al-ready present in the medium is readily co-metabolized.

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Fig. 21. Acetic acid formation in phenylalanine fed-batch fermentation with E. coli AT2471 de-pendent on specific glucose uptake rate (GUR): a discrimination of the time-field of fermen-tation based on acetic acid excretion, b confirmation of the time-fields in a fed-batch fermen-tation GUR (1); acetic acid (2). After [179]

a

b

In general, action was taken to counteract the onset of overflow metabolismby glucose feed reduction. For C. glutamicum chemostat fermentations were usedto determine the critical growth rate for the onset of by-product formation [180].The strategy of adjusting the sugar feeding in such a way that maximum sugaruptake is established during the whole microbial cultivation has recently been ap-plied to fermentations with yeast [181].

For technical lysine fed-batch fermentations with Corynebacterium glutam-icum, Miwa et al. [182] describe a method to control the substrate feed efficientlyin order to avoid overflow metabolism. To obtain low concentrations of the car-bon source in the fermentation broth, the increase in the pH or in the dissolvedoxygen content is controlled by intermittently adding the feed solution at a com-puter-calculated flow rate. In this way, the limitation signals trigger the next sub-strate dosage (Fig. 22).

A similar method is described by Pfefferle et al. [183] who ensure that duringcarbon feeding, the culture of a technical lysine fermentation is sugar limited.With this method, they are able to achieve high coefficients for lysine (total drymatter)–1 indicating elevated lysine yields and low by-product formation.

An extension of physiological state estimation is the online metabolic path-way analysis which has recently been applied to the optimum control of lysineproduction with C. glutamicum [138]. The online metabolic flux analysis for fer-mentation monitoring and control has recently been reviewed [184]. With ad-vances in fuzzy and neuronal network supervisory control systems for lysine pro-duction, trends from experimental data might be extracted, production rulesmight be defined, and proper feed profiles might be chosen as has been describedfor glutamate production with C. glutamicum [185]. For leucine auxotrophic ly-sine production strains, a central feature of the improvement of lysine produc-tion is the optimization of the initial concentration of leucine [186, 187]. The optimization of the subsequent feed-profile for leucine addition resulted in anideal course of growth rate and led to improved process performance [187]. Fluc-tuations in the composition of complex media components, e.g., corn steepliquor, which are used for the large-scale production of amino acids, might pre-vent a stable and efficient industrial process performance because of the input ofgrowth limiting components as leucine fluctuates. Recently, this problem was ob-served for threonine production with E. coli where the iron input from corn steepliquor varied with different corn steep liquor lots [188]. The growth level wasmade independent of this iron input fluctuation by addition of iron to themedium.At the same time, the growth of the methionine-auxotrophic threonineproduction strain had to be restricted in order to ensure maximum threonineproduction. A reproducible restriction of growth was achieved by reduction ofthe methionine concentration in the medium. As a consequence, an efficient in-dustrial process with high productivity and stability was established which wasno longer dependent on iron limitation or fluctuations of corn steep liquor qual-ity. This kind of process adaptation in order to compensate fluctuations in rawmaterial quality might be useful for the stabilization of lysine productionprocesses as well.

Optimization of medium composition using a genetic algorithm and controlof glucose concentration at about 9 g L–1 by online high performance liquid chro-

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Fig. 22. In fed-batch fermentation the dosage of the feed solution is controlled by substrate de-pletion signals (according to [182]). The increase of pH and pO2 over a threshold value triggeranother feed pulse, the resulting sugar concentration in the fermentation broth remains below2 g L–1

Time

1st

addition2nd

addition3rd

addition

Feed pulsecalculated on

demand

pH

pO2

Substrate depletion

Substrate concentration inthe fermenter

Time

Time

matography resulted in a volumetric productivity of up to 8.3 mmol L–1 h–1 anda yield of lysine on glucose of 0.32 mol mol–1 [189]. Further improvement of theproduct yield to 0.38 mol mol–1 as a consequence of increased growth linked ly-sine formation was achieved by introduction of glucose limitation in the precul-ture which might lead to the activation of glucose-starvation-stimulons as wasobserved for E. coli [190]. Here it becomes obvious that also the control of precul-tures might be a target for the improvement of lysine overproduction. Especially,when the analysis of transcriptome and fluxome is established for process mon-itoring.

4.3.2Single Cell Analysis by Flow Cytometry

Recently, flow cytometry has been used to quantify the response of C. glutamicumto antibiotics [191] and to measure intracellular pH as well as transmembranepotential [192]. This kind of monitoring of single cell properties might, in future,improve process monitoring and control especially because changes in the com-position of a bacterial population might even be detected at an early stage of fer-mentation so that early decisions to counteract them can be made. Since singlecells are analyzed it might be possible to observe physiological state changesmuch earlier than for conventional measurements. The reason is that the uptakeof oxygen or sugar can only be measured on conventional devices once a suffi-cient cell density has been achieved to be in the measurement range of the con-ventional measurement devices. Applications of flow cytometry to E. coli fer-mentations for the investigation of sheer stress phenomena have been describedrecently [193]. First results for lysine producing C. glutamicum strain MH20–22Bare available and these clearly indicate distinct subpopulations of single cells andcell aggregates of different sizes (Fig. 23). On the single-cell level, intact cells, de-polarized cells, and permeabilized cells were differentiated by staining with pro-pidium iodide and bis-oxonole as described in detail for E. coli [193]. For real-time process monitoring, it might be interesting to measure the size of thesubpopulation of aggregates since these are formed when adverse effects appearin lysine fermentation. In conclusion, advances in the monitoring of the physio-logical state of lysine producing C. glutamicum cultures by online metabolic fluxanalysis and optimization of nutrient supply in precultures and main culturescontributed to the improvement of lysine production. Extended physiologicalstate estimation by fluxomics, transcriptomics, and by cytometry analysis mighthelp to achieve further process intensification.

5Large Scale Lysine Manufacture

Corynebacterium glutamicum is able to utilize as substrate sucrose and dextrose.Therefore, the major C-sources for lysine fermentation are pure sucrose, beetmolasses, cane molasses, high test molasses (inverted cane molasses), anddextrose prepared from starch hydrolysate. In addition, technologies for the uti-lization of acetic acid and ethanol as co-substrates are available. The utilization

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Fig. 23. Single cell analysis of C. glutamicum strain MH20–22B. In graph a and b on the x-axissideward scatter and on the y-axis forward scatter is plotted. In graph a, the total populationis split into a “comet tail” (A1) and a bimodal subpopulation which is typical for rod-shapedbacteria where cells are detected which crossed the laser beam with a maximum (A2) or min-imum (A3) cross-section. In graph b, the “comet tail” subpopulation is reduced which indicatesthat a smaller part of the population is made up by aggregates. In graph c, fluorescence signalsfrom propidium iodide (PI; x-axis) and bis-oxonole (BOX; y-axis) stained cells are indicated(cf. text). C. glutamicum cells were stained with 0.1 mg BOX mL–1 and 0.05 mg PI mL–1. Theanalysis was restricted to cells of the bimodal subpopulation as indicated by the box in graphb. Notice that most of the C. glutamicum cells are depolarized (C2) and only a few cells are clas-sified as live (C1) or permeabilized (C3)

a

b

c

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of molasses is gradually declining because its waste burden leads to increasedcosts for waste disposal in the lysine plant.

Because of the relatively low market price for lysine of around 1.3–2 $ (kg lysine · HCl)–1 there is great pressure to reduce costs.

The most important part of the manufacturing costs of lysine production isthe C-source; therefore it is not astonishing that sugar suppliers and lysine pro-ducers are subject to close alliances: either the plants are situated close togetherso that the sugar is provided across the fence so to speak, or they form joint ven-tures, or C-source suppliers install their own lysine technology.

Beside the price, the economical utilization of the C-source is very important.In technical processes conversion yields of 0.45–0.50 g lysine · HCl (g sugar)–1

have been reported (e.g., FR2645172 [194]). A typical fermentation graph of a lysine fed-batch fermentation is shown in Fig. 24.

Looking at lysine manufacturers world-wide, there is a clear tendency towardbigger plants with very high capacities and thus using their economy of scale.Nowadays, lysine plants use large scale fermenters with up to 500-m3 volume oreven more.

Finally, work up has a major impact on manufacturing costs in lysine pro-duction. Lysine fermentation usually yields lysine sulfate at the end of fermen-tation. The classical way of work up follows a series of steps leading to a crys-talline product of lysine · HCl.

For lysine producers, reduced investment and running costs of downstream-ing are decisive, low costs for waste disposal are also of great importance.

Since lysine is predominantly used as a feed additive, various more econom-ical downstream processes have been developed yielding new lysine preparationswhich are well established on the market (Table 6) indicating that the advantage

Biotechnological Manufacture of Lysine 105

Fig. 24. Technical lysine fed-batch fermentation; sugar feeding is lower than the maximal sugarconsumption rate resulting in a carbon limitation during the feed period

40 500

5

10

15

20

25Su

gar

[g/l

]

0

40

80

120

160

200

0 10 20 30Fermentation time [h]

Lys*

HCl [

g/l];

Opt

ical

Den

sity

[rel

U]

Lysine OD Sugar

of the new product preparations exists on the supplier’s side but also on the cus-tomer’s side.

6Outlook

Recently in the field of microbiology, an enormous amount of data based ongenome sequencing became available for strain development and bioengineering.

Therefore, it is not surprising that the genome of the sole economically im-portant lysine producer Corynebacterium glutamicum has also been sequenced.The availability of these data gives access to post genome technologies such as ex-pression profiling with DNA chips and investigation of protein patterns with 2D-gelelectrophoresis. Metabolic flux analysis in combination with metabolomeanalysis will help to characterize the actual in-vivo conversion rates through theactive metabolic network. In this way, process control will be adjusted in a cor-rect way and new strategies for metabolic engineering will be developed.

These multi-parallel working technologies will accelerate the generation ofknowledge and elucidate new global regulatory networks. For the first time, thereis a chance of us being able to understand the overall picture of the physiologi-cal state of lysine overproduction in a technical environment.

Additionally, technologies like flow cytometry will give deep insights into theheterogeneity of microbial population under lysine producing conditions basedon information at the single-cell level. Especially the possibility for online con-trolling of technical fermentations will be very attractive for further process en-gineering.

Combination of the knowledge emerging from these new technologies willsurely lead to further improvements in lysine production. Therefore, despite thefact that efforts have already been made for four decades for optimizing lysinetechnology, additional major improvements can be expected.

106 W. Pfefferle et al.

Table 6. Commercially available lysine preparations for feed use based on different down-stream technologies

Product Lysine-base Downstreaming Wastes Advantagespreparation content [%] steps

Lysine· HCl [195] 78.8 Biomass separation, Biomass, No Ion exchange, Drying Organic acids, crystallization

Salts, Ammonia,Water

Liquid lysine Approx. 50 Biomass separation, Biomass, Salts No drying step[196] EvaporationLiquid lysine 20–30 Evaporation Almost none No drying,sulfate [197] only a concen-

tration stepGranulated lysine 40–50 Evaporation, Almost none No wastes,sulfate [198] Spray granulation No separation

step

Acknowledgement. The authors thank Dr. C. J. Hewitt for experimental advice and discussionson C. glutamicum flow cytometry analysis and Dr. L. Eggeling for the contribution of unpub-lished results. Mrs. C. Augé is acknowledged for excellent technical assistance. The authorsthank Dr. M. Farwick and Dr. R. Krämer for their helpful comments. Dr. R. Krämer contributedFig. 14.

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