Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.With an annual market volume of more than 1,000,000 tons, lysine is one of the dominating products in biotechnology. In recent years, rational metabolic engineering has emerged as a powerful tool for lysine production (16, 18, 22). Hereby, different target enzymes and pathways in the central metabolism could be identified and successfully modified to create superior production strains (1, 2, 5, 8, 10, 17-20). The tricarboxylic acid (TCA) cycle has not been rationally engineered so far, despite its major role in Corynebacterium glutamicum (6). From metabolic flux studies, however, it seems that the TCA cycle might offer a great potential for optimization (Fig. ​(Fig.1),1), which is also predicted from in silico pathway analysis (13, 22). Experimental evidence comes from studies with Brevibacterium flavum exhibiting increased lysine production due to an induced bottleneck toward the TCA cycle (21). In the present work, we performed TCA cycle engineering by downregulation of isocitrate dehydrogenase (ICD). ICD is the highest expressed TCA cycle enzyme in C. glutamicum (7). Downregulation was achieved by start codon exchange, controlling ICD expression on the level of translation.Open in a separate windowFIG. 1.Stoichiometric correlation of lysine yield (%), biomass yield (g/mol) and TCA cycle flux (%; entry flux through citrate synthase) determined by ¹³C metabolic flux analysis achieved by paraboloid fitting of the data set (parameters were determined with Y0 = 105.1, a = −1.27, b = 0.35, c = −9.35 × 10⁻³, d = −11.16 × 10⁻³). The data displayed represent values from 18 independent experiments with different C. glutamicum strains taken from previous studies (1-3, 11, 12, 15, 23).     

Lysine uptake and exchange in Corynebacterium glutamicum.

Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in fact represents a homologous antiport between extracellular [14C]lysine and intracellular unlabeled lysine. Intracellular [14C]lysine could only be released by the addition of unlabeled lysine to the bacterial suspension. In contrast to this homologous antiport reaction, we observed net uptake of lysine in lysine-depleted cells of a lysine auxotrophic strain. This net uptake was found to be electrogenic and could also be observed as a heterologous antiport reaction in wild-type cells under particular conditions. In this case exchange was mediated between internal lysine and external alanine, isoleucine, or valine. This antiport was electrogenic, since the substrates differ in charge. The cells can switch between electroneutral homologous exchange and electrogenic heterologous antiport mode during fermentation because of changing metabolic conditions.     

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, ¹³C metabolic flux analysis with parallel studies on [1-¹³C^Fru]sucrose, [1-¹³C^Glc]sucrose, and [¹³C₆^Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS^Man or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Activating Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxykinase in Combination for Improvement of Succinate Production

Phosphoenolpyruvate (PEP) carboxylation is an important step in the production of succinate by Escherichia coli. Two enzymes, PEP carboxylase (PPC) and PEP carboxykinase (PCK), are responsible for PEP carboxylation. PPC has high substrate affinity and catalytic velocity but wastes the high energy of PEP. PCK has low substrate affinity and catalytic velocity but can conserve the high energy of PEP for ATP formation. In this work, the expression of both the ppc and pck genes was modulated, with multiple regulatory parts of different strengths, in order to investigate the relationship between PPC or PCK activity and succinate production. There was a positive correlation between PCK activity and succinate production. In contrast, there was a positive correlation between PPC activity and succinate production only when PPC activity was within a certain range; excessive PPC activity decreased the rates of both cell growth and succinate formation. These two enzymes were also activated in combination in order to recruit the advantages of each for the improvement of succinate production. It was demonstrated that PPC and PCK had a synergistic effect in improving succinate production.     

Phosphoenolpyruvate carboxylase in Corynebacterium glutamicum is dispensable for growth and lysine production

Abstract The symbiotic plasmid pRHc1J and the helper plasmid pJB3JI were transferred from Rhizobium "hedysari" strain RJ77 to Agrobacterium tumefaciens strain GMI9023. Transconjugants harboured recombinant plasmids (R-prime plasmids) consisting of pJB3JI carrying DNA fragments, of different sizes, surrounding the Tn 5 mob insert in pRHc1J. Two of these R-prime plasmids (pR1 and pR2) carried nod genes and were able to restore the Nod⁺ phenotype of pSym⁻ derivatives of R. "hedysari" . The R. "hedysari" nod genes harboured by both R-primes were expressed in R. leguminosarum biovar trifolii wild-type but not in a pSym⁻ derivative.     

Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria. In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate (shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.     

Strains of Corynebacterium glutamicum with Different Lysine Productivities May Have Different Lysine Excretion Systems

The lysine excretion systems of three different lysine-producing strains of Corynebacterium glutamicum were characterized in intact cells. Two strains (DG 52-5 and MH 20-22B) are lysine producers of different efficiency. They were bred by classical mutagenesis and have a feedback-resistant aspartate kinase. The third strain (KK 25) was constructed from the wild type by introducing the feedback-resistant aspartate kinase gene of strain MH 20-22B into its genome. The three strains were shown to possess different excretion systems. Export in strain KK 25 is much slower than in the two mutants. The differences between the two lysine-producing strains are more subtle. K(m) and V(max) are similar, but pH dependence and membrane potential dependence reveal differences in the intrinsic properties of the carrier system.     

Feedback Inhibition of Lysine Biosynthesis in Yeast

The pathway for the biosynthesis of lysine is feedback-inhibited by lysine in growing yeast.     

Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.     

Removal of Feedback Inhibition of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> Phosphoenolpyruvate Carboxylase by Addition of a Short Terminal Peptide

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the carboxylation of phosphoenolpyruvate (PEP) in the presence of bicarbonate to form oxaloacetate (OAA), and it plays an important role in high-efficient production of OAA-derived metabolites such as lysine, glutamate and succinate. However, PEPCs often suffered from serious feedback inhibition by various metabolic effectors like aspartate. Here, the feedback inhibition of PEPC from Corynebacterium glutamicum was removed by adding a short terminal peptide like His-tag. The effect of His-tag location on the structure and important properties such as activity, thermostability and feedback inhibition of PEPC has been investigated. The purified untagged PEPC, Nterminal His-tagged PEPC (PEPC-N-His) and C-terminal His-tagged PEPC (PEPC-C-His) were characterized. PEPCN- His (439.71/sec/mM) showed a 1.26 and 186-fold higher catalytic efficiency than untagged PEPC (348.59/sec/mM) and PEPC-C-His (2.36/sec/mM), respectively. Both PEPCN- His and untagged PEPC were significantly inhibited by aspartate at the concentrations above 4 mM (residual activities < 10%), while PEPC-C-His was almost desensitized to aspartate within 10 mM (around 90% of residual activity). Structural analysis showed that the extension of C-terminus may cause steric hindrance for aspartate binding with enzymes, leading to the deregulation of feedback inhibition of PEPC-C-His. This study provides a deeper understanding of the effect of terminal fragments on the structure and function of PEPCs, and helps to engineer the feedback inhibition of PEPCs and structurally similar enzymes.     

Production of Native-Type Streptoverticillium mobaraense Transglutaminase in Corynebacterium glutamicum

We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a result, native-type transglutaminase was secreted.     

Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum

We previously observed secretion of active-form transglutaminase in Corynebacterium glutamicum by coexpressing the subtilisin-like protease SAM-P45 from Streptomyces albogriseolus to process the prodomain. However, the N-terminal amino acid sequence of the transglutaminase differed from that of the native Streptoverticillium mobaraense enzyme. In the present work we have used site-directed mutagenesis to generate an optimal SAM-P45 cleavage site in the C-terminal region of the prodomain. As a result, native-type transglutaminase was secreted.     

Production of l-valine from metabolically engineered Corynebacterium glutamicum

Applied Microbiology and Biotechnology - l-Valine is one of the three branched-chain amino acids (valine, leucine, and isoleucine) essential for animal health and important in metabolism;...     

Salt-stimulated Phosphoenolpyruvate Carboxylase in Cakile maritima

The effects of NaCl and other salts, in vivo and in vitro, on the activity of phosphoenolpyruvate carboxylase from the coastal C₃ halophyte Cakile maritima Scop, were investigated. Plants grown with 100 mM NaCl in their growth medium yielded some 30% higher rates of phosphoenolpyruvate carboxylase activity than did salt-depleted plants. Activity of the enzyme was stimulated when NaCl was added to the reaction mixture in concentrations of up to 200 mM. The magnitude of this in vitro stimulation was similar for plants grown in the presence or absence of NaCl. The effect seems to be caused by chloride rather than by sodium ions.     

Lysine secretion by Corynebacterium glutamicum wild type: regulation of secretion carrier activity

Lysine secretion in wild-type Corynebacterium glutamicum was investigated by means of dipeptide feeding during short-term fermentation. It could be shown that important properties of lysine excretion, e. g. dependence on membrane potential and the internal Michaelis constant (K _m), are not different for the producing strain DG 52-5 and the wild type. The main difference seems to refer to regulatory properties of the lysine excretion carrier activity. The transport of lysine in the wild type is regulated by the presence and kind of carbon sources. These differences in transport activity are not due to changes in the driving force. A possible distinction between phosphotransferase system (PTS) and non-PTS carbon sources with respect to the observed regulatory phenomena is discussed.     

Improved electrotransformation frequencies of Corynebacterium glutamicum using cell-surface mutants

The electrotransformation efficiency for homologously- and heterologously-derived plasmid DNA was determined for two families of Corynebacterium glutamicum strains derived from ATCC13059 (AS019 and auxotrophic, cell surface mutants MLB133 and MLB194) and ATCC13032 (parent strain and restriction-minus mutants RM3 and RM4), following their growth in LBG supplemented with glycine plus isonicotinic acid hydrazide (INH). Electrotransformation efficiencies of MLB133 were up to 100-fold higher than for strain ASO19 and, when using heterologously-derived plasmid DNA, MLB133 showed efficiencies comparable to or better than strains RM3 and RM4, demonstrating the relative importance of cell surface structures in impeding DNA uptake in C. glutamicum. Transmission electron microscopy analysis of cell surface structures showed that strain MLB133 has a thin cell wall relative to AS019 and growth in either glycine or INH further diminished its thickness. Both RM3 and RM4 were more sensitive to INH than ATCC13032 and growth in glycine plus INH further improved transformation efficiency. The mycolic acid composition of these strains is described and the impact of glycine and INH on this is reported.     

Involvement of Ubiquitin in Phosphoenolpyruvate Carboxylase Degradation

Western immunoblot analysis of protein extracts prepared from epidermal peels, whole leaves, and mesophyll protoplasts with ubiquitin and PEPCase antibodies indicated ubiquitinated PEPCase bands and degradation products only in crude extracts which have been obtained in the presence of the proteolysis inhibitors leupeptin and hemin. After ammonium sulfate precipitation and further purification, PEPCase forms were stable and not ubiquitinated. It is assumed, that only a certain part of PEPCase is degraded via the ubiquitin-dependent proteolysis.