Aspartokinase of a lysine producing mutant ofMicrococcus glutamicus

Aspartokinase fromMicrococcus glutamicus AEC RN-13-6/1 [a homoserine requiring, S-(2-aminoethyl)-L-cysteine resistant, lysine producing strain] was purified 71 fold. The partially purified enzyme was inhibited by L-lysine. L-threonine, L-methionine, L-isoleucine, L-valine and L-phenylalanine activated the enzyme and reversed the inhibition by L-lysine. Aspartokinase activity was not derepressed by growth-limiting concentrations of L-threonine and/or L-methionine. It was not repressed by an excess of L-lysine (20 mM) and/or L-isoleucine (15.3 mM). The degree of activation or inhibition by amino acids was dependant on the composition of the growth medium. This observation is in contrast with the enzyme from the original (non-lysine-producing) strain which was inhibited by lysine or threonine and in a concerted manner by threonine plus lysine.     

Enhancement of L-lysine production in methylotroph Methylophilus methylotrophus by introducing a mutant LysE exporter

The obligate methylotroph Methylophilus methylotrophus AS1 expressing a mutant form of dapA (dapA24) encoding a dihydrodipicolinate synthase desensitized from feedback inhibition by L-lysine could secrete L-lysine into the medium, but also maintained a high concentration of intracellular L-lysine. To improve the yield from excretion, we attempted to introduce an L-lysine/L-arginine exporter (LysE) from Corynebacterium glutamicum 2256 into M. methylotrophus. We were unable to stably transform M. methylotrophus with a plasmid expressing the wild type lysE gene, but happened to obtain a transformant carrying a spontaneously mutated lysE gene (designated lysE24) which could induce L-lysine production even in the wild type strain. The transformant also possessed increased tolerance to S-(2-aminoethyl)-L-cysteine (an L-lysine analog). lysE24 has a single-base insertion mutation in the middle of the lysE gene, and its product is presumably quite different in structure from wild-type LysE. When lysE24 was introduced into an L-lysine producer of M. methylotrophus carrying dapA24, the level of intracellular L-lysine fell. During fermentation, M. methylotrophus carrying both lysE24 and dapA24 produced 10-fold more L-lysine (11.3 gl(-1) in jar-fermentation) than the parent producer carrying only dapA24 or lysE24. These results show the importance of the factor (lysE24) involved in the excretion of L-lysine on its overproduction in M. methylotrophus.     

Aspartokinase of Streptococcus mutans: purification, properties, and regulation.

Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine.     

Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production

L-Threonine is an important biotechnological product and Corynebacterium glutamicum is able to synthesize and accumulate this amino acid to high intracellular levels. We here use four exporters of Escherichia coli and show that three of them operate in C. glutamicum, with RhtA and RhtC being the most effective. Whereas RhtA was unspecific, resulting in L-homoserine together with L-threonine excretion, this was not the case with RhtC. Expression of rhtC reduced the intracellular L-threonine concentration from 140 to 11 mM and resulted in maximal excretion rates of 11.2 nmol min(-1) mg(-1) as compared to 2.3 nmol min(-1) mg(-1) obtained without rhtC expression. In combination with an ilvA mutation generated and introduced into the chromosome, an accumulation of up to 54 mM L-threonine was achieved as compared to 21 mM obtained with the ancestor strain. This shows that expression of rhtC is the pivotal point for industrial relevant L-threonine production with C. glutamicum, and might encourage in general the use of heterologous exporters in the field of white biotechnology to make full use of biosynthesis pathways.     

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine.

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by L-threonine. To investigate the regulation of the C. glutamicum HD enzyme by L-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, homdr. L-Threonine inhibits the wild-type (wt) enzyme with a Ki of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM L-threonine. The homdr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned homdr gene is genetically unstable. The cloned homdr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM L-threonine. Sequence analysis revealed that the homdr mutation is a single nucleotide (G1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the L-threonine allosteric response. The homdr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.     

Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum

Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete L-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since L-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize L-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for D-pantothenate. When the resulting strain, C. glutamicum 13032DeltailvADeltapanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM L-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the L-valine biosynthesis pathway.     

Overproduction of L-Lysine from methanol by Methylobacillus glycogenes derivatives carrying a plasmid with a mutated dapA gene.

The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by L-lysine, was cloned from an L-threonine- and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2- to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M(r) of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.     

Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

To identify potential L-lysine export limitations by Corynebacterium glutamicum in the L-lysine production process, the excretion of L-lysine was studied in continuous and fed-batch operated stirred tank reactors. A structured biochemical model of the L-lysine excretion mechanism was used to determine the activity of the export carrier and to calculate a cell-specific concentration of the export carrier. For the biochemical characterization of this specific carrier concentration a standardized L-lysine efflux test was developed. Carrier activity, cell-specific carrier concentration, and the specific L-lysine export rate were identified as a function of pH value and L-lysine concentration in the reactors. Also, the correlation of these parameters to the metabolic state of C. glutamicum was determined. The pH value in the reactor governs the carrier activity (maximum at pH 6.5) and the specific carrier concentration (maximum at pH 8.0). The specific L-lysine export rate, as the product of carrier activity and specific carrier concentration, revealed a maximum at pH 7.0. Decreasing L-lysine productivities also correlated with decreasing specific carrier concentrations. The L-lysine concentration in the reactor had no effect on the specific carrier concentration but strongly inhibited the carrier activity. The specific export rate was reduced to 50% at 400 mM L-lysine compared to the specific export rate at 80 mM L-lysine. (c) 1996 John Wiley & Sons, Inc.     

Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum

A series of experiments reported in the literature using fluxomics as an efficient functional genomics tool revealed that the L-lysine production of the Corynebacterium glutamicum strain MH20-22B correlates with the extent of intracellular NADPH supply. Some alternative metabolic engineering strategies to increase intracellular NADPH supply in the C. glutamicum strain DSM5715 were considered and finally the redirection of carbon flux through the pentose phosphate pathway with two NADPH generating enzymatic reactions was favored. Elsewhere, the construction of a phosphoglucose isomerase (Pgi) null mutant of the C. glutamicum strain DSM5715 has been described by utilizing genetic engineering as well as some aspects of its metabolic phenotype. Most interestingly, it was shown that not only could the L-lysine formation be increased by 1.7-fold but the by-product concentration for the null mutant strain was also able to be drastically reduced. In this publication we discuss this metabolic phenotype in detail and present additional data on by-product formation as well as yield considerations. Results from isotope based metabolic flux analysis in combination with considerations on NADPH metabolism clearly exclude the existence of Pgi isoenzymes in C. glutamicum strain DSM5715. The genome region containing the pgi gene was analyzed. It cannot be excluded that polar effects might have been caused by the disruption of the pgi gene and might have contributed to the observed metabolic phenotype of C. glutamicum Pgi mutants. We illustrate growth characteristics of a Pgi mutant of an industrial L-lysine production strain. A reduced growth rate and a biphasic growth behavior was observed. The importance of NADPH reoxidation for well balanced growth in Pgi mutants is discussed. Another phosphoglucose isomerase mutant of C. glutamicum has been described in literature with which an increase in L-lysine yield from 42 to 52% was observed. This finding highlights the general potential of metabolic flux redirection towards the pentose phosphate pathway, which could be used for metabolic engineering of the biotechnological synthesis of (1) aromatic amino acids and (2) chemicals whose synthesis depends on intracellular NADPH supply.     

Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli

The biochemical basis for paraquat tolerance was investigated using one of the paraquat-resistant Escherichia coli mutants previously isolated. When grown in the absence of paraquat (PQ2+), the specific activities of glucose-6-phosphate dehydrogenase and NADPH:PQ2+-diaphorase, both required for the expression of PQ2+ toxicity, were comparable in the wild type and the mutant. However, growth in the presence of 1 mM PQ2+ resulted in greater induction of these two enzymes in the wild type than in the mutant. Nevertheless, when the mutant was grown in 50 mM PQ2+, the activities of these two enzymes were comparable to those of the wild type grown in the presence of 1 mM PQ2+. Measurement of cyanide-resistant respiration, an indication of intracellular superoxide generation, showed that the intracellular flux of superoxide mediated by subsaturating concentrations of paraquat was significantly lower in the mutant than in the wild type. Extracellular superoxide formation, as measured by superoxide dismutase-inhibitable cytochrome c reduction, was higher in the wild type than in the mutant whether grown in the absence or the presence of PQ2+. The mutant did not show cross-resistance toward juglone or plumbagin, compounds known to exacerbate superoxide generation. The kinetics of [14C]PQ2+ uptake showed that the wild type accumulated PQ2+ against a concentration gradient, whereas the mutant seemed to do so only by facilitated diffusion. The results indicate that the impaired paraquat uptake system in the mutant results in the physiological and biochemical differences observed between the wild type and mutant.     

Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.

Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.     

抗反馈抑制的天冬氨酸激酶基因在钝齿棒杆菌中的表达

将来自钝齿棒杆菌（Corynebacterium crenatum）CD945具有AEC抗性的天冬氨酸激酶(AKfbr)基因克隆到穿梭载体pJC1上,构建重组质粒pLY153。用电击法将质粒pLY153转化到野生型菌株C. crenatum AS1.542及其突变株C. crenatum CD945中。携带AKfbr基因的C. crenatum AS1.542菌株能抗浓度皆为12mg/mL的AEC和苏氨酸。AKfbr基因在C. crenatum CD945中得到表达,天冬氨酸激酶活性提高4倍。摇瓶发酵实验结果表明,重组菌在对数前期和中期生长正常,不受抑制;与对照菌相比,赖氨酸终产量提高22％,赖氨酸生产率提高23％。     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain Corynebacterium glutamicum YILW

Previous studies have shown that the deletion of brnQ from the Corynebacterium glutamicum chromosome results in a significant reduction in L-isoleucine uptake rates, while overexpression of brnFE leads to enhanced L-isoleucine export rates. Given that net excretion rates would be an important factor for high titers of L-isoleucine accumulation, we have tested the notion that decreased L-isoleucine uptake combined with increased L-isoleucine excretion will further improve high-yield strains that are currently used for the industrial-scale production of L-isoleucine. To examine the effect of the two carriers on L-isoleucine accumulation in L-isoleucine producer C. glutamicum YILW, we constructed a brnQ deletion mutant (C. glutamicum YILW?brnQ) and two brnFE overexpressors (C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE). Compared to the original strain, the efflux rate of the brnQ mutant increased from 19.0 to 23.6?nmol?min(-1) mg (dry wt)(-1) and its L-isoleucine titer increased from 154.3?mM (20.2?g?l(-1)) to 170.3?mM (22.3?g?l(-1)). The efflux rates of C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE were 33.5 and 39.1?nmol?min(-1) mg (dry wt)(-1), and their L-isoleucine production titers were 197.2?mM (25.9?g?l(-1)) and 221.0?mM (29.0?g?l(-1)), respectively. Our results suggest that modifications of the transport system could provide a promising avenue for further increasing L-isoleucine yield in the L-isoleucine producer.     

Aspartokinase isoenzymes of the fruiting myxobacterium Myxoccus xanthus.

Two isoenzymes of aspartokinase can be found in extracts of the differentiating bacterium Myxococcus xanthus. Aspartokinase I is repressed by L-lysine and feedback is inhibited by meso-diaminopimelate and by low concentrations of L-lysine. However, the inhibition by L-lysine is no longer observed at high concentration of this amino acid. Aspartokinase II is repressed and feedback inhibited specifically by L-threonine. Both enzymes are stimulated significantly by L-methionine and L-isoleucine; the effect is greater with aspartokinase I. The role of these enzymes in relation to growth conditions of the organism is discussed and a correlation with life cycle activity is indicated.     

Culture instability of auxotrophic amino acid producers

The long-term dynamic characteristics of the L-lysine producer Corynebcterium glutamicum in continuous culture were studied over a range of specific growth rates. The double-auxotroph parent strain was found to be susceptible to a back mutation, or reversion, which negated the regulatory bypass that allows this strain to accumulate L-lysine in culture but also gives rise to a L-threonine auxotrophic requirement. Consequently, the revertant cells no longer over-produced L-lysine, nor were they limited in their growth by the low levels of L-threonine in the medium. All continuous culture experiments were enventually taken over by these revertants. The instability of the culture was found to be primarily due to the growth rate differential between the two competing populations, the (productive) parent auxotrophs and the (nonproductive) revertants. A deterministic mathematical model of the culture dynamics, incoroporating two limiting-substrate balances, satisfactorily described the takeover profiles. A linear stability analysis of the model equations identified that although long-term culture demise is inevitable, the dimensionless ratio of the two limiting substrates controls the rate of takeover by nonproductive cells. The anslysis further demonstrated the importance of proper medium design in delaying the onset of takeover in cultures of this double-auxotroph strain. The theoretical medium design criterion was then confirmed experimentally by the stabilization of a fed-batch culture against revertant takeover for an extended fermentation time.