Phosphotransferase-dependent glucose transport in Corynebacterium glutamicum

G.M. MALIN AND G.I. BOURD. 1991. The transport system for glucose and its non-metabolizable analogue methyl-α-D-glucoside (MG) has been described in Corynebacterium glutamicum. The initial product of the transport reaction was shown to be a phosphate ester of MG (MGP). Free MG appeared inside the cells as a result of MGP dephosphorylation. The bacteria transported MG with an apparent K_m of 0.08 ± 0.017 mmol/l and V_max of 21 ± 2.3 nmol/(min × mg dry wt). Toluenized cells and crude cell extracts catalysed phosphoenolpyruvate (PEP)-dependent phosphorylation of MG and glucose. Both the membrane and the cytoplasmic fractions of bacterial extracts were required for phosphotransferase reaction. Most of the spontaneous mutants resistant to 2-deoxyglucose (DG), xylitol and 5-thioglucose were defective both in transport and in PEP-dependent phosphorylation of MG. Some strains were defective only in glucose utilization and some were also unable to grow on a number of other sugars. The phosphotransferase activity in extracts from mutant cells was restored by the addition of either membrane or cytoplasmic fraction from wild type bacteria. It was concluded that Corynebacterium glutamicum accumulated glucose and MG by means of a PEP-dependent phosphotransferase system (PTS).     

Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation

As a response to hyperosmotic stress bacterial cells accumulate compatible solutes by synthesis or by uptake. Beside the instant activation of uptake systems after an osmotic upshift, transport systems show also a second, equally important type of regulation. In order to adapt the pool size of compatible solutes in the cytoplasm to the actual extent of osmotic stress, cells down-regulate solute uptake when the initial osmotic stress is compensated. Here we describe the role of the betaine transporter BetP, the major uptake carrier for compatible solutes in Corynebacterium glutamicum, in this adaptation process. For this purpose, betP was expressed in cells (C. glutamicum and Escherichia coli), which lack all known uptake systems for compatible solutes. Betaine uptake mediated by BetP as well as by a truncated form of BetP, which is deregulated in its response to hyperosmotic stress, was dissected into the individual substrate fluxes of unidirectional uptake, unidirectional efflux and net uptake. We determined a strong decrease of unidirectional betaine uptake by BetP in the adaptation phase. The observed decrease in net uptake was thus mainly due to a decrease of Vmax of BetP and not a consequence of the presence of separate efflux system(s). These results indicate that adaptation of BetP to osmotic compensation is different from activation by osmotic stress and also different from previously described adaptation mechanisms in other organisms. Cytoplasmic K+, which was shown to be responsible for activation of BetP upon osmotic stress, as well as a number of other factors was ruled out as triggers for the adaptation process. Our results thus indicate the presence of a second type of signal input in the adaptive regulation of osmoregulated carrier proteins.     

Sodium and proline accumulation in Corynebacterium glutamicum as a response to an osmotic saline upshock

In order to understand the role of the medium osmolality on the metabolism of glumate-producing Corynebacterium glutamicum, effects of saline osmotic upshocks from 0.4 osnol. kg^–1 to 2 osmol. kg^–1 have been investigated on the growth kinetics and the intracellular content of the bacteria. Addition of a high concentration of NaCl after a few hours of batch culture results in a temporary interruption of the cellular growth. Cell growth resumes after about 1 h but at a specific rate that decreases with increasing medium osmolality. Investigation of the intracellular content showed, during the first 30 min following the shock, a rapid but transient influx of sodium ions. This was followed by a strong accumulation of proline, which rose from 5 to 110 mg/g dry weight at the end of the growth phase. A slight accumulation of intracellular glutamate from 60 to 75 mg/g dry weight was also observed. Accordingly, for Corynebacterium glutamicum an increased osmolality in the glutamate and proline synthesis during the growth phase.     

Kinetics of volume variation of Corynebacterium glutamicum following saline osmotic upshifts

Summary The variation over time of the volume of Corynebacterium glutamicum following osmotic upshocks produced by the addition of increasing NaCl quantities was measured by a light-scattering method. During the first seconds the cell volume rapidly decreased. The extent of volume reduction varied between 30 % at 1.5 osmol/kg and 65 % at 3.3 osmo/kg. In a second phase the cell volume increased again over a time period lasting 10 minutes or more depending on the magnitude of the saline upshock.     

Siderophore-mediated iron transport in Bacillus subtilis and Corynebacterium glutamicum

Hexadentate bacillibactin is the siderophore of Bacillus subtilis and is structurally similar to the better known enterobactin of Gram-negative bacteria such as Escherichia coli. Although both are triscatecholamide trilactones, the structural differences of these two siderophores result in opposite metal chiralities, different affinity for ferric ion, and dissimilar iron transport behaviors. Bacillibactin was first reported as isolated from Corynebacterium glutamicum and called corynebactin. However, failure of iron-starved C. glutamicum to transport ⁵⁵Fe bacillibactin and lack of required bacillibactin biosynthetic genes suggest that bacillibactin is not the siderophore produced by this organism. Iron transport mediated by siderophores in B. subtilis occurs through a transport process that is specific for the iron chelating moiety, with parallel pathways for catecholates and hydroxamates. For bacillibactin, enterobactin, and their analogs, neither chirality nor presence of an amino acid spacer affects the uptake and transport process, but alteration of the net charge and size of the molecule impedes the recognition.Paper number 77 in the series Coordination Chemistry of Microbial Iron Transport Compounds. See Abergel et al. [1].     

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.     

Growth of Corynebacterium glutamicum in ammonium- and potassium-limited continuous cultures under high osmotic pressure

 In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower than previously reported in glucose-limited continuous cultures (50 mg/g and 10 mg/g dry weight respectively). On the other hand, the internal trehalose pool was much higher at 40 mg/g dry weight. When the medium osmolality was increased to 1.2 osmol/kg by NaCl addition, under ammonia limitation, the proline content rose from 5 mg/g to 20 mg/g dry weight and the trehalose content from 40 mg/g to 70 mg/g dry weight, whereas the intracellular pool of glutamate remained essentially constant. An increase in the internal sodium content was also observed. Similar results were found for the internal pool of glutamate, proline and trehalose when C. glutamicum was grown under potassium limitations at an osmolality of 1.2 osmol/kg. There were also higher levels of sodium ions, glutamine and alanine. According to the present results, whereas proline was previously reported to be the dominantly accumulated osmoprotectant in C. glutamicum grown under glucose limitations, under ammonia and potassium limitations trehalose represented the dominantly synthesized metabolite. Received: 19 December 1995/Received revision: 9 April 1996/Accepted: 15 April 1996     

Study of mycoloyl transferase transport across the cell envelope of Corynebacterium glutamicum

A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes

Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.     

OdhI dephosphorylation kinetics during different glutamate production processes involving Corynebacterium glutamicum

In Corynebacterium glutamicum, the activity of the 2-oxoglutarate dehydrogenase complex was shown to be controlled by the phosphorylation of a 15-kDa protein OdhI by different serine/threonine protein kinases. In this paper, the phosphorylation status and kinetics of OdhI dephosphorylation were assessed during glutamate producing processes triggered by either a biotin limitation or a temperature upshock from 33°C to 39°C. A dephosphorylation of OdhI in C. glutamicum 2262 was observed during the biotin-limited as well as the temperature-induced glutamate-producing process. Deletion of pknG in C. glutamicum 2262 did not affect the phosphorylation status of OdhI during growth and glutamate production phases triggered by a temperature upshock, though a 40% increase in the specific glutamate production rate was measured. These results suggest that, under the conditions analyzed, PknG is not the kinase responsible for the phosphorylation of OdhI in C. glutamicum 2262. The phosphorylation status of OdhI alone is, as expected, not the only parameter that determines the performance of a specific strain, as no clear relation between the specific glutamate production rate and OdhI phosphorylation level was demonstrated.     

Impact of osmotic stress on volume regulation,cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.     

Impact of a new glucose utilization pathway in amino acid-producing Corynebacterium glutamicum

Corynebacterium glutamicum imports and phosphorylates glucose, fructose and sucrose by the phosphoenolpyruvate-dependent phosphotransferase carbohydrate uptake system (PTS). Recently, we have discovered how glucose can be utilized by C. glutamicum in a PTS-independent manner. PTS-independent glucose uptake is mediated by one of two inositol permeases (IolT1 or IolT2) and the second function of PTS, substrate phosphorylation, is catalyzed by one of two glucokinases (Glk or PpgK). PTS-deficient C. glutamicum strains exclusively utilizing glucose via this system grew comparably well on glucose minimal media as the parental strain. Furthermore, PTS-deficient L-lysine producing C. glutamicum strains overexpressing genes for inositol permease and glucokinase showed increased L-lysine production and reduced formation of by-products derived from pyruvate. Here, we discuss the impact of our findings on engineering strategies of C. glutamicum strains used in various biotechnological production processes.     

Ethanol catabolism in Corynebacterium glutamicum

Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids as single or combined sources of carbon and energy. Here we show the ability of C. glutamicum to grow on ethanol with growth rates up to 0.24 h(-1) and biomass yields up to 0.47 g dry weight (g ethanol)(-1). Mutants of C. glutamicum deficient in phosphotransacetylase (PTA), isocitrate lyase (ICL) and malate synthase (MS) were unable to grow on ethanol, indicating that acetate activation and the glyoxylate cycle are essential for utilization of this substrate. In accordance, the expression profile of ethanol-grown C. glutamicum cells compared to that of glucose-grown cells revealed an increased expression of genes encoding acetate kinase (AK), PTA, ICL and MS. Furthermore, the specific activities of these four enzymes as well as those of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) were found to be high in ethanol-grown and low in glucose-grown cells. Growth of C. glutamicum on a mixture of glucose and ethanol led to a biphasic growth behavior, which was due to the sequential utilization of glucose before ethanol. Accordingly, the specific activities of ADH, ALDH, AK, PTA, ICL and MS in cells grown in medium containing both substrates were as low as in glucose-grown cells in the first growth phase, but increased 5- to 100-fold during the second growth phase. The results indicate that ethanol catabolism in C. glutamicum is subject to carbon source-dependent regulation, i.e., to a carbon catabolite control.     

Protein secretion in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. Due to several characteristics – its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity – C. glutamicum is also a very favorable host cell for the secretory production of heterologous proteins, important enzymes, and pharmaceutical proteins. The target proteins are secreted into the culture medium, which has attractive advantages over the manufacturing process for inclusion of body expression – the simplified downstream purification process. The secretory process of proteins is complicated and energy consuming. There are two major secretory pathways in C. glutamicum, the Sec pathway and the Tat pathway, both have specific signal peptides that mediate the secretion of the target proteins. In the present review, we critically discuss recent progress in the secretory production of heterologous proteins and examine in depth the mechanisms of the protein translocation process in C. glutamicum. Some successful case studies of actual applications of this secretory expression host are also evaluated. Finally, the existing issues and solutions in using C. glutamicum as a host of secretory proteins are specifically addressed.     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

Gene expression of Corynebacterium glutamicum in response to the conditions inducing glutamate overproduction

AIM: The ultimate aim is to elucidate the molecular mechanisms for glutamate overproduction by Corynebacterium glutamicum. METHODS AND RESULTS: Gene expression in response to the conditions inducing glutamate overproduction was investigated by using a DNA microarray technique. Most genes involved in the EMP pathway, the PPP, and the TCA cycle were downregulated, while five genes that were highly upregulated (NCgl0917, NCgl2944, NCgl2945, NCgl2946, and NCgl2975) were identified under all the three conditions for overproduction that are studied here. Gene products of NCgl2944, NCgl2945, and NCgl2946 were highly homologous to each other, did not resemble any other protein, and have remained uncharacterized thus far. The product of NCgl0917 showed a similarity to a few hypothetical and uncharacterized proteins. NCgl2975 was homologous to metal-binding proteins. CONCLUSIONS: The decrease in the activity of 2-oxoglutarate dehydrogenase complex, a key enzyme that is downregulated during glutamate overproduction, can be mainly attributed to the downregulation of odhA and sucB. Five highly upregulated genes were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Although fermentative production of glutamate has been carried out for more than 45 years, information on the molecular mechanisms of glutamate overproduction is still limited. This study further elucidates these mechanisms.