The effect of AmtR on growth and amino acids production in Corynebacterium glutamicum

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less alpha-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, Ile, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val,and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.     

The respiratory chain of Corynebacterium glutamicum

Corynebacterium glutamicum is an aerobic bacterium that requires oxygen as exogenous electron acceptor for respiration. Recent molecular and biochemical analyses together with information obtained from the genome sequence showed that C. glutamicum possesses a branched electron transport chain to oxygen with some remarkable features. Reducing equivalents obtained by the oxidation of various substrates are transferred to menaquinone via at least eight different dehydrogenases, i.e. NADH dehydrogenase, succinate dehydrogenase, malate:quinone oxidoreductase, pyruvate:quinone oxidoreductase, D-lactate dehydrogenase, L-lactate dehydrogenase, glycerol-3-phosphate dehydrogenase and L-proline dehydrogenase. All these enzymes contain a flavin cofactor and, except succinate dehydrogenase, are single subunit peripheral membrane proteins located inside the cell. From menaquinol, the electrons are passed either via the cytochrome bc(1) complex to the aa(3)-type cytochrome c oxidase with low oxygen affinity, or to the cytochrome bd-type menaquinol oxidase with high oxygen affinity. The former branch is exceptional, in that it does not involve a separate cytochrome c for electron transfer from cytochrome c(1) to the Cu(A) center in subunit II of cytochrome aa(3). Rather, cytochrome c(1) contains two covalently bound heme groups, one of which presumably takes over the function of a separate cytochrome c. The bc(1) complex and cytochrome aa(3) oxidase form a supercomplex in C. glutamicum. The phenotype of defined mutants revealed that the bc(1)-aa(3) branch, but not the bd branch, is of major importance for aerobic growth in minimal medium. Changes of the efficiency of oxidative phosphorylation caused by qualitative changes of the respiratory chain or by a defective F(1)F(0)-ATP synthase were found to have strong effects on metabolism and amino acid production. Therefore, the system of oxidative phosphorylation represents an attractive target for improving amino acid productivity of C. glutamicum by metabolic engineering.     

Cell growth and cell division in the rod-shaped actinomycete Corynebacterium glutamicum

Bacterial cell growth and cell division are highly complicated and diversified biological processes. In most rod-shaped bacteria, actin-like MreB homologues produce helicoidal structures along the cell that support elongation of the lateral cell wall. An exception to this rule is peptidoglycan synthesis in the rod-shaped actinomycete Corynebacterium glutamicum, which is MreB-independent. Instead, during cell elongation this bacterium synthesizes new cell-wall material at the cell poles whereas the lateral wall remains inert. Thus, the strategy employed by C. glutamicum to acquire a rod-shaped morphology is completely different from that of Escherichia coli or Bacillus subtilis. Cell division in C. glutamicum also differs profoundly by the apparent absence in its genome of homologues of spatial or temporal regulators of cell division, and its cell division apparatus seems to be simpler than those of other bacteria. Here we review recent advances in our knowledge of the C. glutamicum cell cycle in order to further understand this very different model of rod-shape acquisition.     

Secretion of human epidermal growth factor by Corynebacterium glutamicum

AIMS: To examine the secretion of human epidermal growth factor (hEGF) by Corynebacterium glutamicum. METHODS AND RESULTS: We recently showed that a novel protein-secretion system in C. glutamicum could produce Streptomyces mobaraensis transglutaminase. In the present study, the industrially important protein hEGF was secreted into the culture medium in a fully active form by C. glutamicum and accumulated at a rate of up to 156 mg l(-1) day(-1). CONCLUSIONS: These results demonstrated that the hEGF protein could be secreted in an active form by C. glutamicum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data confirmed that the pharmaceutically important human protein hEGF could be efficiently secreted in an active form by the C. glutamicum protein-expression system. Moreover, we demonstrated that this bacterium has potential as a host for the industrial-scale production of human proteins.     

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.     

NCgl2620 encodes a class II polyphosphate kinase in Corynebacterium glutamicum

Corynebacterium glutamicum is able to accumulate up to 600 mM cytosolic phosphorus in the form of polyphosphate (poly P). Granular poly P (volutin) can make up to 37% of the internal cell volume. This bacterium lacks the classic enzyme of poly P synthesis, class I polyphosphate kinase (PPK1), but it possesses two genes, ppk2A (corresponds to NCgl0880) and ppk2B (corresponds to NCgl2620), for putative class II (PPK2) PPKs. Deletion of ppk2B decreased PPK activity and cellular poly P content, while overexpression of ppk2B increased both PPK activity and cellular poly P content. Neither deletion nor overexpression of ppk2A changed specific activity of PPK or cellular poly P content significantly. Purified PPK2B of C. glutamicum is active as a homotetramer and formed poly P with an average chain length of about 125, as determined with (31)P nuclear magnetic resonance. The catalytic efficiency of C. glutamicum PPK2B was higher in the poly P-forming direction than for nucleoside triphosphate formation from poly P. The ppk2B deletion mutant, which accumulated very little poly P and grew as C. glutamicum wild type under phosphate-sufficient conditions, showed a growth defect under phosphate-limiting conditions.     

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.     

Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum

L-Glutamate is made with Corynebacterium glutamicum on a scale of more than 106 tons/year. Nevertheless, formation of this amino acid is enigmatic and there is very limited molecular information available to unravel the apparently complex conditions leading to L-glutamate efflux. Here, we report the isolation and overexpression of the genes involved in lipid synthesis: acp, fadD 15, cma, cls, pgsA2, cdsA, gpsA, and plsC, and the inactivation of cma and cls. In addition, the consequences for phospholipid content, temperature sensitivity, as well as detergent-independent and detergent-dependent L-glutamate efflux were quantified. An in part strong alteration of the phospholipid composition was achieved; for instance, overexpression offadD15 encoding an acyl-CoA ligase resulted in an increase of phosphatidyl inositol from 12.6 to 30.2%. All strains, except that overexpressing acp (acyl carrier protein), exhibited increased temperature sensitivity, with the strongest sensitivity present upon cls (cardiolipin synthetase) inactivation. As a consequence of the genetically modified lipid synthesis, L-glutamate efflux changed quite dramatically; for instance, overexpression of plsC (acylglycerolacyl transferase) resulted in a detergent-triggered increase of L-glutamate accumulation from 92 mM to 108 mM, whereas acp overexpression reduced the accumulation to 24 mM. With some of the overexpressed genes, substantial L-glutamate excretion even without detergent addition was obtained when the fermentation temperature was elevated. These data show that the chemical and physical properties of the cytoplasmic membrane are altered and suggest that this is a necessary precondition to achieve L-glutamate efflux.     

Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

ABSTRACT: BACKGROUND: Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. RESULTS: Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 epsilon-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum DeltacrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum DeltacrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 +/- 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum DeltacrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 +/- 0.3 mg/g CDW were obtained. CONCLUSION: C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.     

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).     

Carrier-mediated acetate uptake in Corynebacterium glutamicum

Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K _m of acetate uptake was 50 μM and the V _max 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by propionate with a K _i of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane potential, but independent of the presence of Na⁺-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism.     

Role of cytochrome bd oxidase from Corynebacterium glutamicum in growth and lysine production

Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa3 and cytochrome bd. Cytochrome aa3 forms a supercomplex with the cytochrome bc1 complex, which contains an unusual diheme cytochrome c1. Both the bc1 -aa3 supercomplex and cytochrome bd transfer reducing equivalents from menaquinol to oxygen; however, they differ in their proton translocation efficiency by a factor of three. Here, we analyzed the role of cytochrome bd for growth and lysine production. When cultivated in glucose minimal medium, a cydAB deletion mutant of C. glutamicum ATCC 13032 grew like the wild type in the exponential phase, but growth thereafter was inhibited, leading to a biomass formation 40% less than that of the wild type. Constitutive overproduction of functional cytochrome bd oxidase in ATCC 13032 led to a reduction of the growth rate by approximately 45% and of the maximal biomass by approximately 35%, presumably as a consequence of increased electron flow through the inefficient cytochrome bd oxidase. In the L-lysine-producing C. glutamicum strain MH20-22B, deletion of the cydAB genes had only minor effects on growth rate and biomass formation, but lysine production was increased by approximately 12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum.