Reinhard Krämer

Genetic and physiological approaches for the production of amino acids

Abstract The purposeful design of metabolic pathways and networks in order to increase the yield and productivity of microorganisms capable of amino acid production is still a major goal of modem biotechnology. Knowledge of fundamental aspects of physiology, biochemistry, molecular biology and bioprocess engineering is described which must be optimized further in order to reach this goal. These fundamentals refer to the following topics, (a) Improved knowledge of the relevant pathways in biotechnologically relevant amino-acid-producing microorganisms. This must include metabolic regulation and integration into global regulatory networks of the cell, (b) Detailed flux analysis of all relevant pathways. This refers to substrate uptake, to the central and peripheral carbon, redox and energy fluxes, as well as product excretion. A network of data is thus established, which provides a basis for a purposeful metabolic design, (c) Redirection of the carbon, redox and energy flux in a coordinated and fine-tuned way, thus, limiting precursor availability, by-product formation and imbalanced pool sizes, as well as avoiding problems with the redox and energy balance.

Functional and Genetic Characterization of the (Methyl)ammonium Uptake Carrier of Corynebacterium glutamicum

Under nitrogen starvation conditions, Corynebacterium glutamicum was found to take up methylammonium at a rate of 20 ± 5 nmol•min•(mg dry weight). The specific activity of this uptake was 10-fold lower when growing the cells under sufficient nitrogen supply, indicating a tight regulation on the expression level. The methylammonium uptake showed Michaelis-Menten kinetics with an K of 44 ± 7 μM and was completely inhibited by the addition of 10 μM ammonium. This finding and the fact that methylammonium was not metabolized by C. glutamicum strongly suggests that the uptake carrier actually represents an ammonium uptake system. Methylammonium uptake was strictly dependent on the membrane potential. From the pH optimum and the accumulation of methylammonium in equilibrium, it could be deduced that only one net charge is transported and, thus, that methylammonium is taken up in its protonated form via an uniport mechanism. The amt gene encoding the (methyl)ammonium uptake system was isolated and characterized. The predicted gene product of amt consists of 452 amino acids (M = 47,699) and shows 26-33% identity to ammonium transporter proteins from Saccharomyces cerevisiae and Arabidopsis thaliana. According to the hydrophobicity profile, it is an integral membrane protein containing 10 or 11 membrane-spanning segments.

Functional reconstitution of carrier proteins by removal of detergent with a hydrophobic ion exchange column

A method has been developed for the functional reconstitution of membrane proteins in phospholipid vesicles. This method is an extension of a previously published procedure (Ueno, M., Tanford, C. and Reynolds, A. (1984) Biochemistry 23, 3070-3076) for the formation of unilamellar vesicles from mixed micelles of egg phosphatidylcholine and dodecyl octaoxyethylene ether. Mixed micelles are formed from detergent-solubilized protein and egg-yolk phospholipid vesicles. These micelles are subjected to repeated passage through small columns filled with Amberlite XAD-2 beads. Several carrier proteins from the inner mitochondrial membrane have been reconstituted in this way; experimental data are shown for the aspartate/glutamate carrier and the ADP/ATP carrier. Certain parameters proved to be important for optimal efficiency of reconstitution: the ratio of detergent/phospholipid in the mixed micelles, the concentration of phospholipid during the hydrophobic chromatography, the ratio of phospholipid/protein, (d) the ratio of detergent/Amberlite XAD 2 beads, the number of column passages, and the type of detergent. After optimization of these parameters, phospholipid vesicles with a diameter of about 150 nm were obtained. The main advantage of this procedure, however, lies in the fact that high amounts of membrane protein can be incorporated into the phospholipid vesicles, i.e. up to 15% (w/w).

Carrier-mediated glutamate secretion by Corynebacterium glutamicum under biotin limitation

Previous studies have demonstrated the involvement of a carrier system in glutamate secretion by Corynebacterium glutamicum under biotin limitation (Hoischen, C. and Krämer, R. (1989) Arch. Microbiol. 151, 342-347). In a detailed analysis of the export process we found secretion to be independent of secondary forces: (i) glutamate was secreted at high rate even when external glutamate exceeded the internal concentration, (ii) movement of neither protons nor potassium or chloride ions was found to be coupled to glutamate secretion, and (iii) secretion continued unaffected after breakdown of the membrane potential. Instead, under conditions leading to variation of glutamate secretion activity, a correlation of secretion rate and the intracellular ATP-pool was observed. Thus, ATP or a related high-energy metabolite is thought to be involved in the activity of the glutamate secretion system.

Molecular basis of age-dependent changes in the activity of adenine nucleotide translocase ☆

(1) Rat-heart mitochondria from 30-month-old animals are 40% less active in translocating adenine nucleotides across the inner membrane than 3-month-old rats. (2) The number of sites available for binding the specific ligands to the adenine nucleotide carrier remains unchanged during aging. (3) The endogenous pool of the adenine nucleotides exhibits an age-dependent fall by more than 25%, essentially at the expense of ATP. The amount of ATP + ADP representing the exchangeable pool for adenine nucleotide translocation is decreased to the same extent. (4) Negatively charged phospholipids as well as polyunsaturated fatty acids of membrane lipids were found to be reduced with aging. (5) The results are discussed in terms of changes in the phospholipid-protein interactions due to the observed alterations in the physical state of the bulk phase of membrane lipids.

Pore-like and carrier-like properties of the mitochondrial aspartate/glutamate carrier after modification by SH-reagents: evidence for a preformed channel as a structural requirement of carrier-mediated transport

Upon modification of the reconstituted aspartate/glutamate carrier by mercury reagents the antiporter was converted into a unidirectional efflux carrier (Dierks, T., Salentin, A., Heberger, C. and Krämer, R. (1990) Biochim. Biophys. Acta 1028, 268). In addition to this basic change in the mechanism, the mercurials, reacting with exofacial cysteines, also affected the internal binding site of the carrier leading to an unmeasurable high Km and to a drastically reduced substrate specificity. The spectrum of efflux substrates comprised small anions from chloride to glutamate, but not cationic amino acids and ATP, hence resembling pore-like properties. However, in the efflux state important carrier properties were also observed. The activation energy (86 kJ/mol) was as high as for the antiport. Furthermore, efflux was inhibited by the presence of external substrate. This trans-inhibition strongly suggests that the external binding site of the carrier, prerequisite in the antiport mechanism, also is involved in conformational transitions during efflux function. However, antiport no longer is catalyzed after switching to the efflux state. Reversion of the induced efflux carrier to the antiport state was achieved using dithioerythritol, thereby further restoring substrate specificity and saturation kinetics. A model for antiport-efflux interconversion is presented suggesting that two reactive cysteines have to be modified in order to uncouple the inward and outward directed component of antiport. The pore-type characteristics of efflux are taken as evidence that a channel-like structure determines the selectivity of unidirectional transport. This intrinsic channel of the protein then is required for substrate translocation also during antiport function.

Evidence for an efflux carrier system involved in the secretion of glutamate by Corynebacterium glutamicum

Corynebacterium glutamicum effectively secretes L-glutamate when growing under biotin limitation. The secretion of glutamate was studied with respect to kinetic and energetic parameters: rate of glutamate uptake and efflux, specificity of transport, dependence of efflux on the energy state of the cell, concentration gradient of glutamate and ions, and membrane potential. By comparing these parameters when measured in biotin-limited, i.e. “producer” cells, and biotin-supplemented, i.e. “non-producer” cells, respectively, the following conclusions could be drawn: 1. The efflux of L-glutamate in C. glutamicum cannot be explained by passive permeation of this amino acid through the plasma membrane, as it has been assumed in the generally accepted model of glutamate secretion in biotin-limited cells. 2. It is unlikely that the efflux of glutamate occurs via an inversion of the glutamate uptake system. 3. Based on our results concerning the specificity and the kinetics of glutamate transport as well as the observed regulation phenomena, we conclude that secretion of glutamate in C. glutamicum occurs by a special efflux carrier system.

Reconstitution of adenine nucleotide transport with purified ADP, ATP-carrier protein

1. Introduction Starting with our first report in 1974-[ l] , the ADP, ATP carrier of beef heart mitochondria was solubilized and purified in an undenatured form [2,3]. The protein was stabilized in the native state by com- plexing with high affinity ligands carboxyatractylate (CAT) or bongkrekate (BKA) enabling easy purifica- tion and characterization of chemical and physical parameters [4,5]. It seems to be very important to incorporate the isolated and purified protein into artificial membranes in order to study the catalytic activity of this protein, separate from the complexi- ties in the mitochondrial membrane. For this purpose we isolated the unliganded protein with sufficient purity with a special rapid procedure, described else- where [6,7], in order to overcome the denaturation of the unprotected protein. By applying a simple method for reincorporating the protein into the phos- pholipid vesicles, the original binding activity towards BKA and CAT was reconstituted [7]. Extending this procedure further to a three step reincorporation method, we have now been able to reconstitute transport activity with the purified protein. This reconstitution with the purified carrier protein permits to study parameters pertinent to the catalytic activity of the carrier and to relate these parameters to the molecular level of the pure protein incorporated into the artificial membrane. Recently also a reconstitution of ADP, ATP trans-

Adaptation of Corynebacterium glutamicum to Ammonium Limitation: a Global Analysis Using Transcriptome and Proteome Techniques

ABSTRACT Theresponse of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for FoF1-ATP synthase subunits.

Reaction mechanism of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria

A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Krämer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.