UniversityFacultyInstitute

Substrate uptake and product excretion in Corynebacteria
Stress physiology in C. glutamicum und E. coli

A. Wittmann, S. Huhn, C. Lange, A. Uhde, K. Kirsch, A. Bartsch, K. Schlidt
and R. Krämer

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Scientific Background

Cells exchange matter, energy and information with their surroundings and membrane-bound solute transport systems are essential for these processes. In bacteria a broad variety of different transporter families are known. Beside primary transporters, which use ATP for energatization, secondary transporters depend on electrochemical gradient across the membrane. For metabolic engineering of bacteria in order to produce biotechnologically relevant compounds, optimization of nutrient uptake (precursors) or excretion of metabolic products as well as the prevention of product reuptake are important parameters for strain improvement. We focus on the “workhorses” of white biotechnology C. glutamicum and E. coli. Product excretion and overflow metabolism in microorganisms are analyzed by biochemical and physiological approaches. We focus on particular aspects of biosynthesis, accumulation and transmembrane transport of selected solutes. While in previous projects L-glutamate, L-isoleucine, L-lysine and riboflavin transport was studied, recently, also transport of other amino acids, e.g. methionine and tryptophan is investigated.
Moreover also import and export of sugars and citric acid cycle intermediates are analyzed in C. glutamicum. Furthermore, transporters are essential components during stress adaptation and for homeostasis of major bioenergetic parameters like internal pH, membrane potential or energy charge and reduction state of the cytoplasm. We want to understand the impact of transport processes on cellular parameters during stress acclimatization and during production processes. In order to quantify the impact of several transporters on cell physiology and production we compare major metabolic parameters in wild type and mutant cells with respect to known or predicted components involved in pH or ion homeostasis.

Aims

  • Understanding and quantifying the impact of transport processes on production of amino acids and other compounds in Corynebacterium glutamicum and Escherichia coli.
  • Elucidation of mechanism and regulation of solute excretion systems as well as their physiological significance in order to use this knowledge for approaches of metabolic design.
  • Quantification of the impact of transport processes on the cellular physiology and homeostasis.

Corynebacterium glutamicum Basic metabolic situations during production of metabolites by bacteria

Besides metabolic conversions in the cytoplam uptake of nutrients and excretion of metabolic products are major processes of the cellular metabolism in C. glutamicum. This is underlined by the number of genes encoding secondary transporter (units) (at least 130) and primary transporter units (185).

Basic metabolic situations during production of metabolites by bacteria (A). Three physiological models have been proposed:

  1. Metabolic overflow (example: glutamate in C. glutamicum),
  2. limited catabolism (example: lysine in C. glutamicum), and
  3. deregulated anabolism (example: threonine in E. coli).
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Current Team

Corynebacterium glutamicum

Katja Kirsch, Carolin Lange, Benjamin Roenneke, Anna Bartsch, Andreas Uhde, Stephanie Huhn, Kay Marin, Ines Ochrombel, Natalie Brühl

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Selected Projects

Amino acid export in C. glutamicum

Project 1 The characterization of methionine uptake in C. glutamicum revealed two uptake systems (A). The first system is the high-affinity transporter MetD with a Km of ~ 0.1 µM and a Vmax of ~ 0.7 nmol/min (mg dw) encoded by the genes metNIQ. The expression of the gene cluster is regulated by the repressor McbR. The second methionine uptake system is the secondary medium-affinity transporter MetP with a Km of ~ 88 µM and a Vmax of ~ 1.65 nmol/min (mg dw) for which the encoding gene(s) are unknown and under investigation. In order to characterize methionine export, an L-methionine containing dipeptide loading system was established. The dipeptide is taken up by the cell and hydrolyzed in the cytoplasm, resulting in a strong increase of the internal methionine concentration, followed by an decrease of the internal concentration (B). It was shown that BrnFE is the main export system for methionine, and the expression of brnFE depends on the cytoplasmic methionine concentration. Since methionine export was still detectable in the brnE deletion strain, a further export system is present which was named MetE. The biochemical properties of MetE and the encoding gene(s) are under investigation.

Components involved in methionine transport in C. glutamicum (A) and internal methionine concentration in the delta metD strain, the brnFE deletion as well as overexpressing strain after addition of Met-Met (B).

Impact of transport processes on pH homeostasis in C. glutamicum and E. coli

Project pH A variation of the external pH value is a common stress situation that bacteria have to cope with in their natural habitat as well as during biotechnological production processes. Since maintenance of a stable cytoplasmic pH is crucial for function and integrity of proteins and membranes, pH homeostasis is essential for survival and growth. Passive mechanisms of pH homeostasis are the low permeability of the cytoplasmic membrane for protons and the buffering capacity of the cytoplasm that is mainly due to the amino acid side chains of proteins. Active pH homeostasis in bacteria includes the controlled transport of protons by the H+ ATPase the activity of the urease or the uptake of amino acids followed by the decarboxylation and export of the product as shown for the Gab-system in E. coli (B). Besides that cations transport across the membrane is an important factor and is facilitated by a variety of transporters like the potassium uptake systems Kdp, Ktr, TrK or Kup (B) as well as sodium proton antiporters like MdfA, CPA1 or CpA3 (C). Our aim is to identify new components of the pH homeostais and to quantify the impact of the individual systems in E. coli and C. glutamicum by comparing growth, survival, major metabolic parameters and transport activities in wild type and selected mutants.

Active pH homeostasis in C. glutamicum was observed at external pH values in the range between 6 and 9, where internal pH values between 7 and 8 were determined (A). Known and proposed components involved in pH homeostasis in bacteria like E. coli and C. glutamicum during acidic (B) or alkaline challenge (C) are listed.

Transport of central metabolites in C. glutamicum

Project pH Metabolites of the central metabolism like succinate are not only of vital importance for bacterial cellular physiology, but also play an important role in food, medical and industry and are used as food and feed additives, in pharmaceuticals or in the production of different solvents, resins and plastics. On the other hand carbon flux to undesired products like lactate or acetate leads to a loss of carbon, energy and to the acidification of media. The production of these carboxylic acids is frequently induced by unfavorable cultivation conditions. This diminishes the product yield and therefore reduces the production efficiency. The anabolic and catabolic reactions involving organic acids are well know for C. glutamicum, but their uptake and excretion were not considered to a similar extent in spite being important parameters in substrate flux. By bioinformatic analysis of protein sequences putative transporters can by proposed (A). We aim to identify genes encoding these transporters for central metabolites and to characterize the biochemical properties by inactivation mutagenesis, overexpression strategies as well as the time dependent analysis of substrate uptake and product excretion (B).

Known and proposed components involved in the transport of substrate of the central metabolism (A) and excretion of lactate and succinate during anaerobiosis in C. glutamicum (B).

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Methods

By a combination of methods from physiology, biochemistry, molecular biology and biotechnology the significance of transport processes and the structure/function of transport systems is investigated in different microorganisms.

Molecular methods

Corynebacterium glutamicum
For generation of mutants we use insertions of antibiotic resistance markers (A) or deletions of genes (B). The activity of export systems can be investigated by overexpression of genes encoding putative candidates (C). For screening procedures systematic and random transposon mutant collections are generated (D).
Corynebacterium glutamicum
All routine molecular biology techniques for cloning and modification of genes (A) are established in C. glutamicum. Several heterologous expression systems are in use (B).
Gene expression can be monitored by Northern-Blot as well as Western-Blot techniques (C) and in collaboration with other groups by DNA microarray (D) or 2D-PAGE analysis (E).

Biochemical methods

Corynebacterium glutamicum
For identification of substrates and quantification of external and internal substrate concentration we use HPLC, GC as well as GC-MS.
By time dependent substrate concentration analysis the kinetic parameters of transport process can be determined.

Biotechnological methods

Corynebacterium glutamicum
Besides the cultivation of C. glutamicum or E. coli on agar plates or in Erlenmeyer flasks (A) screening procedures are performed with Tn mutant collections under challenging conditions on agar plates (B) or in microtiter plates (C). For the analysis of production parameters we scale up the culture volume up to 5 (D) and 10 l.

Bioinformatic methods

Corynebacterium glutamicum
For the genome wide analysis of gene length, orientation and neighborhood in different annotations of the C. glutamicum genome the data bank CoryneBase was developed as an in-house application (A). Besides gene related information, protein data like molecular weight, pI value, number of predicted transmembrane helices as well as the occurrence of known functional domains can be compared (B). Moreover, predicted transporter classes as well as substrates are included and can be updated by the addition of new in-lab data regarding the functional characterization of proteins or mutants.
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Funding

    Evonik Degussa AG
    Ajinomoto Co., inc.
    BMBF
    International Max-Planck Research School (MPI f. Züchtungsforschung)
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Cooperation

    Prof. V. Wendisch, Universität Bielefeld
    Dr. J. Kalinowski, CeBiTec Bielefeld
    Prof. B. Eikmanns, Universität Ulm
    Prof. M. Bott, Dr. L. Eggeling, Forschungszentrum Jülich
    Dr. R. Kelle, Dr. B. Bathe, Dr. M. Rieping, EVONIK DEGUSSA AG
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Selected Papers

  • Ochrombel, I., Ott, L. Krämer, R., Burkovski, A. and Marin, K. (2011) Impact of improved potassium accumulation on pH homeostasis, membrane potential adjustment and survival of Corynebacterium glutamicum. BBA-Bioenergetics accepted
  • Huhn, S., Jolkver, E., Krämer, R. Marin, K. (2010) Identification of the membrane protein SucE and its role in succinate transport in Corynebacterium glutamicum. Appl. Microbiol Biotechn. 89: 327-335
  • Börngen K, Battle AR, Möker N, Morbach S, Marin K, Martinac B, Krämer R. (2010) The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation. Biochim Biophys Acta. DOI: 10.1016/j.bbamem.2010.06.022
  • Marin, K. and Krämer, R. (2009) Im- und Export von Aminosäuren in Bakterien. BIOSPEKTRUM, 6: 600-4
  • Follmann, M., Ochrombel, I., Krämer, R., Trötschel, C., Poetsch, A., Rückert, C., Hüser, A., Persicke, M., Seiferling, D., Kalinowski, J. and Marin, K. (2009) Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis. BMC Genomics, 10: 621, doi: 10.1186/1471-2164-10-621
  • Follmann, M., Becker, M., Ochrombel, I., Ott, V., Krämer, R., Marin, K. (2009) Potassium transport in Corynebacterium glutamicum is facilitated by the putative channel protein CglK, which is essential for pH homeostasis and growth at acidic pH. J Bacteriol. 191:2944-52 (Faculty of the 1000, recommended)
  • Heuser, F., Marin, K., Kaup, B., Bringer, S., Sahm, H. (2009) Improving D-mannitol productivity of Escherichia coli: Impact of NAD, CO(2) and expression of a putative sugar permease from Leuconostoc pseudomesenteroides. Metab. Eng. 11:178-183
  • Youn, J.W., Jolkver, E., Krämer, R., Marin, K., Wendisch, V.F. (2009) Characterization of the dicarboxylate transporter DctA in Corynebacterium glutamicum. J Bacteriol. 191:5480-8
  • Nentwich, S. S., Brinkrolf, K., Gaigalat, L., Hüser, A. T., Rey, D. A., Mohrbach, T., Marin, K., Pühler, A., Tauch, A., Kalinowski, J. (2008) Characterization of the LacI-type transcriptional repressor RbsR controlling ribose transport in Corynebacterium glutamicum ATCC 13032. Microbiology. 155, 150-64
  • Jolkver, E., Emer, D., Ballan, S., Krämer, R., Eikmanns, B., and Marin, K. (2008) Identification and characterization of a bacterial transport system for the uptake ov pyruvate, propionate, and acetate in Corynebacterium glutamicum. J. Bacteriol, 191:940-8
  • Youn, J.-W., Jolkver, E., Krämer, R., Marin, K., and Wendisch, V.F. (2008) Identification and characterization of the dicarboxylate uptake system DccT in Corynebacterium glutamicum. JBac. 190: 6458-66
  • Trötschel, C., Follmann, M., Nettekoven, J.A., Mohrbach, T., Forrest, L.R., Burkovski, A., Marin, K., Krämer, R. (2008) Methionine Uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a Novel Methionine and Alanine Importer of the NSS Neurotransmitter Transporter Family. Biochemistry. 47:12698–12709
  • Marin, K., Krämer, R. (2006) Amino acid transport systems in biotechnologically relevant bacteria. In Amino Acid Biosynthesis – Pathways, Regulation and Metabolic Engineering, Vol. 5, Wendisch, V. ed., series: Microbiology Monographs , Steinbüchel, A. ed.
  • Trötschel, C., Deutenberg, D., Bathe, B., Burkovski, A., and Krämer, R. (2005) Identification and characterization of methionine export systems in Corynebacterium glutamicum J. Bacteriol. 187, 3786-3794
  • Krämer, R. (2005) Production of amino acids: physiological and genetic approaches.in: Handbook of Food Biotechnology (Shetty, K., ed.), M. Dekker, in press
  • Schneider, F., Krämer, R., and Burkovski, A. (2004) Identification of the main b-alanine uptake system in Escherichia coli. Appl. Microbiol Biotechnol. 65, 576-582
  • Trötschel, C., Burkovski, A., Krämer, R., and Bathe, B. (2004) Verfahren zur fermentativen Herstellung von L-Aminosäuren unter Verwendung rekombinanter Mikroorganismen. Deutsche und Internationale Patentanmeldung 10 2004 009 454.3
  • Burkovski, A., Krämer, R. (2002). Bacterial amino acid transport proteins:occurence, functions, and significance for biotechnological applications. Appl. Microbiol. Biotechnol., 58, 265-274.
    [Abstract]
  • Krämer, R. (1996). Genetic and physiological approaches for the production of amino acids. J. Biotechnol., 45, 1-21.
  • Hermann, T., Krämer, R. (1996). Mechanism and regulation of isoleucine excretion in Corynebacterium glutamicum. Appl. Environ. Microbiol., 62, 3238-3244.
  • complete list
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