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Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII2CIV2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the QN and QP sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme bL. A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme bL. Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV.

l-Arabinonic acid is a five carbon sugar acid valuable for various potential applications. Based on previous observations that Gluconobacter oxydans 621H oxidizes l-arabinose in the periplasm by the membrane-bound glucose dehydrogenase GdhM yielding l-arabinonic acid, we tested its ability for production. In fed-batch shake flasks without pH control, 17.4 g/L l-arabinonic acid and 5.7 g/L l-arabino-1,4-lactone were obtained. A large fraction of l-arabinose remained unused since acidification of the medium prevented further substrate oxidation. In pH 6-controlled fed-batch bioreactors, up to 120 g/L l-arabinonic acid and 13 g/L l-arabino-1,4-lactone could be obtained with strain 621H (144 h). Constitutive overexpression of gdhM did not increase product titers and resulted in reduced biomass formation, yet the gdhM overexpression strain showed higher biomass-specific production (169 g/L/gcdw) compared to 621H (131 g/L/gcdw). The high combined product titer (133 g/L, 814 mM) makes G. oxydans a very promising host for high-level production of l-arabinonic acid.

Plant polyphenols receive significant attention due to their anti-oxidative and health-promoting properties, and several microorganisms are currently engineered towards producing these valuable compounds. Previously, Corynebacterium glutamicum has been engineered for synthesizing polyphenol core structures such as the stilbene resveratrol and the (2S)-flavanone naringenin. Decoration of these compounds by O-methylation or hydroxylation would provide access to polyphenols of even higher commercial interest. In this study, introduction of a heterologous O-methyltransferase into a resveratrol-producing C. glutamicum strain allowed synthesis of 42mg/L (0.16mM) of the di-O-methylated pterostilbene from p-coumaric acid. A prerequisite for reaching this product titer was a fusion of O-methyltransferase with the maltose-binding protein of Escherichia coli lacking its signal peptide, thereby increasing the solubility of the O-methyltransferase. Furthermore, expression of heterologous dioxygenase genes in (2S)-flavanone-producing C. glutamicum strains enabled the production of flavanonols and flavonols starting from the phenylpropanoids p-coumaric acid and caffeic acid. For the flavonols kaempferol and quercetin, maximum product titers of 23mg/L (0.08mM) and 10mg/L (0.03mM) could be achieved, respectively. The obtained results demonstrate that C. glutamicum is a suitable host organism for the production of more complex plant polyphenols.

β-Oxidation is the ubiquitous metabolic strategy to break down fatty acids. In the course of this four-step process, two carbon atoms are liberated per cycle from the fatty acid chain in the form of acetyl-CoA. However, typical β-oxidative strategies are not restricted to monocarboxylic (fatty) acid degradation only, but can also be involved in the utilization of aromatic compounds, amino acids and dicarboxylic acids. Each enzymatic step of a typical β-oxidation cycle is reversible, offering the possibility to also take advantage of reversed metabolic pathways for applied purposes. In such cases, 3-oxoacyl-CoA thiolases, which catalyze the final chain-shortening step in the catabolic direction, mediate the condensation of an acyl-CoA starter molecule with acetyl-CoA in the anabolic direction. Subsequently, the carbonyl-group at C3 is stepwise reduced and dehydrated yielding a chain-elongated product. In the last years, several β-oxidation pathways have been studied in detail and reversal of these pathways already proved to be a promising strategy for the production of chemicals and polymer building blocks in several industrially relevant microorganisms. This review covers recent advancements in this field and discusses constraints and bottlenecks of this metabolic strategy in comparison to alternative production pathways.

Biosensors offer exciting possibilities for improving cells or enzymes as biocatalysts for the synthesis of small molecules. We here review recent progress in the development and the screening applications of transcription-factor-based biosensors. An example is a cofactor-dependent biosensor which provides a generalizable screen for NADPH-dependent enzymes. Another example is the use of a biosensor in combination with recombineering for strain development, thereby expanding the genome engineering techniques to deliver directly bacteria producing small molecules of interest. Biosensor-based techniques in combination with fluorescence-activated cell sorting demonstrate that the gap regarding throughput capabilities of existing methods for the generation of genetic diversity and methods for the subsequent screening can be closed.

Corynebacterium glutamicum, like Mycobacterium tuberculosis, is a member of the Corynebacteriales, which have linear fatty acids and as branched fatty acids the mycolic acids. We identified accD1 and fasA as key genes of fatty acid synthesis, encoding the β-subunit of the acetyl-CoA carboxylase and a type-I fatty acid synthase, respectively, and observed their repression during growth on minimal medium with acetate. We also identified the transcriptional regulator FasR and its binding sites in the 5′ upstream regions of accD1 and fasA. In the present work we establish by co-isolation and gel-mobility shifts oleoyl-CoA and palmitoyl-CoA as effectors of FasR, and show by DNA microarray analysis that in presence of exogeneous fatty acids accD1 and fasA are repressed. These results are evidence that acyl-CoA derivatives derived from extracellular fatty acids interact with FasR to repress the genes of fatty acid synthesis. This model also explains the observed repression of accD1 and fasA during growth on acetate, where apparently the known high intracellular acetyl-CoA concentration during growth on this substrate requires reduced accD1 and fasA expression for fine control of de novo fatty acid synthesis. Consequently, this mechanism ensures that membrane lipid homeostasis is maintained when specific nutrients are available resulting in increased acetyl-CoA concentration, as is the case with acetate, or when fatty acids are directly available from the extracellular environment. However, the genes specific to mycolic acid synthesis, which are in part shared with linear fatty acid synthesis, are not controlled by FasR, which is in agreement with the fact that they can not be supplied from the extracellular environment but that their synthesis fully depends on a constant supply of linear fatty acid chains.

Pyruvate dehydrogenase and oxoglutarate dehydrogenase catalyze key reactions in central metabolism. In Corynebacterium glutamicum and related bacteria like Mycobacterium tuberculosis both activities reside in a novel protein supercomplex with the fusion protein OdhA catalyzing the conversion of oxoglutarate to succinyl-coenzyme A. This activity is inhibited by the forkhead-associated (FHA) domain of the small autoinhibitory protein OdhI. Here we used a biological screen which enabled us to isolate suppressor mutants that are influenced in OdhA-OdhI interaction. Five mutants carrying an OdhI mutation were isolated and one with an OdhA mutation. The OdhA mutein OdhA-C704E and three additional C704 variants were constructed. They exhibited unaltered or even slightly enhanced OdhA activity but showed reduced inhibition and interaction with OdhI. The FHA domain of OdhI was crystallized and its structure found in full agreement with previously determined NMR structures. Based on further structural studies, OdhA-OdhI crosslinking experiments, and modeling we discuss the experimental data generated on OdhA-OdhI interaction, with the latter protein representing a rare example of an FHA domain also recognizing a non-phosphorylated interaction partner.

Adipic acid is the most important commercial aliphatic dicarboxylic acid in the chemical industry and is primarily used for the production of nylon-6,6 polyamide. The current adipic acid market volume is about 2.6 million tons/y and the average annual demand growth rate forecast to stay at 3-3.5% worldwide. Hitherto, the industrial production of adipic acid is carried out by petroleum-based chemo-catalytic processes from non-renewable fossil fuels. However, in the past years, efforts were made to find alternative routes for adipic acid production from renewable carbon sources by biotechnological processes. Here we review the approaches and the progress made toward bio-based production of adipic acid.

Corynebacterium glutamicum is an important industrial bacterium as well as a model organism for the order Corynebacteriales, whose citric acid cycle occupies a central position in energy and precursor supply. Expression of aconitase, which isomerizes citrate into isocitrate, is controlled by several transcriptional regulators, including the dimeric aconitase repressor AcnR, assigned by sequence identity to the TetR family. We report the structures of AcnR in two crystal forms together with ligand binding experiments and in vivo studies. First, there is a citrate-Mg(2+) moiety bound in both forms, not in the canonical TetR ligand binding site but rather in a second pocket more distant from the DNA binding domain. Second, the citrate-Mg(2+) binds with a KD of 6 mM, within the range of physiological significance. Third, citrate-Mg(2+) lowers the affinity of AcnR for its target DNA in vitro. Fourth, analyses of several AcnR point mutations provide evidence for the possible involvement of the corresponding residues in ligand binding, DNA binding, and signal transfer. AcnR derivatives defective in citrate-Mg(2+) binding severely inhibit growth of C. glutamicum on citrate. Finally, the structures do have a pocket corresponding to the canonical tetracycline site, and although we have not identified a ligand that binds there, comparison of the two crystal forms suggests differences in the region of the canonical pocket that may indicate a biological significance.

Of more than 200,000 plant natural products known to date, many demonstrate important pharmacological activities or are of biotechnological significance. However, isolation from natural sources is usually limited by low abundance and environmental, seasonal as well as regional variation, whereas total chemical synthesis is typically commercially unfeasible considering the complex structures of most plant natural products. With advances in DNA sequencing and recombinant DNA technology many of the biosynthetic pathways responsible for the production of these valuable compounds have been elucidated, offering the opportunity of a functional integration of biosynthetic pathways in suitable microorganisms. This approach offers promise to provide sufficient quantities of the desired plant natural products from inexpensive renewable resources. This review covers recent advancements in the metabolic engineering of microorganisms for the production of plant natural products such as isoprenoids, phenylpropanoids and alkaloids, and highlights general approaches and strategies to gain access to the rich biochemical diversity of plants by employing the biosynthetic power of microorganisms.

Corynebacterium glutamicum possesses export systems for various amino acids including BrnFE, a two-component export system for L-methionine and the branched-chain amino acids L-valine, L-isoleucine and L-leucine. A gene for a putative transcriptional regulator of the Lrp family is transcribed divergently to the brnFE operon and is required for L-isoleucine export. By comparing global gene expression changes due to L-isoleucine addition we revealed increased brnFE expression in response to L-isoleucine in C. glutamicum wild type but not in an lrp deletion mutant. ChIP-to-chip analysis, band shift experiments and DNAse footprint analysis demonstrated that Lrp binds to the intergenic region between lrp and brnF. Expression analysis of transcriptional fusions with the lrp and brnFE promoters indicated that branched-chain amino acids and L-methionine when added to the growth medium stimulated brnFE expression in the order L-leucine > L-methionine > L-isoleucine > L-valine and that Lrp was required for activation of brnFE expression. Thus, regulation of brnFE by Lrp ensures that BrnFE is synthesized only if its substrate amino acids accumulate in cells which is commensurate with its role to counteract such situations of metabolic imbalance.

Citrate synthase (CS) is located at a major branch point in the metabolism and is required for both tricarboxylic acid and glyoxylic acid cycle activity. Here we show that the CS gene gltA of Corynebacterium glutamicum is monocistronic, but that two transcripts are formed with their transcript initiation sites located 121 bp and 357 bp upstream of the translational start codon, respectively. Northern blot analyses revealed that during growth on acetate the short transcript prevails, whereas during growth on glucose the long transcript is dominant. Further Northern blots, reporter gene fusions, and CS activity measurements in mutants devoid of the transcriptional regulators RamA and RamB or with the global regulator GlxR overexpressed revealed a complex involvement of these regulators in gltA transcription. This was confirmed by demonstrating the direct interaction of isolated RamA, RamB and GlxR proteins with specific gltA promoter regions in vitro. The combined analyses point to an elaborate control of gltA transcript formation, which is possibly required as a dedicated mechanism to balance the total CS activity according to the physiological requirements.

Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.

Corynebacterium glutamicum, a Gram-positive soil bacterium, is used for the production of l-glutamate and l-lysine, both of which are derived from intermediates of the tricarboxylic acid (TCA) cycle. Recent studies have revealed that this amphibolic pathway is subject to complex regulation not only at the transcriptional level, but also at the post-transcriptional level. The latter involves serine/threonine protein kinase G and its target protein OdhI. Depending on its phosphorylation state, OdhI inhibits the 2-oxoglutarate dehydrogenase complex. This new type of TCA cycle control will be reviewed here together with regulation at the level of gene expression.

In Corynebacterium glutamicum, the activity of aconitase is 2.5-4-fold higher on propionate, citrate, or acetate than on glucose. Here we show that this variation is caused by transcriptional regulation. In search for putative regulators, a gene (acnR) encoding a TetR-type transcriptional regulator was found to be encoded immediately downstream of the aconitase gene (acn) in C. glutamicum. Deletion of the acnR gene led to a 5-fold increased acn-mRNA level and a 5-fold increased aconitase activity, suggesting that AcnR functions as repressor of acn expression. DNA microarray analyses indicated that acn is the primary target gene of AcnR in the C. glutamicum genome. Purified AcnR was shown to be a homodimer, which binds to the acn promoter in the region from -11 to -28 relative to the transcription start. It thus presumably acts by interfering with the binding of RNA polymerase. The acn-acnR organization is conserved in all corynebacteria and mycobacteria with known genome sequence and a putative AcnR consensus binding motif (CAGNACnnncGTACTG) was identified in the corresponding acn upstream regions. Mutations within this motif inhibited AcnR binding. Because the activities of citrate synthase and isocitrate dehydrogenase were previously reported not to be increased during growth on acetate, our data indicate that aconitase is a major control point of tricarboxylic acid cycle activity in C. glutamicum, and they identify AcnR as the first transcriptional regulator of a tricarboxylic acid cycle gene in the Corynebacterianeae.

The genes specifically required for citrate fermentation in Klebsiella pneumoniae form a cluster on the chromosome consisting of two divergently transcribed groups, citCDEFG and citS-oadGAB-citAB. Northern blot analyses described here and elsewhere indicate that each group forms an operon. The transcriptional start sites of citC and citS, which were mapped in this work by primer extension, are separated by a stretch of 193 bp with an extraordinary high A + T content of 67%. Expression of the citrate fermentation genes was recently shown to be positively controlled by a two-component signal transduction system encoded by the promoter-distal genes of the citS operon, citA (sensor kinase) and citB (response regulator). As a first step towards the functional characterization of CitB, we analysed its DNA-binding properties. To this end, the entire CitB, its N-terminal receiver domain (CitBN), and its C-terminal output domain (CitBC), all modified by a (His)6-tag, were purified. CitB(His) and CitBN(His) could be phosphorylated either with acetylphosphate or with ATP plus MalE-CitAC. The latter protein contains the kinase domain of CitA fused to the C terminus of the maltose-binding protein. Upon phosphorylation, CitB(His) became more resistant towards limited proteolysis by trypsin, reflecting substantial changes in tertiary structure. In gel retardation assays, CitB(His) bound specifically to the citC-citS intergenic region. The retardation pattern changed significantly upon phosphorylation and the apparent binding affinity increased 10 to 100-fold. Depending on the protein concentration, four different phospho-CitB(His)-DNA complexes could be resolved, suggesting the presence of multiple binding sites between citC and citS. DNase I footprints revealed two protected regions extending maximally from -55 to -89 relative to the citS transcription start and from -50 to -96 relative to the citC transcription start. Gel retardation and DNase I footprint assays with CitBC(His) showed that the C-terminal domain is sufficient for specific DNA binding. Since its properties were similar to that of unphosphorylated CitB(His), an essential role of the N-terminal receiver domain in high-affinity DNA binding was indicated. The positions of the binding sites for CitB and of putative recognition sequences for the cAMP receptor protein suggested a model for the interaction of these activators with RNA polymerase.

The detection and quantification of specific metabolites in single bacterial cells is a major goal for industrial biotechnology. We have developed a biosensor based on the transcriptional regulator Lrp that detects intracellular l-methionine and branched-chain amino acids in Corynebacterium glutamicum. In assays, fluorescence output showed a linear relationship with cytoplasmic concentrations of the effector amino acids. In increasing order, the affinity of Lrp for the amino acids is l-valine, l-isoleucine, l-leucine and l-methionine. The sensor was applied for online monitoring and analysis of cell-to-cell variability of l-valine production by the pyruvate dehydrogenase-deficient C. glutamicum strain ΔaceE. Finally, the sensor system was successfully used in a high-throughput (HT) FACS screen for the isolation of amino acid-producing mutants after random mutagenesis of a non-producing wild type strain. These applications illustrate how one of nature's sensor devices – transcriptional regulators – can be used for the analysis, directed evolution and HT screening for microbial strain development. Highlights ► Development of a single cell biosensor for the intracellular detection of amino acids. ► Biosensor-based online monitoring of valine production of a C. glutamicum strain. ► Visualization of cell-to-cell variability of microbial production strains. ► Establishment of a FACS high-throughput screening.

The homoepitaxial growth of Pt on Pt(111) has been investigated by STM and the results have been compared to recent thermal He scattering (TEAS) data obtained on the same system. Additional information on the growth modes is obtained and the real space aspect of the growing surface, which results in TEAS and RHEED oscillations is evidenced. The three different growth modes, including the reentrant layer-by-layer growth at low temperatures, are confirmed. The limited diffusion along the adatom island edges, which causes their fractal aspect with dendritic structures, appears to play a significant role in the appearance of the low temperature layer-by-layer growth.