Energie- und Redoxstoffwechsel von Corynebacterium glutamicum\textit{Corynebacterium glutamicum}

In this work three different aspects of the energy and redox metabolism of the aerobic Grampositive soil bacterium $\textit{Corynebacterium glutamicum}$ were investigated. This species is used industrially for the large-scale production of amino acids, in particular the flavour-enhancer Lglutamate (1.5 million tons per year) and the feed-additive L-lysine (0.8 million tons per year). In the main part of the work, the role of cytochrome $\textit{bd}$ oxidase for growth and lysine production was analysed. Previous investigations suggested that electron transfer to oxygen by the wild type ATCC 13032 is mainly catalysed by the cytochrome $bc_{1}-aa_{3}$ supercomplex and that cytochrome $\textit{bd}$ oxidase is of minor importance. In the current work it could be shown, however, that the deletion of the cytochrome $\textit{bd}$ oxidase genes $\textit{cydAB}$ in strain 13032 led to a drastic growth defect, starting in the late exponential growth phase. In the lysine-producing strains MH20-22B and DM1730, deletion of the $\textit{cydAB}$ genes had only minor effects on growth, but lysine formation was increased by approximately 10 %, which is probably due to a reduced glucose demand for energy generation and maintenance. Thus, the respiratory chain was shown to be a target for metabolic engineering. The different effects of the $\textit{cydAB}$ deletion on growth might result from different growth rates and different ATP requirements for biomass and lysine formation. Overproduction of an active cytochrome $\textit{bd}$ oxidase, which was verified by an increased cytochrome d content and increased oxygen consumption rates, led to reduced growth rates and reduced biomass formation of all strains mentioned above, whereas lysine production varied strain-specifically. The negative effect of $\textit{bd}$ overproduction on growth might be explained by a reduced P/O ratio due to an increased electron flow through the bd branch of the respiratory chain. In a second part of this work,$\textit{C. glutamicum}$ strains lacking the F$_{1}$F$_{O}$-ATP synthase genes ($\textit{atpBEFHAGDC}$) were constructed and characterised. These mutants can form ATP only via substrate-level phosphorylation and, as expected, showed strongly reduced growth rates and cell yields on glucose minimal medium. Furthermore, they formed large amounts of acetate, indicating a limitation of the carbon flux through the tricarboxylic acid cycle. Deletion of the F$_{O}$ genes ($\textit{atpBFE}$) in strain DM1730 led to a significant decrease in lysine formation, but caused the excretion of alanine. In the third part of this work, the membrane-integral transhydrogenase PntAB from $\textit{Escherichia coli}$ was overproduced in $\textit{C. glutamicum}$ DM1730 in order to improve the NADPH availability, which is critical for lysine formation. PntAB uses the electrochemical proton potential to reduce NADP$^{+}$ with NADH. During growth on glucose, the recombinant strain with a transhydrogenase activity of 0.6 μmol min$^{-1}$ (mg protein)-1 formed 18 % more lysine than the parental strain lacking PntAB. Thus, overproduction of PntAB represents a novel metabolic engineering strategy to improve L-lysine production by $\textit{C. glutamicum}$

Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production

Corynebacterium glutamicum possesses two terminal oxidases, cytochrome aa(3) and cytochrome bd. Cytochrome aa(3) forms a supercomplex with the cytochrome bc(1) complex, which contains an unusual diheme cytochrome c(1). Both the bc(1)-aa(3) 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 ∼45% and of the maximal biomass by ∼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 ∼12%. Thus, the respiratory chain was shown to be a target for improving amino acid production by C. glutamicum

Dynamic CT perfusion imaging of the myocardium: a technical note on improvement of image quality.

OBJECTIVE: To improve image and diagnostic quality in dynamic CT myocardial perfusion imaging (MPI) by using motion compensation and a spatio-temporal filter. METHODS: Dynamic CT MPI was performed using a 256-slice multidetector computed tomography scanner (MDCT). Data from two different patients-with and without myocardial perfusion defects-were evaluated to illustrate potential improvements for MPI (institutional review board approved). Three datasets for each patient were generated: (i) original data (ii) motion compensated data and (iii) motion compensated data with spatio-temporal filtering performed. In addition to the visual assessment of the tomographic slices, noise and contrast-to-noise-ratio (CNR) were measured for all data. Perfusion analysis was performed using time-density curves with regions-of-interest (ROI) placed in normal and hypoperfused myocardium. Precision in definition of normal and hypoperfused areas was determined in corresponding coloured perfusion maps. RESULTS: The use of motion compensation followed by spatio-temporal filtering resulted in better alignment of the cardiac volumes over time leading to a more consistent perfusion quantification and improved detection of the extend of perfusion defects. Additionally image noise was reduced by 78.5%, with CNR improvements by a factor of 4.7. The average effective radiation dose estimate was 7.1±1.1 mSv. CONCLUSION: The use of motion compensation and spatio-temporal smoothing will result in improved quantification of dynamic CT MPI using a latest generation CT scanner

Coloured perfusion maps for original data (a), motion corrected data (b) and combined motion-corrected and spatio-temporal filtered data (c) of Patient 1 in axial orientation.

<p>As a reference, short axis view late enhancement MR images are shown (d) presenting partly transmural infarction of the inferolateral wall. LV endocardium and epicardium are outlined in red. The perfusion defect of the inferior and lateral LV wall is visible in all CT perfusion images, with improved delineation of the hypoperfused and remote myocardium using motion correction (b) and best image quality using combined reconstruction algorithm with motion correction and spatio-temporal filtering (c). Thus the transmural extent of the infarction as diagnosed in MRI (d) can be distinguished only in combined motion-corrected and spatio-temporal filtered images (c).</p

Dynamic contrast enhancement of the LV myocardium apical (orange) and inferolateral (blue) in Patient 2 with a HR varying from 60–110 bpm and several extrasytoles during CT perfusion examination.

<p>Perfusion analysis of original images shows irregular zigzag pattern of the resulting curves (a). A continuous run of the time density curves is illustrated after motion correction and spatio-temporal filtering (b). Both curves (orange and blue) illustrate normal contrast enhancement of the LV myocardium with no evidence of perfusion deficit. Figs. c-f: Shown are the axial images (and the ROIs) at two different instances in time – the original uncorrected (c, d) and after motion correction plus spatio-temporal smoothing (e, f). Images c and e are from cardiac cycle 3; d and f are from cardiac cycle 8. In the original images (c & d), the two ROIs do not cover the same area of the myocardium at both time points, but instead cover adjacent tissue and the contrast-filled lumen of the LV, resulting in several spikes in (a). In contrast, consistent measurements of the same part of the myocardium after motion correction (e & f) leading to coherent time-density curves (b) for both ROIs. All CT images are presented using the same window level and window width.</p

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation

Kabus A, Georgi T, Wendisch VF, Bott M. Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves L-lysine formation. Applied Microbiology and Biotechnology. 2007;75(1):47-53.A critical factor in the biotechnological production of (L)-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) stop via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by similar to 10% on glucose, similar to 70% on fructose, similar to 50% on glucose plus fructose, and even by similar to 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type

A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum

Kranz A, Polen T, Kotulla C, et al. A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum. Scientific Data. 2022;9: 594