Physiology and global gene expression of a Corynebacterium glutamicum ΔF(1)F(O)-ATP synthase mutant devoid of oxidative phosphorylation.

A mutant of Corynebacterium glutamicum ATCC 13032 with a deletion of the atpBEFHAGDC genes encoding F(1)F(O)-ATP synthase was characterized. Whereas no growth was observed with acetate as sole carbon source, the ΔF(1)F(O) mutant reached 47% of the growth rate and 65% of the biomass of the wild type during shake-flask cultivation in glucose minimal medium. Initially, the mutant strain showed a strongly increased glucose uptake rate accompanied by a high oxygen consumption rate and pyruvate secretion into the medium. When oxygen became limiting, the glucose consumption rate was reduced below that of the wild type and pyruvate was consumed again. The ΔF(1)F(O) mutant had increased levels of b- and d-type cytochromes and a significantly increased proton motive force. Transcription of genes involved in central carbon metabolism was essentially unchanged, whereas genes for cytochrome bd oxidase, pyruvate:quinone oxidoreductase, oxidative stress response, and others showed increased mRNA levels. On the other hand, genes for amino acid biosynthesis and ribosomal proteins as well as many genes involved in transport displayed decreased mRNA levels. Several of the transcriptional changes were reflected at the protein level, but there were also discrepancies between the mRNA and protein levels suggesting some kind of posttranscriptional regulation. The results prove for the first time that F(1)F(O)-ATP synthase and oxidative phosphorylation are in general not essential for growth of C. glutamicum.

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

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum.

Corynebacterium glutamicum, an established industrial amino acid producer, has been genetically modified for efficient succinate production from the renewable carbon source glucose under fully aerobic conditions in minimal medium. The initial deletion of the succinate dehydrogenase genes (sdhCAB) led to an accumulation of 4.7 g l(-1) (40 mM) succinate as well as high amounts of acetate (125 mM) as by-product. By deleting genes for all known acetate-producing pathways (pta-ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l(-1) (66 mM). Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l(-1) (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)(-1) h(-1). This value represents the highest productivity among currently described aerobic bacterial succinate producers. Optimization of the production conditions by decoupling succinate production from cell growth using the most advanced producer strain (C. glutamicumΔpqoΔpta-ackAΔsdhCABΔcat/pAN6-pyc(P458S) ppc) led to an additional increase of the product yield to 0.45 mol succinate mol(-1) glucose and a titre of 10.6 g l(-1) (90 mM) succinate.

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.

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

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(+) 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 approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 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.

Corynebacterial protein kinase G controls 2-oxoglutarate dehydrogenase activity via the phosphorylation status of the OdhI protein.

A novel regulatory mechanism for control of the ubiquitous 2-oxoglutarate dehydrogenase complex (ODH), a key enzyme of the tricarboxylic acid cycle, was discovered in the actinomycete Corynebacterium glutamicum, a close relative of important human pathogens like Corynebacterium diphtheriae and Mycobacterium tuberculosis. Based on the finding that a C. glutamicum mutant lacking serine/threonine protein kinase G (PknG) was impaired in glutamine utilization, proteome comparisons led to the identification of OdhI as a putative substrate of PknG. OdhI is a 15-kDa protein with a forkhead-associated domain and a homolog of mycobacterial GarA. By using purified proteins, PknG was shown to phosphorylate OdhI at threonine 14. The glutamine utilization defect of the delta pknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a delta odhI mutant with a plasmid encoding OdhI-T14A caused a defect in glutamine utilization. Affinity purification of OdhI-T14A led to the specific copurification of OdhA, the E1 subunit of ODH. Because ODH is essential for glutamine utilization, we assumed that unphosphorylated OdhI inhibits ODH activity. In fact, OdhI was shown to strongly inhibit ODH activity with a Ki value of 2.4 nM. The regulatory mechanism described offers a molecular clue for the reduced ODH activity that is essential for the industrial production of 1.5 million tons/year of glutamate with C. glutamicum. Moreover, because this signaling cascade is likely to operate also in mycobacteria, our results suggest that the attenuated pathogenicity of mycobacteria lacking PknG might be caused by a disturbed tricarboxylic acid cycle.