Physiology and global gene expression of a Corynebacterium glutamicum ΔF1FO-ATP synthase mutant devoid of oxidative phosphorylation

Abstract

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.

Metabolic engineering of Corynebacterium glutamicum for the production of itaconate

“…Under the cultivation conditions chosen, C. glutamicum wild type transiently accumulates lactate as major and acetate, succinate and malate as minor by-products. The excretion of these organic acids starts when the cells become oxygen-limited and they are later consumed again (Koch-Koerfges et al, 2012). To induce overexpression of plasmid-encoded target genes, 0.5 mM isopropyl-β-D-thiogalactopyranosid (IPTG) was added to the culture.…”

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

“…By SLP and ETP with the cytochrome bc 1 -aa 3 branch, complete aerobic oxidation of glucose and acetate yields 26.7 and 7.3 mol of ATP, respectively (29,30). ETP has been shown to be essential for growth with substrates that do not allow ATP generation by SLP (e.g., acetate); however, C. glutamicum mutants devoid of ETP grew with substrates allowing SLP, such as glucose (31). Glucose catabolism by C. glutamicum ⌬F 1 F o was slow and biphasic under oxygen-limiting conditions.…”

“…Glucose catabolism by C. glutamicum ⌬F 1 F o was slow and biphasic under oxygen-limiting conditions. In the first growth phase, ATP was generated via SLP in glycolysis, while the second phase was characterized by the formation of organic acids such as acetate, and ATP was apparently generated by SLP via an acetate kinase reaction (31).…”

Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

“…An OD 600 of 1 corresponds to 0.25 g cell dry weight (CDW) per liter (30). The culture samples were centrifuged twice (10,000 ϫ g, 4°C, 5 min) to remove the cells, and the resulting supernatants were analyzed for the presence of sugars and organic acids by high-pressure liquid chromatography (HPLC) (Agilent Technologies, Waldbronn, Germany) as described previously (31). Sucrose was measured with the Enzytec kit (Biopharm, Darmstadt, Germany) following the manufacturer's manual.…”