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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 lysineproducing 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 ∼10% on glucose, ∼70% on fructose, ∼50% on glucose plus fructose, and even by ∼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.

Corynebacterium glutamicum can grow on L-lactate as a sole carbon and energy source. The NCgl2816-lldD operon encoding a putative transporter (NCgl2816) and a quinone-dependent L-lactate dehydrogenase (LldD) is required for L-lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCgl2814) binds to the upstream region of NCgl2816-lldD. Overexpression of lldR resulted in strongly reduced NCgl2816-lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on L-lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCgl2816-lldD mRNA levels and high LldD activity in the presence and absence of L-lactate. Lactate is a major product of anaerobic metabolism, but it also serves as a carbon and energy source for anaerobic and aerobic microorganisms. Lactate can be fermented to acetate, propionate, or butyrate by, e.g., sulfate-reducing bacteria, propionibacteria, or Eubacterium hallii (11). When oxygen becomes available but glucose is limiting, Lactobacillus plantarum converts its fermentation product, lactate, to acetate (18). Aerobic growth with L-lactate as the sole carbon and energy source has been studied in Escherichia coli in some detail. L-Lactate is taken up into the E. coli cell either by the L-lactate permease LldP or by the glycolate permease GlcA (39). LLactate is oxidized to the central metabolite pyruvate by quinone-dependent L-lactate dehydrogenase (LldD; EC 1.1.2.3) (10). For growth on L-lactate, E. coli requires lldD, which forms an operon with lldP and the putative lactate regulator gene lldR (10). Transcription of lldDRP is repressed by ArcAB under anaerobic reducing conditions (24) and is maximal in the presence of L-lactate (10); however, regulation of lldDRP by the putative regulator LldR encoded in this operon has not been analyzed yet in detail.Recently, we identified the L-lactate utilization operon in Corynebacterium glutamicum, a nonpathogenic gram-positive soil bacterium that is widely used for biotechnological production of amino acids such as L-glutamate and L-lysine. C. glutamicum can grow aerobically on a variety of sugars, sugar alcohols, and organic acids, including L-lactate, as sole carbon and energy sources (9,17,27,31,36,59). C. glutamicum forms L-lactate with the soluble NAD ϩ -dependent L-lactate dehydrogenase (EC 1.1.1.27) encoded by ldhA (3, 22) under oxygen deprivation (22) and as a by-product during glutamate and lysine production (27,28,53). For L-lactate utilization, on the other hand, C. glutamicum requires the quinone-dependent L-lactate dehydrogenase LldD (EC 1.1.2.3) (53), which is a peripheral membrane protein (51) catalyzing oxidation of Llactate to pyruvate (3, 53).The C. glutamicum L-lactate utilization operon comprises the quinone-dependent L-lactate dehydrogenase gene lldD and a gene encoding a putative permease (NCgl281...

Corynebacterium glutamicum, a Gram-positive soil bacterium employed in the industrial production of various amino acids, is able to use a number of different nitrogen sources, such as ammonium, urea or creatinine. This study shows that l-glutamine serves as an excellent nitrogen source for C. glutamicum and allows similar growth rates in glucose minimal medium to those in ammonium. A transcriptome comparison revealed that the nitrogen starvation response was elicited when glutamine served as the sole nitrogen source, meaning that the target genes of the global nitrogen regulator AmtR were derepressed. Subsequent growth experiments with a variety of mutants defective in nitrogen metabolism showed that glutamate synthase is crucial for glutamine utilization, while a putative glutaminase is dispensable under the experimental conditions used. The gltBD operon encoding the glutamate synthase is a member of the AmtR regulon. The observation that the nitrogen starvation response was elicited at high intracellular l-glutamine levels has implications for nitrogen sensing. In contrast with other Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Salmonella enterica serovar Typhimurium and Klebsiella pneumoniae, a drop in glutamine concentration obviously does not serve as a nitrogen starvation signal in C. glutamicum.

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