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The wild type of Corynebacterium glutamicum does not excrete l-lysine, but mutants are developed to use C. glutamicum for the large-scale production of this amino acid. l-lysine is derived from oxaloacetate and pyruvate. A reduced entry of these precursors into the citric acid cycle triggers l-lysine synthesis, but there is simultaneously an unfavorable reduction in growth rate. Here, we further investigated strains with reduced citrate synthase activity and observed that proline supplementation partly reversed the growth rate reduction, and simultaneously increased l-lysine accumulation by 10%. The combined effect of faster growth and higher l-lysine formation lead to an increase in l-lysine productivity of about 14%. Small scale bioreactor cultivations showed an increase of the glucose uptake rate from 3.96 to 5.12 mmol gCDW−1 h−1 as the effect of proline addition. Deletion of the proline-utilization gene putA demonstrated that proline did not serve to refill the citric acid cycle. The combined data show that proline addition has a favorable effect on l-lysine formation and sugar uptake, which indicates that engineering sugar uptake is a future option for increased product formation.

We here developed a series of Corynebacterium glutamicum strains with gradual decreased specific citrate synthase (CS) activity and quantified in a multifaceted approach the consequences of residual activity on the transcriptome, metabolome, and fluxome level as well as on L-lysine formation and growth. We achieved an intended gradual L-lysine yield increase and recognized and overcame further new limitations in the L-lysine biosynthesis pathway to result in a strain with the highest yield reported so far when assayed under comparable conditions. As a non-intended outcome, a detailed flux analysis revealed an almost constant flux through CS at 10% remaining CS activity, whereas the metabolome data revealed an increase in the oxaloacetate and acetyl-CoA concentrations. Hence reduced CS activity is apparently efficiently buffered by increased concentrations of CS substrates, implying a certain robustness of the central metabolism in response of the imposed gene expressions.

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grunberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is mu = 0.42 ± 0.03 h(-1) even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of mu = 0.62 ± 0.02 h(-1). When in the same perfusion system cell-free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to mu = 0.47 ± 0.02 h(-1). Likewise, when fresh medium is additionally supplied with 5 mM acetate, a growth rate of mu = 0.51 ± 0.01 h(-1) is determined. These results prove that higher growth rates of C. glutamicum than known from typical batch cultivations are possible, and that growth is definitely impaired by very low concentrations of byproducts such as acetate.

The addition of fatty acids to either Escherichia coli or Bacillus subtilis elicits an elaborate cellular response of the lipid metabolism. We found that in Corynebacterium glutamicum the expression of accD1 encoding the β-subunit of the essential acetyl-CoA carboxylase is repressed in acetate-grown cells without the addition of fatty acids. The TetR-type transcriptional regulator NCgl2404, termed FasR, was identified and deleted. During growth on acetate, but not on glucose, 17 genes are differentially expressed in the deletion mutant, among them accD1, and fasA and fasB both encoding fatty acid synthases, which were upregulated. Determination of the 5' ends of accD1, fasA, fasB and accBC together with the use of isolated FasR protein identified the FasR binding site, fasO, which is located within the accD1 and fasA transcript initiation site thus blocking transcription by RNA polymerase binding directly. The identified fasO motif is present in C. efficiens or C. diphtheriae, too, and it is actually similarly positioned in these bacteria within the 5' ends of the accD1 and fasA transcripts, and a fasR orthologue is also present. The identification of the FasR-fasO system in Corynebacteriaceae might indicate a conserved transcriptional control of the unique lipid synthesis in these mycolic acid-containing bacteria.