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Advancing Dual-Phase Production Strategies with Corynebacterium Glutamicum through Systems Biology

Lange, J., Institute of Biochemical Engineering
Kalinowski, J., University of Bielefeld
Busche, T., University of Bielefeld

Background. Bacteria encounter varying oxygen concentrations in manifold situations e.g. in their natural habitat and especially in large scale industrial processes generating viability and production deficiencies. In particular, dual-phase approaches, e.g. for isobutanol or succinic acid production, evoked yield problems due to an abrupt shift from aerobic cultivation to anaerobic production (1, 2). In order to adapt to the changing environment bacteria have to remodel their entire metabolism (3, 4). Despite its overarching relevance for pathogenicity and pharmaceutical and bio-based production processes, the molecular events during these transitions are poorly understood. To address this question, we systematically investigate the adaptation of the industrially relevant Corynebacterium glutamicum to altering conditions that range from aerobiosis via microaerobiosis to anaerobiosis.

Methods. A “triple-phase” batch bioprocess with C. glutamicum was established that depicts the three successive phases (aerobiosis, microaerobic interface and anaerobiosis) in a single bioreactor. Throughout the process, samples were collected and analyzed for substrate consumption and organic acid production and additionally used for whole transcriptome analysis by RNA-sequencing.

Results. A definition of the three phases was directly feasible by the bacterium’s physiological changes, i.e. a decreasing growth rate with increasing oxygen limitation. Furthermore, L‑lactic acid, succinic acid and acetic acid were the main fermentation products secreted to the culture supernatant, interestingly in a manner, that their respective differential product yields clearly bordered each process phase. A closed carbon balance indicated that all significant products were analyzed. RNA‑sequencing analysis revealed differential expression of an abundance of genes of the central and peripheral metabolism matching the expectations of previous work (5) but also delivered novel insights into the regulatory regime required for adaptation.

Conclusion. The established bioprocess is an elegant approach for the systematic understanding of C. glutamicum’s adaptation to a progressive oxygen deprivation. Deeper analysis of the RNA-sequencing data especially focusing on regulatory networks and their hierarchy could reveal novel targets for strain optimization to increase productivities in a dual-phase production process. A comprehensive understanding of the molecular events during the transitions might be also projected to other organisms and applications.

1.   Blombach B, Riester T, Wieschalka S, Ziert C, Youn J-W, Wendisch VF, Eikmanns BJ. 2011. Appl. Environ. Microbiol. 77:3300–10.

2.   Vemuri GN, Eiteman MA, Altman E. 2002. J. Ind. Microbiol. Biotechnol. 28:325–32.

3.   Patschkowski T, Bates DM, Kiley PJ. 2000. p. 61–78. In Storz, G, Hengge-Aronis, R (eds.), Bacterial stress responses. ASM Press, Washington, D.C.

4.   Bunn HF, Poyton RO. 1996. Physiol. Rev. 76:839–885.

5.   Inui M, Suda M, Okino S, Nonaka H, Puskás LG, Vertès AA, Yukawa H. 2007. Microbiology 153:2491–504.