Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum

Schwentner A, Feith A, Muench E, Busche T, Rückert C, Kalinowski J, Takors R, Blombach B (2018)
METABOLIC ENGINEERING 47: 31-41.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Abstract / Bemerkung
Evolutionary approaches are often undirected and mutagen-based yielding numerous mutations, which need elaborate screenings to identify relevant targets. We here apply Metabolic engineering to Guide Evolution (MGE), an evolutionary approach evolving and identifying new targets to improve microbial producer strains. MGE is based on the idea to impair the cell's metabolism by metabolic engineering, thereby generating guided evolutionary pressure. It consists of three distinct phases: (i) metabolic engineering to create the evolutionary pressure on the applied strain followed by (ii) a cultivation phase with growth as straightforward screening indicator for the evolutionary event, and (iii) comparative whole genome sequencing (WGS), to identify mutations in the evolved strains, which are eventually re-engineered for verification. Applying MGE, we evolved the PEP and pyruvate carboxylase-deficient strain C. glutamicum Delta ppc Delta pyc to grow on glucose as substrate with rates up to 0.31 +/- 0.02 h(-1) which corresponds to 80% of the growth rate of the wildtype strain. The intersection of the mutations identified by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in three independently evolved mutants. Upon re-engineering in C. glutamicum Delta ppc Delta pyc, the identified mutations led to diminished ICD activities and activated the glyoxylate shunt replenishing oxaloacetate required for growth. Intracellular relative quantitative metabolome analysis showed that the pools of citrate, isocitrate, cis-aconitate, and L-valine were significantly higher compared to the WT control. As an alternative to existing L-valine producer strains based on inactivated or attenuated pyruvate dehydrogenase complex, we finally engineered the PEP and pyruvate carboxylase-deficient C. glutamicum strains with identified ICD mutations for L-valine production by overexpression of the L-valine biosynthesis genes. Among them, C. glutamicum Delta ppc Delta pyc ICDG407S (pJC4ilvBNCE) produced up to 8.9 +/- 0.4 g L-valine L-1, with a product yield of 0.22 +/- 0.01 g L-valine per g glucose.
Stichworte
Directed evolution; Metabolic engineering; Isocitrate dehydrogenase; Glyoxylate shunt; L-valine production; Corynebacterium glutamicum
Erscheinungsjahr
2018
Zeitschriftentitel
METABOLIC ENGINEERING
Band
47
Seite(n)
31-41
ISSN
1096-7176
eISSN
1096-7184
Page URI
https://pub.uni-bielefeld.de/record/2920690

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Schwentner A, Feith A, Muench E, et al. Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum. METABOLIC ENGINEERING. 2018;47:31-41.
Schwentner, A., Feith, A., Muench, E., Busche, T., Rückert, C., Kalinowski, J., Takors, R., et al. (2018). Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum. METABOLIC ENGINEERING, 47, 31-41. doi:10.1016/j.ymben.2018.02.015
Schwentner, A., Feith, A., Muench, E., Busche, T., Rückert, C., Kalinowski, J., Takors, R., and Blombach, B. (2018). Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum. METABOLIC ENGINEERING 47, 31-41.
Schwentner, A., et al., 2018. Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum. METABOLIC ENGINEERING, 47, p 31-41.
A. Schwentner, et al., “Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum”, METABOLIC ENGINEERING, vol. 47, 2018, pp. 31-41.
Schwentner, A., Feith, A., Muench, E., Busche, T., Rückert, C., Kalinowski, J., Takors, R., Blombach, B.: Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum. METABOLIC ENGINEERING. 47, 31-41 (2018).
Schwentner, Andreas, Feith, Andre, Muench, Eugenia, Busche, Tobias, Rückert, Christian, Kalinowski, Jörn, Takors, Ralf, and Blombach, Bastian. “Metabolic engineering to guide evolution - Creating a novel mode for L-valine production with Corynebacterium glutamicum”. METABOLIC ENGINEERING 47 (2018): 31-41.

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