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Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum.

Litsanov B, Kabus A, Brocker M, Bott M - Microb Biotechnol (2011)

Bottom Line: By deleting genes for all known acetate-producing pathways (pta-ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l(-1) (66 mM).Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l(-1) (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)(-1) h(-1).This value represents the highest productivity among currently described aerobic bacterial succinate producers.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, D-52425 Jülich, Germany.

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Scheme of the central metabolism of C. glutamicum showing the genetic modifications used in this work to construct a strain for aerobic succinate production. Enzymes whose genes were deleted are indicated by ‘X’. The reactions affected by these deletions and their products are displayed in grey. Enzymes whose genes were overexpressed are highlighted in grey boxes and the arrows for the corresponding reactions are thickened. Abbreviations: ACN, aconitase; AK, acetate kinase; CoAT, acetyl‐CoA:CoA transferase; CS, citrate synthase; FUM, fumarase; ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; MQO, malate:menaquinone oxidoreductase; MS, malate synthase; OAA, oxaloacetate; ODHC, 2‐oxoglutarate dehydrogenase complex; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PEPCx, PEP carboxylase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PTA, phosphotransacetylase; PQO, pyruvate:menaquinone oxidoreductase; SCS, succinyl‐CoA synthetase; SDH, succinate dehydrogenase.
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f1: Scheme of the central metabolism of C. glutamicum showing the genetic modifications used in this work to construct a strain for aerobic succinate production. Enzymes whose genes were deleted are indicated by ‘X’. The reactions affected by these deletions and their products are displayed in grey. Enzymes whose genes were overexpressed are highlighted in grey boxes and the arrows for the corresponding reactions are thickened. Abbreviations: ACN, aconitase; AK, acetate kinase; CoAT, acetyl‐CoA:CoA transferase; CS, citrate synthase; FUM, fumarase; ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; MQO, malate:menaquinone oxidoreductase; MS, malate synthase; OAA, oxaloacetate; ODHC, 2‐oxoglutarate dehydrogenase complex; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PEPCx, PEP carboxylase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PTA, phosphotransacetylase; PQO, pyruvate:menaquinone oxidoreductase; SCS, succinyl‐CoA synthetase; SDH, succinate dehydrogenase.

Mentions: In view of these facts, we started to explore the potential of C. glutamicum for aerobic succinate production. Corynebacterium glutamicum is a facultatively anaerobic, Gram‐positive soil bacterium with GRAS status (generally regarded as safe), which is used for the large‐scale production of more than 2 million tons of l‐glutamate and 1.1 million tons of l‐lysine annually. In addition, C. glutamicum strains were developed for the production of several other industrially relevant products such as putrescine (Schneider and Wendisch, 2010), isobutanol (Blombach et al., 2011) or ethanol (Inui et al., 2004a). The genome of C. glutamicum is known (Ikeda and Nakagawa, 2003; Kalinowski et al., 2003; Yukawa et al., 2007) and numerous genetic tools are available allowing genetic engineering (Kirchner and Tauch, 2003). Moreover, extensive knowledge on the central metabolism of C. glutamicum is available due to 60 years of research on amino acid production (Eggeling and Bott, 2005; Burkovski, 2008). Novel results relevant for succinate production represent the identification of two genes coding for succinate importers, dccT (Youn et al., 2008) and dctA (Youn et al., 2009), and of a gene coding for a succinate exporter, sucE (Huhn et al., 2011). Based on the extensive knowledge available and the fact that anaerobic succinate production with this species has been demonstrated, a C. glutamicum strain for efficient aerobic succinate production from glucose was constructed in this work by metabolic engineering, using similar strategies as described for E. coli (Lin et al., 2005a,b; Wendisch et al., 2006). The key mutation required for succinate production was the deletion of the succinate dehydrogenase genes. Subsequent deletion of all known acetate‐producing pathways as well as overproduction of phosphoenolpyruvate carboxylase and pyruvate carboxylase led to significantly increased succinate production (Fig. 1). Finally, the production process was optimized by limiting biomass formation, thereby providing more carbon for succinate production. In summary, our results demonstrate for the first time the potential of C. glutamicum for aerobic succinate production in minimal medium.


Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum.

Litsanov B, Kabus A, Brocker M, Bott M - Microb Biotechnol (2011)

Scheme of the central metabolism of C. glutamicum showing the genetic modifications used in this work to construct a strain for aerobic succinate production. Enzymes whose genes were deleted are indicated by ‘X’. The reactions affected by these deletions and their products are displayed in grey. Enzymes whose genes were overexpressed are highlighted in grey boxes and the arrows for the corresponding reactions are thickened. Abbreviations: ACN, aconitase; AK, acetate kinase; CoAT, acetyl‐CoA:CoA transferase; CS, citrate synthase; FUM, fumarase; ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; MQO, malate:menaquinone oxidoreductase; MS, malate synthase; OAA, oxaloacetate; ODHC, 2‐oxoglutarate dehydrogenase complex; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PEPCx, PEP carboxylase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PTA, phosphotransacetylase; PQO, pyruvate:menaquinone oxidoreductase; SCS, succinyl‐CoA synthetase; SDH, succinate dehydrogenase.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815278&req=5

f1: Scheme of the central metabolism of C. glutamicum showing the genetic modifications used in this work to construct a strain for aerobic succinate production. Enzymes whose genes were deleted are indicated by ‘X’. The reactions affected by these deletions and their products are displayed in grey. Enzymes whose genes were overexpressed are highlighted in grey boxes and the arrows for the corresponding reactions are thickened. Abbreviations: ACN, aconitase; AK, acetate kinase; CoAT, acetyl‐CoA:CoA transferase; CS, citrate synthase; FUM, fumarase; ICD, isocitrate dehydrogenase; ICL, isocitrate lyase; MQO, malate:menaquinone oxidoreductase; MS, malate synthase; OAA, oxaloacetate; ODHC, 2‐oxoglutarate dehydrogenase complex; PEP, phosphoenolpyruvate; PK, pyruvate kinase; PEPCx, PEP carboxylase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PTA, phosphotransacetylase; PQO, pyruvate:menaquinone oxidoreductase; SCS, succinyl‐CoA synthetase; SDH, succinate dehydrogenase.
Mentions: In view of these facts, we started to explore the potential of C. glutamicum for aerobic succinate production. Corynebacterium glutamicum is a facultatively anaerobic, Gram‐positive soil bacterium with GRAS status (generally regarded as safe), which is used for the large‐scale production of more than 2 million tons of l‐glutamate and 1.1 million tons of l‐lysine annually. In addition, C. glutamicum strains were developed for the production of several other industrially relevant products such as putrescine (Schneider and Wendisch, 2010), isobutanol (Blombach et al., 2011) or ethanol (Inui et al., 2004a). The genome of C. glutamicum is known (Ikeda and Nakagawa, 2003; Kalinowski et al., 2003; Yukawa et al., 2007) and numerous genetic tools are available allowing genetic engineering (Kirchner and Tauch, 2003). Moreover, extensive knowledge on the central metabolism of C. glutamicum is available due to 60 years of research on amino acid production (Eggeling and Bott, 2005; Burkovski, 2008). Novel results relevant for succinate production represent the identification of two genes coding for succinate importers, dccT (Youn et al., 2008) and dctA (Youn et al., 2009), and of a gene coding for a succinate exporter, sucE (Huhn et al., 2011). Based on the extensive knowledge available and the fact that anaerobic succinate production with this species has been demonstrated, a C. glutamicum strain for efficient aerobic succinate production from glucose was constructed in this work by metabolic engineering, using similar strategies as described for E. coli (Lin et al., 2005a,b; Wendisch et al., 2006). The key mutation required for succinate production was the deletion of the succinate dehydrogenase genes. Subsequent deletion of all known acetate‐producing pathways as well as overproduction of phosphoenolpyruvate carboxylase and pyruvate carboxylase led to significantly increased succinate production (Fig. 1). Finally, the production process was optimized by limiting biomass formation, thereby providing more carbon for succinate production. In summary, our results demonstrate for the first time the potential of C. glutamicum for aerobic succinate production in minimal medium.

Bottom Line: By deleting genes for all known acetate-producing pathways (pta-ackA, pqo and cat) acetate production could be strongly reduced by 83% and succinate production increased up to 7.8 g l(-1) (66 mM).Whereas overexpression of the glyoxylate shunt genes (aceA and aceB) or overproduction of the anaplerotic enzyme pyruvate carboxylase (PCx) had only minor effects on succinate production, simultaneous overproduction of pyruvate carboxylase and PEP carboxylase resulted in a strain that produced 9.7 g l(-1) (82 mM) succinate with a specific productivity of 1.60 mmol g (cdw)(-1) h(-1).This value represents the highest productivity among currently described aerobic bacterial succinate producers.

View Article: PubMed Central - PubMed

Affiliation: Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, D-52425 Jülich, Germany.

Show MeSH
Related in: MedlinePlus