Efficient aerobic succinate production from glucose in minimal medium with Corynebacterium glutamicum.
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.
Affiliation: Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, D-52425 Jülich, Germany.Show MeSH
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Mentions: For the succinate production experiments, strain BL‐1 was transformed with each of the three above mentioned plasmids resulting in the C. glutamicum strains BL‐1/pAN6‐pycP458S, BL‐1/pAN6‐pycP458Sppc and BL‐1/pAN6‐aceAaceB. These strains were analysed and compared in independent batch cultivations with modified CGXII medium containing 4% (w/v) glucose, 25 mg l−1 kanamycin and 0.5 mM IPTG at pH 7.0 and pO2 > 30% saturation (Fig. 3). Surprisingly, overproduction of PCx had no influence on the glucose uptake rate and on succinate production compared with the parental strain BL‐1, as no significant increase of the succinate titre (68 mM), the succinate production rate [1.44 mmol g (cdw)−1 h−1] or the succinate yield (0.28 mol mol−1 glucose) was detected (Table 1). Overproduction of PCx led to a decreased growth rate (0.30 h−1) and biomass formation [12.2 g (cdw) l−1]. Acetate accumulation was decelerated, but the final acetate concentration (20 mM) reached the level of the BL‐1 strain. The combined overexpression of pycP458S and ppc in the strain BL‐1/pAN6‐ pycP458Sppc led to an increased succinate production rate [1.60 mmol g (cdw)−1 h−1] and the final succinate titre was raised about 25% to 82 mM compared with the parental strain (Table 1), which is also reflected in a 28% higher succinate yield (0.36 mol mol−1 glucose). Interestingly, although the succinate production rate was increased by 15%, the glucose consumption rate was reduced by 7% to 5.15 mmol g (cdw)−1 h−1. Overexpression of pycP458S and ppc had no effect on acetate accumulation (Table 1). The functional overexpression of aceA and aceB in BL‐1/pAN6‐aceAaceB was confirmed by in vitro enzymatic activity measurements. The specific activity of ICL (encoded by aceA) was 0.79 U mg−1 protein, the specific activity of MS (aceB) was 0.51 U mg−1 protein. The overexpression of aceA and aceB in strain BL‐1/pAN6‐aceAaceB had a negative influence on the growth rate (0.27 h−1) and on biomass formation [10.5 g (cdw) l−1] compared with the ΔsdhCAB mutant (Table 1). The glucose uptake rate was reduced compared with the BL‐1 strain [5.19 versus 5.54 mmol g (cdw)−1 h−1], whereas the succinate production rate was the same as for the BL‐1 strain [1.38 versus 1.39 mmol g (cdw)−1 h−1]. The final succinate titre (72 versus 66 mM) as well as the succinate yield (0.30 versus 0.28 mol mol−1) were slightly increased compared with the BL‐1 strain. Overproduction of ICL and MS had no influence on the final acetate titre in the supernatant although its accumulation was delayed probably due to the slower growth.
Affiliation: Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich, D-52425 Jülich, Germany.