Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink

Boecker, Simon; Espinel-Ríos, Sebastián; Bettenbrock, Katja; Klamt, Steffen

doi:10.1016/j.ymben.2022.05.006

Cited by 5 publications

(7 citation statements)

References 44 publications

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“…1 ) and produces ATP, we hypothesized that this infeasibility is due to a redox imbalance induced by the net production of some NADH equivalents when biomass is synthesized from glucose. Similar to our approach employed in [ 14 ], we introduced an artificial reaction (R_NADHox) to the network that oxidizes NADH to NAD + (R_NADHox: 1 M_nadh_c + 1 M_h_c → 1 M_nad_c) in order to verify our hypothesis and to calculate the amount of NADH that is causing this imbalance. We then repeated the calculation, this time fixing the growth rate to 0.1 h −1 and minimizing R_NADHox.…”

Section: Resultsmentioning

confidence: 99%

“…After identifying the likely cause of impaired cell growth, we aimed to find a practical mechanism that (a) resolves the redox imbalance; (b) does not reduce the high iBuOH/glucose yield; and (3) can be easily applied in E.coli . Recently, we solved a similar issue with unbalanced redox equivalents (anaerobic growth of E. coli on glycerol in minimal medium [ 14 ]) by allowing co-uptake of small amounts of acetate. Acetate was converted via acetyl-CoA to ethanol, consuming 1 mol of ATP per 1 mol of acetate and oxidizing 2 mol of NADH to NAD + .…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly (and in contrast to anaerobic growth of E. coli on glycerol [ 14 ]), the model predicts that the uptake of small amounts of acetate without its conversion to ethanol would already suffice to enable redox-balanced growth-coupled iBuOH production. In this case, uptake of acetate is only used to synthesize the biomass precursor acetyl-CoA (or α-ketoglutarate lying downstream of it), which produces one less NADH per acetyl-CoA synthesized compared to generation of acetyl-CoA from glucose.…”

Section: Resultsmentioning

confidence: 99%

“…To restore cell growth, small amounts of oxygen, other external electron acceptors (e.g., nitrate, fumarate), or complex media additives need to be provided. Recently, it was shown that the addition of small amounts of acetate also resolve the redox imbalance in minimal medium and enables growth [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

“…In line with Trinh and co-workers [ 24 , 25 ], we hypothesized that, even though the iBuOH pathway itself is redox balanced, biomass formation leads to a redox imbalance that needs to be resolved for cell growth. As already mentioned above, recently we could show that small amounts of acetate taken up by E. coli and converted to ethanol via acetyl-CoA enabled for the first time anaerobic growth of this bacterium on defined minimal medium with glycerol as substrate [ 14 ]. Here, the uptake of one molecule of acetate costs one molecule of ATP but via conversion to ethanol, two molecules of NADH are consumed.…”

Section: Introductionmentioning

confidence: 99%

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Growth-coupled anaerobic production of isobutanol from glucose in minimal medium with Escherichia coli

Boecker,

Schulze,

Klamt

2023

Biotechnol Biofuels

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Background The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs. Results In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h−1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW−1 h−1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium. Conclusions This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate–product combinations.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%