Overflow metabolism is a prevalent problem for aerobic cultivations of Escherichia coli. Although several process and molecular approaches have been applied to prevent overflow metabolism, these approaches often result in reductions in growth rate, biomass yield or accumulation of other byproducts. In this report, we present an alternative approach based on increasing the efficiency of aerobic metabolism by the expression of the Vitreoscilla stercoraria hemoglobin (VHb) to avoid overflow metabolism. VHb is expected to increase the consumption of NADH in the respiratory chain, leading to increased activity of the tricarboxylic acid (TCA) cycle. This would result in a faster consumption of acetyl Co-A and a decrease in acetate production. When this strategy was tested in E. coli strains, acetate production decreased by 50% in MG1655 and more than 90% in W3110, without affecting growth rates or biomass yields. VHb expression in mutant strains with higher TCA activity and reduced acetate formation resulted in a significant increase in growth and glucose consumption rates, whereas acetate production did not increase. The results presented here show that enhancing the efficiency of aerobic metabolism is a valuable approach to avoid overflow metabolism in E. coli and to attain high cell densities in batch mode.

Operational and economic constraints in large-scale bioreactors often result in local or global microaerobic conditions, which lead to less efficient bioprocesses. Escherichia coli adapts to microaerobicity by activating fermentation pathways that accumulate acidic by-products, in detriment of growth rate (μ) and biomass yield on glucose (Y X/S ). In this study, the metabolism of E. coli was modified to better cope with microaerobicity. For that purpose, genes coding for global regulators like carbon source responsive B protein and aerobic respiratory control A protein, or for fermentative pathways were inactivated. The performance of a wild-type (W3110) and engineered E. coli strains was evaluated in batch cultures at constant low dissolved oxygen tension (3% air sat.). By combining the partial elimination of fermentation pathways and the expression of the Vitreoscilla hemoglobin (VHb), a 32% decrease on carbon waste as by-products, 24 % increase on Y X/S and 13% increase of μ were obtained. Flux balance analysis of the best strain estimated major differences in the fluxes through the pentose phosphate pathway and tricarboxylic acid cycle as consequence of VHb presence. Overall, our results show that E. coli can be genetically modified to overcome some of the disadvantages of microaerobic growth, which is potentially useful for better bioreactor scale-up and operation.

High cell-density cultivations are the preferred system for biomolecules production by Escherichia coli. It has been previously demonstrated that a strain of E. coli with a modified substrate transport system is able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. The use of elevated amounts of glucose from the beginning of the cultivation, eliminates the existence of substrate gradients due to deficient mixing at large-scale. However, the large amounts of oxygen demanded resulted in microaerobic conditions after some hours of cultivation, even at small-scale. In this work, the effect of expressing the Vitreoscilla hemoglobin (VHb) in the engineered strain during batch cultures using high-glucose concentrations was tested. Together, the expression of VHb and the modified substrate transport system resulted in a 33% increase of biomass production compared to the parental strain (W3110) lacking the VHb in batch cultivations using 25 g/L of glucose. When 50 g/L of glucose were used, expression of VHb in the modified strain led to 11% higher biomass production compared to W3110. The VHb also increased the growth rates of the strains by about 30% in the aerobic phase and more than 200% in the microaerobic phase of batch cultivation.

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