Metabolism of sucrose in a non-fermentative Escherichia coli under oxygen limitation

Karel Olavarria,Sebastian Aljoscha Wahl,Albert Fina,Mark C M Van Loosdrecht,Mariana I Velasco

doi:10.1007/s00253-019-09909-6

Karel Olavarria, Sebastian Aljoscha Wahl + Show 3 more

Open Access

https://doi.org/10.1007/s00253-019-09909-6

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Abstract

Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.

Highlights

The design of efficient and sustainable biotechnological processes demands the maximization of product titer, production rate, and yield, and the reduction of fixed and variable costs (Porro et al 2014)
E. coli K-12–derived strains can anaerobically grow using sucrose as the sole carbon source (Hoefel et al 2012), the bio-energetic impact of the oxygen limitation while using this carbon source has not been studied in detail
For enabling the sucrose uptake and catabolism in E. coli K-12, we chose the episomal expression of the foreign genes cscB and cscK from E. coli W combined with the cscA gene from E. coli W or the sucP gene from B. adolescentis

Summary

Introduction

The design of efficient and sustainable biotechnological processes demands the maximization of product titer, production rate, and yield, and the reduction of fixed and variable costs (Porro et al 2014). For the growth experiments under anaerobic conditions, the medium was supplemented with 0.24 mg/L of Na2SeO3 × 5 H2O, 0.28 mg/L Na2MoO4, and 2.3 mg/L Ni(NO3)2 × 6 H2O to provide the metals required for the activity of the formate hydrogen lyase (Soini et al 2008), and it was supplemented with 50 mM of 3-(N-morpholino)propanesulfonic acid (MOPS) to increase the buffering capacity of the medium. For these experiments, sealed bottles were used. The initial optical density of the cultures at 600 nm was 0.1

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