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BackgroundThe autotransporter (AT) system can potentially be used in the secretion of saccharolytic enzymes for the production of lignocellulosic biofuels and chemicals using Escherichia coli. Although ATs share similar structural characteristics, their capacity for secreting heterologous proteins widely varies. Additionally, the saccharolytic enzyme selected to be secreted should match the cell growth or cell fermentation conditions of E. coli.ResultsIn the search for an AT that suits the physiological performance of the homo-ethanologenic E. coli strain MS04, an expression plasmid based on the AT antigen 43 (Ag43) from E. coli was developed. The β-glucosidase BglC from the thermophile bacterium Thermobifida fusca was displayed on the outer membrane of the E. coli strain MS04 using the Ag43 system (MS04/pAg43BglC). This strain was used to hydrolyze and ferment 40 g/L of cellobiose in mineral media to produce 16.65 g/L of ethanol in 48 h at a yield of 81% of the theoretical maximum. Knowing that BglC shows its highest activity at 50°C and retains more than 70% of its activity at pH 6, therefore E. coli MS04/pAg43BglC was used to ferment crystalline cellulose (Avicel) in a simultaneous saccharification and fermentation (SSF) process using a commercial cocktail of cellulases (endo and exo) at pH 6 and at a relatively high temperature for E. coli (45°C). As much as 22 g/L of ethanol was produced in 48 h.ConclusionsThe Ag43-BglC system can be used in E. coli strains without commercial β-glucosidases, reducing the quantities of commercial enzymes needed for the SSF process. Furthermore, the present work shows that E. coli cells are able to ferment sugars at 45°C during the SSF process using 40 g/L of Avicel, reducing the gap between the working conditions of the commercial saccharolytic enzymes and ethanologenic E. coli.

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The production of biofuels, such as bioethanol from lignocellulosic biomass, is an important task within the sustainable energy concept. Understanding the metabolism of ethanologenic microorganisms for the consumption of sugar mixtures contained in lignocellulosic hydrolysates could allow the improvement of the fermentation process. In this study, the ethanologenic strain Escherichia coli MS04 was used to ferment hydrolysates from five different lignocellulosic agroindustrial wastes, which contained different glucose and xylose concentrations. The volumetric rates of glucose and xylose consumption and ethanol production depend on the initial concentration of glucose and xylose, concentrations of inhibitors, and the positive effect of acetate in the fermentation to ethanol. Ethanol yields above 80% and productivities up to 1.85 gEtOH/Lh were obtained. Furthermore, in all evaluations, a simultaneous co-consumption of glucose and xylose was observed. The effect of deleting the xyIR regulator was studied, concluding that it plays an important role in the metabolism of monosaccharides and in xylose consumption. Moreover, the importance of acetate was confirmed for the ethanologenic strain, showing the positive effect of acetate on the co-consumption rates of glucose and xylose in cultivation media and hydrolysates containing sugar mixtures.