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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BACKGROUND: This study evaluated the influence of different micro-aerated conditions, including the aeration rate, the volumetric oxygen transfer coefficient (k L a) and the oxygen transfer rate (OTR), on improving ethanol productivity and scale-up of the fermentation step of the ethanologenic Escherichia coli strain MS04 in mineral medium supplemented with xylose, glucose, and sodium acetate. RESULTS: Growth and ethanol production results using 0.75 L of fermenter showed that micro-aeration (0.1 vvm, 400 rpm) improved the volumetric ethanol productivity and sugar consumption rate compared with the anaerobic condition (0 vvm, 400 rpm) or higher aeration rates (>0.2 vvm) without reducing significantly the ethanol yield. The k L a and the OTR were estimated and a k L a value of 7.2 h −1 was used as a criterion to scale-up the fermentation process from 0.75 L to 9.16 L and 110 L. During scale-up, the volumetric ethanol productivity and ethanol yield of consumed sugars were maintained at similar levels to those obtained in the laboratory in the 0.75 L fermenter. CONCLUSIONS: The controlled supply of low levels of oxygen promoted an increase in the concentration of biomass favoring the production and volumetric productivity of ethanol. The use of k L a allowed the fermentation step to be scaled up with ethanologenic E. coli maintaining similar ethanol yields and productivities.

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