High cell density co-culture for production of recombinant hydrolases

► We showed that the concept of co-culture could be applied to produce more than one recombinant enzyme. ► We showed the successful application of high cell density cultivation to produce recombinant enzymes by co-culture. ► We developed a simple method to evaluate clone distribution during co-culture based on an enzymatic panel. Sugarcane bagasse is a residue with great potential as a feedstock for second-generation ethanol production. One of the approaches studied for making use of this material is the utilization of enzymes to hydrolyze the cell wall carbohydrates and generate fermentable sugars. These enzymes can be produced by cultivation of filamentous fungi or bacteria; however, the high production cost still represents a bottleneck to second-generation ethanol production. Expression of recombinant hydrolases through a co-culture strategy could be an interesting alternative for producing a recombinant cocktail at high levels of productivity that is tailor-made for each material to be hydrolyzed. In this study we evaluate the production of hydrolases by co-culturing two recombinant Escherichia coli, each expressing a specific hydrolase, β-1,3-1,4-glucanase or β-1,4-xylanase, both isolated from Bacillus subtilis. The cultures were conducted in bioreactors in batch and fed-batch mode in order to reach high cell densities. Co-culture in batch cultivation reached a dry cell weight of 10.4g/L and volumetric activities of 31.96U/mL and 11.89U/mL for xylanase and endoglucanase, respectively. Fed-batch cultivation reached a dry cell weight of 60g/L and the volumetric activities of xylanase and endoglucanase were respectively up to 5 and 1.3 times higher than those in batch mode. A competition assay indicates that no clone predominates over the other during cultivation. These results suggest that co-culture is a potential technique for producing recombinant hydrolase cocktails at lower cost than those associated with the production of a single culture.