Enhancing the robustness of Clostridium saccharoperbutylacetonicum N1-4 for butanol production through metabolic engineering and cell immobilization strategies

Abstract

n-Butanol (butanol hereafter) has many advantages over ethanol to be used as a biofuel. However, one of the significant drawbacks in bacterial production of butanol is the low productivity and titer due to the low butanol tolerance in the host strain. Lignocellulosic materials are considered as cheap and renewable feedstock for biofuel production through microbial fermentation. Nevertheless, there are by-products generated during the conversion of biomass into sugar-rich hydrolysates that could inhibit bacterial growth and lead to unsatisfying fermentation performance. In this study, combinatory strategies have been investigated for improving the tolerance of strains used for butanol production, particularly focused on Clostridium saccharoperbutylacetonicum N1-4, a hyper-butanol producing strain. Firstly, the effects of two genetic engineering strategies, including the overexpression of the srp efflux pump system from Pseudomonas putida and the identification and deletion of autolysin genes, were investigated. The efflux pump could extrude toxic compounds out of the cells, and increase the maximum levels of furfural and ferulic acid (two representative lignocellulosic inhibitors) in which C. saccharoperbutylacetonicum N1-4 can survive. The deletion of four autolysin genes led to enhanced cell growth and butanol production, and meanwhile increased the plasmid DNA transformation efficiency for the mutant strain. The results also provided a better understanding about the role of the endogenous megaplasmid within C. saccharoperbutylacetonicum N1-4, and supported other recent findings. Finally, a cell immobilization strategy was evaluated for the effects of butanol fermentation, based on the cationic surfaces such as insoluble chitosan and cationized celluloses. Results indicated that chitosan promoted microgranulation, increased the amount of cell biomass present in the liquid phase, elevated the butanol productivity in C. saccharoperbutylacetonicum N1-4, and also improved the butanol titer in another prominent butanol-producing strain C. beijerinckii 8052. Overall, the results of this study provided insights towards the enhancement of biobutanol production from lignocellulosic feedstocks through integrated strain development and bioprocess development strategies.