Abstract
N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.
Highlights
Butanol can be produced from renewable feedstocks of different kinds, including agricultural waste materials, by acetone-butanolethanol (ABE) fermentation using solventogenic clostridia (Lee et al, 2008)
Ethyl methanesulfonate (EMS) was used as a mutagenic agent in combination with selection in butanol (EMS + butanol mutants), secondly, ethyl methanesulfonate (EMS) was used for mutagenesis but selection was carried out in ethidium bromide (EB) (EMS + EB mutants) and, EB was used as a mutagenic agent and strains were directly selected on agar plates containing EB without exposition to EMS (EB mutants)
wild-type strain (WTS) C. beijerinckii NRRL B-598 was tested for its tolerance to butanol, ethanol and EB
Summary
Butanol can be produced from renewable feedstocks of different kinds, including agricultural waste materials, by acetone-butanolethanol (ABE) fermentation using solventogenic clostridia (Lee et al, 2008). In C. acetobutylicum, at inhibitory levels, effects of butanol on cell membrane results in lower ATP generation by the cell, a malfunction in nutrient uptake and an inability of the cell to maintain its internal pH (Bowles and Ellefson, 1985). In Gram-negative bacteria, butanol damages the inner and outer membranes and can result in a change in cell shape (Fletcher et al, 2016). Such damage, at the cellular level, results in low culture viability and decreased growth rate and, as a consequence, low final butanol concentrations achieved after fermentation (Ingram, 1986)
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