Abstract
BackgroundButanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, Bacillus subtilis 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of B. subtilis 168 to butanol and other alcohol biocommodities.ResultsUsing comparative proteomics approach and molecular analysis of butanol-challenged B. subtilis 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline–glutamate–arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of B. subtilis 168 up to 1.8% (vol/vol) under the conditions tested.ConclusionThis work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in B. subtilis 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance B. subtilis 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of B. subtilis 168 for the production of butanol and other alcohol biocommodities.
Highlights
Butanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity
When ProHJ or GltP was overexpressed, the HK′-HJOX and HK-GPOX strains showed less susceptibility to butanol stress as the specific growth-rate inhibition was approximately at 60 and 80% when tested with 1.6 and 1.8% butanol, respectively (Fig. 7). These results indicated that increasing the expression of enzymes that are responsible for the second proline biosynthesis route (ProHJ), or a glutamate-transporting protein (GltP) considerably enhanced butanol tolerance in B. subtilis 168
While a native butanol-producing strain, Clostridium acetobutylicum, responded to butanol-challenged conditions with 102 responsive proteins that are mainly involved in amino acid metabolism and protein synthesis in addition to solvent formation-related proteins [43], other potential heterologous hosts for butanol production exhibited various numbers of differentially expressed proteins when subjected to butanol stress, the majority of which are transporters, oxidative stress response proteins, and proteins related to energy metabolism [41,42,43,44,45,46]; these include E. coli [997] [44], P. putida [138] [41], Staphylococcus warneri SG1 [108] [42], Saccharomyces cerevisiae [>300] [47], and Synechocystis sp
Summary
Butanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Butanol production can be achieved using heterologous bacterial hosts with metabolically engineered butanol synthetic pathway, such as Escherichia coli, Pseudomonas putida, Lactobacillus brevis, and Bacillus subtilis [3,4,5]. These bacteria share common traits as industrial relevant strains, such as high growth rate and genetic competency; one of the most desirable and requisite host characteristics for alcohol bioproduction is microbial tolerance to the alcohol product [6, 7]. While global and general stress response mechanisms in Gram-positive bacteria have been extensively studied by omics-based analysis [13,14,15], comprehensive study on tolerance and adaptation towards solvents as well as alcohol, especially butanol, is limited [16]
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