Butanol Tolerance Research Articles

Performance of Saccharomyces cerevisiae strains against the application of adaptive laboratory evolution strategies for butanol tolerance

Although the industrial production of butanol has been carried out for decades by bacteria of the Clostridium species, recent studies have shown the use of the yeast Saccharomyces cerevisiae as a promising alternative. While the production of n-butanol by this yeast is still very far from its tolerability (up to 2% butanol), the improvement in the tolerance can lead to an increase in butanol production. The aim of the present work was to evaluate the adaptive capacity of the laboratory strain X2180-1B and the Brazilian ethanol-producing strain CAT-1 when submitted to two strategies of adaptive laboratory Evolution (ALE) in butanol. The strains were submitted, in parallel, to ALE with successive passages or with UV irradiation, using 1% butanol as selection pressure. Despite initially showing greater tolerance to butanol, the CAT-1 strain did not show great improvements after being submitted to ALE. Already the laboratory strain X2180-1B showed an incredible increase in butanol tolerance, starting from a condition of inability to grow in 1% butanol, to the capacity to grow in this same condition. With emphasis on the X2180_n100#28 isolated colony that presented the highest maximum specific growth rate among all isolated colonies, we believe that this colony has good potential to be used as a model yeast for understanding the mechanisms that involve tolerance to alcohols and other inhibitory compounds.

Identification of membrane engineering targets for increased butanol tolerance in Clostridium saccharoperbutylacetonicum

There is a growing interest in the use of microbial cell factories to produce butanol, an industrial solvent and platform chemical. Biobutanol can also be used as a biofuel and represents a cleaner and more sustainable alternative to the use of conventional fossil fuels. Solventogenic Clostridia are the most popular microorganisms used due to the native expression of butanol synthesis pathways. A major drawback to the wide scale implementation and development of these technologies is the toxicity of butanol. Various membrane properties and related functions are perturbed by the interaction of butanol with the cell membrane, causing lower yields and higher purification costs. This is ultimately why the technology remains underemployed. This study aimed to develop a deeper understanding of butanol toxicity at the membrane to determine future targets for membrane engineering. Changes to the lipidome in Clostridium saccharoperbutylacetonicum N1–4 (HMT) throughout butanol fermentation were investigated with thin layer chromatography and mass spectrometry. By the end of fermentation, levels of phosphatidylglycerol lipids had increased significantly, suggesting an important role of these lipid species in tolerance to butanol. Using membrane models and in vitro assays to investigate characteristics such as permeability, fluidity, and swelling, it was found that altering the composition of membrane models can convey tolerance to butanol, and that modulating membrane fluidity appears to be a key factor. Data presented here will ultimately help to inform rational strain engineering efforts to produce more robust strains capable of producing higher butanol titres.

Engineering E. coli to synthesize butanol.

Biobutanol is gaining much attention as a potential biofuel due to its superior properties over ethanol. Butanol has been naturally produced via acetone-butanol-ethanol (ABE) fermentation by many Clostridium species, which are not very user-friendly bacteria. Therefore, to improve butanol titers and yield, various butanol synthesis pathways have been engineered in Escherichia coli, a much more robust and convenient host than Clostridium species. This review mainly focuses on the biosynthesis of n-butanol in engineered E. coli with an emphasis on efficient enzymes for butanol production in E. coli, butanol competing pathways, and genome engineering of E. coli for butanol production. In addition, the use of alternate strategies for butanol biosynthesis/enhancement, alternate substrates for the low cost of butanol production, and genetic improvement for butanol tolerance in E. coli have also been discussed.

Enhanced production and in-situ removal of butanol during the fermentation of lignocellulosic hydrolysate of pineapple leaves

The pineapple leaves waste was exploited as a potential biomass for butanol production. The cocktail of lignocellulolytic enzymes secreted by Sphingobacterium sp. ksn- 11 employed for the saccharification of steam pretreated pineapple leaves (100 g) which produced fermentable sugars (42.89 g) and polyphenols in the hydrolysate. The polyphenols were removed selectively by using residual cornhusk bed from the hydrolysate and subjected to fermentation by Clostridium acetobutylicum NCIM 2337. The butanol tolerance and production capacity of Clostridium acetobutylicum NCIM 2337 were enhanced by supplementing histidine and proline (0.5 %) to the fermentation medium by 1.8-fold and observed increase in butanol production (4.63 g/L). In order to minimize the sub-inhibitory effect of butanol, in-situ product recovery was carried out by the addition of Dowex L-493 resin which enhanced the butanol production (6.73 g/L) by 2.6-fold and achieved maximum yield of 0.16 g/g. Thus, the one pot step optimized process for butanol production was achieved.

Clostridium cellulovorans Proteomic Responses to Butanol Stress.

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

Butanol production from Saccharina japonica hydrolysate by engineered Clostridium tyrobutyricum: The effects of pretreatment method and heat shock protein overexpression

Macroalgal biomass is currently considered as a potential candidate for biofuel production. In this study, the effects of pretreatment method and heat shock protein overexpression were investigated for efficient butanol production from Saccharina japonica using engineered Clostridium tyrobutyricum. First, various pretreatment methods including acid hydrolysis, acid hydrolysis and enzymatic saccharification, and ultrasonic-assisted acid hydrolysis were employed to obtain the fermentable sugars, and the resulted hydrolysates were evaluated for butanol fermentation. The results showed that ultrasonic-assisted acid hydrolysate obtained the highest butanol yield (0.26 g/g) and productivity (0.19 g/L⋅h). Then, the effects of homologous or heterologous heat shock protein overexpression on butanol production and tolerance were examined. Among all the engineered strains, Ct-pMA12G exhibited improved butanol tolerance and enhanced butanol production (12.15 g/L butanol with a yield of 0.34 g/g and productivity of 0.15 g/L⋅h) from 1.8-fold concentrated S. japonica hydrolysate, which was the highest level ever reported for macroalgal biomass.

Improvements in the formulation of sugarcane-sweet sorghum juices fermentation media for enhanced isopropanol and butanol production

The production of isopropanol and butanol (IB) has been gaining attention through isopropanol-butanol-ethanol (IBE) fermentation. In this work, the substrate inhibition and butanol tolerance of Clostridium beijerinckii DSM 6423 in isopropanol-butanol (IB) production by isopropanol-butanol-ethanol (IBE) fermentation from an industrial mixture of sugarcane and sweet sorghum juices were assessed. The results showed that an initial sugar concentration of 40 g/L in the industrial juices showed the best kinetic for the fermentation. The formulation of a low-cost medium for IB production from industrial juices by C. beijerinckii DSM 6423 was also studied. Corn steep liquor (CSL), a byproduct of the wet milling of corn, could serve as a nutrient of low cost and replace both yeast extract and vitamin complex without significantly affecting the production of butanol and isopropanol. A CSL concentration of 5 g/L was shown to be adequate to achieve the best results. C. beijerinckii DSM 6423 produced 5.3 and 7.9 g/L of isopropanol and butanol, respectively, with a sugar conversion of 93%. This work provides valuable insights into cost-effective nutritional supplementation of industrial juices for IB production.

Proteomic Analysis Identifies Dysregulated Proteins in Butanol-Tolerant Gram-Positive Lactobacillus mucosae BR0713-33.

Butanol can be produced biologically through fermentation of lignocellulosic biomass-derived sugars by Gram-positive Clostridium species. For cost-effective production, increased butanol fermentation titers are desired. However, the currently available butanol-fermenting microbes do not tolerate sufficiently high butanol concentrations; thus, new butanol-tolerant strains are desired. One promising strategy is to genetically modify Clostridium species by introducing stress tolerance-associated genes. This study was aimed to seek butanol tolerance genes from other Gram-positive species, which might be better suited than those from Gram-negative E. coli or eukaryotic Saccharomyces cerevisiae. Several butanol-tolerant lactobacilli were reported previously, and Lactobacillus mucosae BR0713–33, which showed the most robust anaerobic growth in 4% butanol, was used here for proteomics analyses. Cellular proteins that responded to 2, 3, and 4% butanol were characterized. Twenty-nine proteins that were identified were dysregulated in response to increased concentrations of butanol in L. mucosae. Seventeen genes involved in coding for stress-tolerant proteins GroES, GroEL, and DnaK and genes involved in substrate utilization, fatty acid metabolism, and nucleotide synthesis were induced by increased butanol, and 12 genes involving energy production (F0F1ATP synthases) and redox balance preservation were repressed by increased butanol. These results can help guide targeted engineering strategies to improve tolerance and production of biobutanol.

Butanol Tolerance of Lactiplantibacillus plantarum: A Transcriptome Study.

Biobutanol is a promising alternative fuel with impaired microbial production thanks to its toxicity. Lactiplantibacillus plantarum (L. plantarum) is among the few bacterial species that can naturally tolerate 3% (v/v) butanol. This study aims to identify the genetic factors involved in the butanol stress response of L. plantarum by comparing the differential gene expression in two strains with very different butanol tolerance: the highly resistant Ym1, and the relatively sensitive 8-1. During butanol stress, a total of 319 differentially expressed genes (DEGs) were found in Ym1, and 516 in 8-1. Fifty genes were upregulated and 54 were downregulated in both strains, revealing the common species-specific effects of butanol stress: upregulation of multidrug efflux transporters (SMR, MSF), toxin-antitoxin system, transcriptional regulators (TetR/AcrR, Crp/Fnr, and DeoR/GlpR), Hsp20, and genes involved in polysaccharide biosynthesis. Strong inhibition of the pyrimidine biosynthesis occurred in both strains. However, the strains differed greatly in DEGs responsible for the membrane transport, tryptophan synthesis, glycerol metabolism, tRNAs, and some important transcriptional regulators (Spx, LacI). Uniquely upregulated in the butanol-resistant strain Ym1 were the genes encoding GntR, GroEL, GroES, and foldase PrsA. The phosphoenolpyruvate flux and the phosphotransferase system (PTS) also appear to be major factors in butanol tolerance.

Energy-efficient butanol production by Clostridium acetobutylicum with histidine kinase knockouts to improve strain tolerance and process robustness

Engineering histidine kinases in C. acetobutylicum enhanced cell viability and solventogenesis in ABE fermentation and enabled robust and energy-efficient butanol production.

Effects of Carbon Ion Beam Irradiation on Butanol Tolerance and Production of Clostridium acetobutylicum.

Clostridium acetobutylicum (C. acetobutylicum) has considerable potential for use in bioenergy development. Owing to the repeated use of traditional mutagenesis methods, the strains have developed a certain tolerance. The rheology of the bioprocess and the downstream processing of the product heavily depend on the ability of C. acetobutylicum mutants to produce butanol. Carbon ion beam irradiation has advantages over traditional mutation methods for fermentative production because of its dose conformity and superb biological effectiveness. However, its effects on the specific productivity of the strains have not been clearly understood. In this study, we screened five mutants through carbon ion beam irradiation; mutant Y217 achieved a butanol-production level of 13.67 g/L, exceeding that of wild-type strain ATCC 824 (i.e., 9.77 g/L). In addition, we found that the mutant maintained normal cell membrane integrity under the stimulation of 15 g/L butanol, whereas the intracellular macromolecules of wild-type strain ATCC 824 leaked significantly. Subsequently, we used the response surface methodology (RSM) to determine if the mutant cell membrane integrity improved the butanol tolerance. We verified that with the addition of butanol, the mutant could be fermented to produce 8.35 g/L butanol, and the final butanol concentration in the fermentation broth could reach 16.15 g/L. In this study, we proved that under butanol stress, mutant Y217 features excellent butanol production and tolerance and cell membrane integrity and permeability; no prior studies have attempted to do so. This will serve as an interesting and important illustration of the complexity of genetic control of the irradiation mutation of C. acetobutylicum strains. It may also prove to be useful in the bioengineering of strains of the mutant for use in the predevelopment stage.

Optimization of n-butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter.

N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, β-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria.

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance.

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.

Genetic manipulation of non‐solvent‐producing microbial species for effective butanol production

AbstractButanol is widely used as an important bulk chemical and is a potential biofuel. The depletion of fossil fuels and advances in synthetic biotechnology have led to renewed interest in the biological production of butanol. Solventogenic Clostridium was commonly used to produce butanol through traditional acetone‐butanol‐ethanol (ABE) fermentation. However, its relatively slow growth rate, low butanol tolerance, and poor production efficiency have hindered the further application of this procedure. Recently, other promising industrial hosts, including Escherichia coli, Saccharomyces cerevisiae, and Clostridium tyrobutyricum, have been studied for potential use in the production of butanol. This review comprehensively summarizes the advantages and challenges of different non‐solvent strains for butanol production to identify better the ideal non‐solvent hosts for butanol production. Strategies to further increase butanol production are also proposed. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd.

A Novel Butanol Tolerance-Promoting Function of the Transcription Factor Rob in Escherichia coli.

Producing high concentrations of biobutanol is challenging, primarily because of the toxicity of butanol toward cells. In our previous study, several butanol tolerance-promoting genes were identified from butanol-tolerant Escherichia coli mutants and inactivation of the transcriptional regulator factor Rob was shown to improve butanol tolerance. Here, the butanol tolerance characteristics and mechanism regulated by inactivated Rob are investigated. Comparative transcriptome analysis of strain DTrob, with a truncated rob in the genome, and the control BW25113 revealed 285 differentially expressed genes (DEGs) to be associated with butanol tolerance and categorized as having transport, localization, and oxidoreductase activities. Expression of 25 DEGs representing different functional categories was analyzed by quantitative reverse transcription PCR (qRT-PCR) to assess the reliability of the RNA-Seq data, and 92% of the genes showed the same expression trend. Based on functional complementation experiments of key DEGs, deletions of glgS and yibT increased the butanol tolerance of E. coli, whereas overexpression of fadB resulted in increased cell density and a slight increase in butanol tolerance. A metabolic network analysis of these DEGs revealed that six genes (fadA, fadB, fadD, fadL, poxB, and acs) associated with acetyl-CoA production were significantly upregulated in DTrob, suggesting that Rob inactivation might enhance butanol tolerance by increasing acetyl-CoA. Interestingly, DTrob produced more acetate in response to butanol stress than the wild-type strain, resulting in the upregulation expression of some genes involved in acetate metabolism. Altogether, the results of this study reveal the mechanism underlying increased butanol tolerance in E. coli regulated by Rob inactivation.

A new process for separating biofuel based on the salt + 1-butanol + water system

Compared with bioethanol, biobutanol with higher energy density and lower vapor pressure is more suitable as a fuel for motor engines. However, the typical low acetone-butanol-ethanol (ABE) concentration hinders the competitiveness of biobutanol because of 1-butanol tolerance. In addition, the dehydration of 1-butanol involves the separation of 1-butanol + water heterogeneous azeotrope, which was usually achieved in a two-column distillation + decantation system. A new process based on the determination of the salt + 1-butanol + water system was proposed to separate biobutanol from a 1-butanol + water system. The salting out resulted in the formation of an organic solvent-rich phase and an aqueous phase. All the 1-butanol in the water-rich phase was recovered to the organic phase, and most of the water in the 1-butanol-rich phase was attracted to the aqueous phase by the salting-out effects of the electrolytes, namely potassium carbonate, dipotassium hydrogen phosphate, tripotassium phosphate, and potassium pyrophosphate. This effect produces a top phase containing greater than 97% salt-free 1-butanol. The salting-out effect, the solubility, and the solvation effect of an electrolyte play essential roles in the separation of 1-butanol.

Reverse engineering of fatty acid-tolerant Escherichia coli identifies design strategies for robust microbial cell factories

Adaptive laboratory evolution is often used to improve the performance of microbial cell factories. Reverse engineering of evolved strains enables learning and subsequent incorporation of novel design strategies via the design-build-test-learn cycle. Here, we reverse engineer a strain of Escherichia coli previously evolved for increased tolerance of octanoic acid (C8), an attractive biorenewable chemical, resulting in increased C8 production, increased butanol tolerance, and altered membrane properties. Here, evolution was determined to have occurred first through the restoration of WaaG activity, involved in the production of lipopolysaccharides, then an amino acid change in RpoC, a subunit of RNA polymerase, and finally mutation of the BasS-BasR two component system. All three mutations were required in order to reproduce the increased growth rate in the presence of 20 mM C8 and increased cell surface hydrophobicity; the WaaG and RpoC mutations both contributed to increased C8 titers, with the RpoC mutation appearing to be the major driver of this effect. Each of these mutations contributed to changes in the cell membrane. Increased membrane integrity and rigidity and decreased abundance of extracellular polymeric substances can be attributed to the restoration of WaaG. The increase in average lipid tail length can be attributed to the RpoCH419P mutation, which also confers tolerance to other industrially-relevant inhibitors, such as furfural, vanillin and n-butanol. The RpoCH419P mutation may impact binding or function of the stringent response alarmone ppGpp to RpoC site 1. Each of these mutations provides novel strategies for engineering microbial robustness, particularly at the level of the microbial cell membrane.

A novel regulatory pathway consisting of a two-component system and an ABC-type transporter contributes to butanol tolerance in Clostridium acetobutylicum.

Despite the long-term interest in solventogenic clostridia-based ABE (acetone-butanol-ethanol) fermentation, clostridial butanol tolerance and its underlying mechanism remain poorly understood, which is a major obstacle hindering further improvements of this important fermentative process. In this study, a two-component system (TCS), BtrK/BtrR, was identified and demonstrated to positively regulate butanol tolerance and ABE solvent formation in Clostridium acetobutylicum, a representative species of solventogenic clostridia. The transcriptomic analysis results showed that BtrK/BtrR has a pleiotropic regulatory function, affecting a large number of crucial genes and metabolic pathways. Of the differentially expressed genes, btrTM, encoding a putative ABC-type transporter (named BtrTM), was shown to be under the direct control of BtrR, the response regulator of the BtrK/BtrR TCS. Furthermore, BtrTM was shown to contribute to more butanol tolerance (46.5% increase) by overexpression, revealing a novel regulatory mechanism consisting of the BtrK/BtrR TCS and the BtrTM transporter in C. acetobutylicum. Based on these findings, we achieved faster growth and solvent production of C. acetobutylicum by overexpressing BtrK/BtrR or its direct target BtrTM, although no significant improvement in the final butanol titer and yield. These results further confirm the importance of BtrK/BtrR and BtrTM in this organism. Also, of significance, a specific number of btrR-btrT-btrM-btrK-like gene clusters were identified in other Clostridium species, including the pathogens Clostridium perfringens and Clostridium botulinum, indicating a broad role for this regulatory module in the class Clostridia.

Investigation of secondary metabolism in the industrial butanol hyper-producer Clostridium saccharoperbutylacetonicum N1-4.

Clostridium saccharoperbutylacetonicum N1-4 (Csa) is a historically significant anaerobic bacterium which can perform saccharolytic fermentations to produce acetone, butanol, and ethanol (ABE). Recent genomic analyses have highlighted this organism's potential to produce polyketide and nonribosomal peptide secondary metabolites, but little is known regarding the identity and function of these metabolites. This study provides a detailed bioinformatic analysis of seven biosynthetic gene clusters (BGCs) present in the Csa genome that are predicted to produce polyketides/nonribosomal peptides. An RNA-seq-based untargeted transcriptomic approach revealed that five of seven BGCs were expressed during ABE fermentation. Additional characterization of a highly expressed nonribosomal peptide synthetase gene led to the discovery of its associated metabolite and its biosynthetic pathway. Transcriptomic analysis suggested an association of this nonribosomal peptide synthetase gene with butanol tolerance, which was supported by butanol challenge assays.

The biological mechanisms of butanol tolerance and the application of solvent‐tolerant bacteria for environmental protection

AbstractToxicity caused by the accumulation of butanol in fermentation media is an important factor limiting the concentration of butanol. There is currently no systematic research in place investigating the butanol tolerance mechanism of bacteria such as Clostridium acetobutylicum, which adapts to butanol stress and regulates its growth and metabolism. Here, research results related to the butanol tolerance of C. acetobutylicum are reviewed to understand the molecular basis of changes in butanol‐tolerant strains. Organic solvent‐tolerant bacteria play an important role in the fields of biofuel production, enzyme preparation and bioremediation. An analysis of limitations of the application of organic solvent‐tolerant bacteria has revealed that future research should focus on combining the microbial tolerance phenotype with specific utilization to achieve an optimal balance between organic solvent tolerance and production. This review serves as a reference for the improvement and engineering of strains that tolerate organic solvents. © 2019 Society of Chemical Industry