Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress.

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

Design of electron-donating group substituted 2-PAM analogs as antidotes for organophosphate insecticide poisoning.

The use of organophosphate (OPs) pesticides is widespread in agriculture and horticulture, but these chemicals can be lethal to humans, causing fatalities and deaths each year. The inhibition of acetylcholinesterase (AChE) by OPs leads to the overstimulation of cholinergic receptors, ultimately resulting in respiratory arrest, seizures, and death. Although 2-pralidoxime (2-PAM) is the FDA-approved drug for treating OP poisoning, there is difficulty in blood-brain barrier permeation. To address this issue, we designed and evaluated a series of 2-PAM analogs by substituting electron-donating groups on the para and/or ortho positions of the pyridinium core using in silico techniques. Our PCM-ONIOM2 (MP2/6-31G*:PM7//B3LYP/6-31G*:UFF) binding energy results demonstrated that 13 compounds exhibited higher binding energy than 2-PAM. The analog with phenyl and methyl groups substituted on the para and ortho positions, respectively, showed the most favorable binding characteristics, with aromatic residues in the active site (Y124, W286, F297, W338, and Y341) and the catalytic residue S203 covalently bonding with paraoxon. The results of DS-MD simulation revealed a highly favorable apical conformation of the potent analog, which has the potential to enhance reactivation of AChE. Importantly, newly designed compound demonstrated appropriate drug-likeness properties and blood-brain barrier penetration. These results provide a rational guide for developing new antidotes to treat organophosphate insecticide toxicity.

Co-application of citric acid and Nocardiopsis sp. strain RA07 enhances phytoremediation potentiality of Sorghum bicolor L.

This study investigated the combined effects of citric acid (CA) and Nocardiopsis sp. RA07 on the phytoremediation potential of lead (Pb)- and copper (Cu)-contaminated soils by Sorghum bicolor L. The strain RA07 was able to tolerate Pb and Cu, and exhibited plant growth-promoting features like siderophore production, indole-3-acetic acid (IAA) synthesis, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and phosphate solubilization. The combined application of CA and strain RA07 significantly increased S. bicolor growth, chlorophyll content and antioxidant enzymatic activity, and decreased oxidative stress (hydrogen peroxide and malondialdehyde content) under Pb and Cu stress circumstances as compared to individual treatments (i.e., CA and strain RA07). Furthermore, the combined application of CA and RA07 significantly enhanced S. bicolor ability to accumulate Pb and Cu by 64.41% and 60.71% in the root and 188.39% and 125.56% in the shoot, respectively, as compared to the corresponding uninoculated plants. Our results indicate that inoculation of Nocardiopsis sp. together with CA could be a useful practical approach to mitigate Pb and Cu stress on plant growth and increase the effectiveness of phytoremediation in Pb- and Cu-polluted soils.

Bioengineering of biowaste to recover bioproducts and bioenergy: A circular economy approach towards sustainable zero-waste environment.

The inevitable need for waste valorisation and management has revolutionized the way in which the waste is visualised as a potential biorefinery for various product development rather than offensive trash. Biowaste has emerged as a potential feedstock to produce several value-added products. Bioenergy generation is one of the potential applications originating from the valorisation of biowaste. Bioenergy production requires analysis and optimization of various parameters such as biowaste composition and conversion potential to develop innovative and sustainable technologies for most effective utilization of biowaste with enhanced bioenergy production. In this context, feedstocks, such as food, agriculture, beverage, and municipal solid waste act as promising resources to produce renewable energy. Similarly, the concept of microbial fuel cells employing biowaste has clearly gained research focus in the past few decades. Despite of these potential benefits, the area of bioenergy generation still is in infancy and requires more interdisciplinary research to be sustainable alternatives. This review is aimed at analysing the bioconversion potential of biowaste to renewable energy. The possibility of valorising underutilized biowaste substrates is elaborately presented. In addition, the application and efficiency of microbial fuel cells in utilizing biowaste are described in detail taking into consideration of its great scope. Furthermore, the review addresses the significance bioreactor development for energy production along with major challenges and future prospects in bioenergy production. Based on this review it can be concluded that bioenergy production utilizing biowaste can clearly open new avenues in the field of waste valorisation and energy research. Systematic and strategic developments considering the techno economic feasibilities of this excellent energy generation process will make them a true sustainable alternative for conventional energy sources.

Microbial engineering strategies for synthetic microplastics clean up: A review on recent approaches.

Microplastics are the small fragments of the plastic molecules which find their applications in various routine products such as beauty products. Later, it was realized that it has several toxic effects on marine and terrestrial organisms. This review is an approach in understanding the microplastics, their origin, dispersal in the aquatic system, their biodegradation and factors affecting biodegradation. In addition, the paper discusses the major engineering approaches applied in microbial biotechnology. Specifically, it reviews microbial genetic engineering, such as PET-ase engineering, MHET-ase engineering, and immobilization approaches. Moreover, the major challenges associated with the plastic removal are presented by evaluating the recent reports available.

Rhodotorula mucilaginosa CAM4 improved selenium uptake in Spinacia oleracea L. and soil enzymatic activities under abiotic stresses.

This study aimed to examine selenium (Se) acquisition by spinach (Spinacia oleracea L.) plants growing under salinity and drought stress through the inoculation of Rhodotorula mucilaginosa strain CAM4. Under abiotic stress conditions, strain CAM4 with Se inoculation increased the shoot length, root length, shoot dry weight and root dry weight by 75.8-93.7%, 47.7-80.9%, 101.9-109.8% and 130.5-270.2%, respectively compared to uninoculated Se-treated plants grown under the same conditions. Under abiotic stresses, the Se-treated CAM4 inoculated plants showed a significant increase in Se concentration in the edible leaves of spinach, which was 227.3-234.5% higher than uninoculated Se-treated control plants. Likewise, strain CAM4 treatment significantly enhanced the plant nutrition of both micro and macro-nutrients. Under normal and abiotic stresses, CAM4 inoculation enhanced soil activities of acid phosphatase, alkaline phosphatase, dehydrogenase, ß-glucosidase and urease in the post harvested soil up to 28-47.5%, 62.6-121.8%, 69-177.1%, 16.2-37.9% and 19.8-41.2%, respectively over corresponding uninoculated soil.

Prediction of the Glass Transition Temperature in Polyethylene Terephthalate/Polyethylene Vanillate (PET/PEV) Blends: A Molecular Dynamics Study.

Polyethylene terephthalate (PET) is one of the most common polymers used in industries. However, its accumulation in the environment is a health risk to humans and animals. Polyethylene vanillate (PEV) is a bio-based material with topological, mechanical, and thermal properties similar to PET, allowing it to be used as a PET replacement or blending material. This study aimed to investigate some structural and dynamical properties as well as the estimated glass transition temperature (Tg) of PET/PEV blended polymers by molecular dynamics (MD) simulations with an all-atom force field model. Four blended systems of PET/PEV with different composition ratios (4/1, 3/2, 2/3, and 1/4) were investigated and compared to the parent polymers, PET and PEV. The results show that the polymers with all blended ratios have Tg values around 344-347 K, which are not significantly different from each other and are close to the Tg of PET at 345 K. Among all the ratios, the 3/2 blended polymer showed the highest number of contacting atoms and possible hydrogen bonds between the two chain types. Moreover, the radial distribution results suggested the proper interactions in this system, which indicates that this is the most suitable ratio model for further experimental studies of the PET/PEV polymer blend.

Biodegradation of 4-nitroaniline by novel isolate Bacillus sp. strain AVPP64 in the presence of pesticides.

In this study, Bacillus sp. strain AVPP64 was isolated from diuron-contaminated soil. It showed 4-nitroaniline (4-NA) degradation, pesticide tolerance, and self-nutrient integration via nitrogen (N)-fixation and phosphate (P)-solubilization. The rate constant (k) and half-life period (t1/2) of 4-NA degradation in the aqueous medium inoculated with strain AVPP64 were observed to be 0.445 d-1 and 1.55 d, respectively. Nevertheless, in the presence of chlorpyrifos, profenofos, atrazine and diuron pesticides, strain AVPP64 degraded 4-NA with t1/2 values of 2.55 d, 2.26 d, 2.31 d and 3.54 d, respectively. The strain AVPP64 fixed 140 µg mL-1 of N and solubilized 103 µg mL-1 of P during the presence of 4-NA. In addition, strain AVPP64 produced significant amounts of plant growth-promoting metabolites like indole 3-acetic acid, siderophores, exo-polysaccharides and ammonia. In the presence of 4-NA and various pesticides, strain AVPP64 greatly increased the growth and biomass of Vigna radiata and Crotalaria juncea plants. These results revealed that Bacillus sp. strain AVPP64 can be used as an inoculum for bioremediation of 4-NA contaminated soil and sustainable crop production even when pesticides are present.

Plant growth-promoting actinobacterial inoculant assisted phytoremediation increases cadmium uptake in Sorghum bicolor under drought and heat stresses.

In this study, two proficient Cadmium (Cd) resistant and plant growth-promoting actinobacterial strains were isolated from metal-polluted soils and identified as Streptomyces sp. strain RA04 and Nocardiopsis sp. strain RA07. Multiple abiotic stress tolerances were found in these two actinobacterial strains, including Cd stress (CdS), drought stress (DS) and high-temperature stress (HTS). Both actinobacterial strains exhibited multifarious plant growth-promoting (PGP) traits such as phosphate solubilization, and production of indole-3-acetic acid, siderophores and 1-aminocyclopropane-1-carboxylate deaminase under CdS, DS and HTS conditions. The inoculation of strains RA04 and RA07 significantly increased Sorghum bicolor growth and photosynthetic pigments under CdS, DS, HTS, CdS + DS and CdS + HTS conditions as compared to their respective uninoculated plants. The actinobacterial inoculants reduced malondialdehyde concentration and enhanced antioxidant enzymes in plants cultivated under various abiotic stress conditions, indicating that actinobacterial inoculants reduced oxidative damage. Furthermore, strains RA04 and RA07 enhanced the accumulation of Cd in plant tissues and the translocation of Cd from root to shoot under CdS, CdS + DS and CdS + HTS treatments as compared to their respective uninoculated plants. These findings suggest that RA04 and RA07 strains could be effective bio-inoculants to accelerate phytoremediation of Cd polluted soil even in DS and HTS conditions.

All-Atom Molecular Dynamics Simulations on a Single Chain of PET and PEV Polymers.

Polyethylene vanillic (PEV), a bio-based material, has mechanical and thermal properties similar to polyethylene terephthalate (PET), the most common polymer used in industries. The present study aimed to investigate and compare their structural dynamics and physical data using a computational approach. The simple model of a single-chain polymer containing 100 repeating units was performed by all-atom molecular dynamics (MD) simulations with refined OPLS-AA force field parameters. As a result, the flexibility of the PEV structure was greater than that of PET. PET and PEV polymers had the predicted glass transition temperature Tg values of approximately 345 K and 353 K, respectively. PEV showed a slightly higher Tg than PET, consistent with current experimental evidence.

Bioaugmentation of triclocarban and its dechlorinated congeners contaminated soil with functional degraders and the bacterial community response.

Partial removal of haloaromatic antimicrobial triclocarban (TCC) during wastewater treatment caused the final introduction of residual TCC into soils. Bioaugmentation has been proposed for the biodegradation of TCC and its dechlorinated congeners 4,4'-dichlorocarbanilide (DCC) and carbanilide (NCC) in soil. The isolated TCC-degrading strain Ochrobactrum sp. TCC-2 and chloroanilines-degrading strain Diaphorobacter sp. LD72 were used to study the removal efficiency of TCC, DCC and NCC mixture and their chloroanilines intermediates, respectively. The potential degradation competition between TCC and its dechlorinated congeners, and the response of bacterial community during the bioremediation were also investigated. The biodegradation of DCC and TCC was significantly enhanced for soil with inoculums compared with sterilized and natural soils. Chloroanilines products could also be effectively removed. For the degradation of combined substrates in the aqueous medium, NCC had negative effect on the degradation of TCC and DCC, while TCC and DCC negatively influenced each other. The bioaugmentation with two degraders obviously changed the phylogenetic composition and function of indigenous soil microbiome. Importantly, the inoculated degraders could be maintained, suggesting their adaptability and potential application in bioaugmentation for such recalcitrant contaminants. This study offers new insights into the enhanced bioremediation of TCC and its dechlorinated congeners contaminated soils by the bioaugmentation of functional degraders and the structure and function response of the indigenous soil microbiome to the bioremediation process.

Aerobic and anoxic degradation and detoxification of profenofos insecticide by Pseudomonas plecoglossicida strain PF1.

Profenofos insecticide is one of the most broadly used organophosphorus pesticides causing the contamination of soil and groundwater. Since dissolved oxygen concentration in groundwater is limited, this study aimed to investigate profenofos biodegradation and detoxification under aerobic and anoxic conditions using the profenofos-degrading Pseudomonas plecoglossicida strain PF1 (PF1). Anoxic biodegradation under the presence of nitrate was the focus. The results showed that profenofos at 10-150 mg/L was degraded under aerobic and anoxic conditions with removal efficiencies of 38-55% and 27-45%, respectively. Kinetic analysis following the Michaelis-Menten model revealed that the maximum substrate degradation rates and the Michaelis constants were 13.07 and 8.92 mg/L/d and 92.07 and 84.76 mg/L under aerobic and anoxic conditions, respectively. The culture preferred an aerobic environment resulting in better biodegradation performance. During the degradation experiment, 4-bromo-2-chlorophenol and 1,1-dimethylethylphenol were detected as profenofos biodegradation intermediate products. Microbial toxicity, phytotoxicity, and cytogenotoxicity assays showed that the toxicity of the contaminated water significantly decreased after both aerobic and anoxic biodegradation by PF1. The results from this study indicated that PF1 has the potential for bioremediation in a profenofos-contaminated environment under the presence or absence of oxygen.

An alternative genome-integrated method for undomesticated Bacillus subtilis and related species.

Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering.

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).

The effects of disruption in membrane lipid biosynthetic genes on 1-butanol tolerance of Bacillus subtilis.

Microbes with enhanced 1-butanol tolerance have the potentials to be utilized in various biotechnological processes. To achieve the rational design of such strains, we previously conducted an untargeted metabolomics analysis of Bacillus subtilis under 1-butanol stress and uncovered a novel type of microbial responses as the alterations in the glycerolipid and phospholipid composition. However, the current knowledge about the relevance of these changes on 1-butanol tolerance remains quite limited. Here, we constructed the B. subtilis mutants with disruption in the pssA, ugtP (U), mprF (M), yfnI, and yfnI/mprF genes in the membrane lipid biosynthetic pathways. The 1-butanol tolerance test indicated markedly increased and decreased 1-butanol resistance in M and U compared to the wild-type strain, respectively, and slight effects in other strains under high stress level. Further examination of the lipid contents of these strains in the presence of 1-butanol by liquid chromatography-mass spectrometry demonstrated an elevated ratio of neutral and anionic to cationic lipids in direct relation with an improved 1-butanol tolerance. Last, cell morphological studies showed the shortening of only the U cells, compared to the wild-type. All strains including U were capable of elongating by 14-24% under 1-butanol stress. Together, the studies indicated the involvement of membrane lipid biosynthetic genes, which regulated glycerolipid and phospholipid composition, on 1-butanol tolerance and allowed for the procurement of M with enhanced 1-butanol tolerance trait, highlighting the usefulness of the overall approaches on discovery of novel biological insights and engineering of microorganisms with desired resistance characteristics.

Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans.

Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452-459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5'-UTR was higher than that using a plasmid with a short 5'-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5'-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.

Efficiency of the formulated plant-growth promoting Pseudomonas fluorescens MC46 inoculant on triclocarban treatment in soil and its effect on Vigna radiata growth and soil enzyme activities.

For bioaugmentation-based treatment of triclocarban (TCC), an emerging soil pollutant that is recalcitrant to biodegradation and phytotransformation, efficient TCC-degrading bacteria with an effective soil-delivering means are required. This work developed the formulated bacterial inoculant, and successfully demonstrated its TCC removal and detoxification performance in pot soil experiment with Vigna radiata plants. The soil bacterium Pseudomonas fluorescens MC46 was isolated as TCC-degrading, plant-growth promoting bacterium. The characterizations were conducted in vitro revealing that it could utilize TCC as a sole carbon source, and at a wide and higher concentration range from 1.6-31.6mgkg-1 than those previously reported, while the detoxification was assessed by cytogenotoxicity and phytotoxicity tests. The developed sawdust-based inoculant formula combined with molasses (5% w/w), and either PEG or CMC-starch blend (1% w/w) could maintain a 20-week shelf-life inoculant stability in terms of cell viability, and TCC-degrading activity. Bioaugmentation of the formulated inoculants into TCC-contaminated soil efficiently removed TCC up to 74-76% of the initial concentration, mitigated toxicity, restored plant growth and health, and enhanced soil enzyme activities. This work is the first to demonstrate potential application of the formulated plant-growth promoting bacterial inoculant for the treatment and detoxification of a persistent TCC contaminated in soil.

Membrane Alterations in Pseudomonas putida F1 Exposed to Nanoscale Zerovalent Iron: Effects of Short-Term and Repetitive nZVI Exposure.

In this study, we report the effect of the commercial nanoscale zerovalent iron (nZVI) on environmental bacteria, emphasizing the importance of nZVI-bacterial membrane interaction on nZVI toxicity as well as the adaptability of bacteria to nZVI. Exposure of Pseudomonas putida F1 to 0.1, 1.0, and 5.0 g/L of nZVI caused the reduction in colony forming units (CFUs) substantially for almost 3 orders of magnitude. However, a rebound in the cell number was observed after the prolonged exposure except for 5.0 g/L nZVI at which bacterial viability was completely inhibited. Upon exposure, nZVI accumulated on and penetrated into the bacterial cell membrane. Cell membrane composition analysis revealed the conversion of the cis to trans isomer of unsaturated fatty acid upon short-term nZVI exposure, resulting in a more rigid membrane counteracting the membrane-fluidizing effect of nZVI. Several cycles of repetitive exposure of cells to 0.1 g/L nZVI induced a persistent phenotype of P. putida F1 as indicated by smaller colony morphology, a more rigid membrane, and higher tolerance to nZVI. A low interaction between nZVI particles and the surface of the nZVI-persistent phenotypic cells reduced the nZVI-induced membrane damage. This study unveils the significance of nZVI-membrane interaction on toxicity of nZVI toward bacteria.

The significance of proline and glutamate on butanol chaotropic stress in Bacillus subtilis 168.

BACKGROUND: Butanol 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. RESULTS: Using 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. CONCLUSION: This 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.

Production of 1,3-diols in Escherichia coli.

To expand the diversity of chemical compounds produced through microbial conversion, a platform pathway for the production of widely used industrial chemicals, 1,3-diols, was engineered in Escherichia coli. The pathway was designed by modifying the previously reported (R)-1,3-butanediol synthetic pathway to consist of pct (propionate CoA-transferase) from Megasphaera elsdenii, bktB (thiolase), phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, bld (butyraldehyde dehydrogenase) from Clostridium saccharoperbutylacetonicum, and the endogenous alcohol dehydrogenase(s) of E. coli. The recombinant E. coli strains produced 1,3-pentanediol, 4-methyl-1,3-pentanediol, and 1,2,4-butanetriol, together with 1,3-butanediol, from mixtures of glucose and propionate, isobutyrate, and glycolate, respectively, in shake flask cultures. To the best of our knowledge, this is the first report of microbial production of 1,3-pentanediol and 4-methyl-1,3-pentanediol.