PHB Production by Bacillus megaterium LSRB 0103 Using Cornstarch and Urea.

Polyhydroxybutyrates (PHBs) are biopolymers that are good green alternative for synthetic carbon-based polymers, and are also one of the most researched members of the Polyhydroxyalkanoates (PHA) family. In this study, a gram-positive bacterial strain Bacillus megaterium LSRB 0103 was isolated from Pallikaranai Marshland, Chennai, India. Primary screening using Sudan Black dye revealed the presence of intracellular PHB granules. Minimal Davis Media (MDM) which was used or PHB production gave a yield of 0.7107 g/L. Subsequently, using response surface methodology (RSM), a central composite design (CCD) model was designed for media optimization having cornstarch, urea, and pH as independent variables. The findings of the CCD model were fitted into a second-order polynomial equation. The RSM model predicted the maximum PHB yield of 0.93 g/L, at these independent variable levels, cornstarch, 5 g/L; urea, 2.1 g/L; and pH 7.0; while the experimental PHB yield was 0.94 g/L, with a percentage error of 1.1%. This study is the first-time report of production of PHB by Bacillus megaterium using cornstarch and urea as substrate.

Structural insights into the interactions of repositioning and known drugs for Alzheimer's disease with hen egg white lysozyme by MM-GBSA.

Six drugs (dapsone, diltiazem, timolol, rosiglitazone, mesalazine, and milnacipran) that were predicted by network-based polypharmacology approaches as potential anti-Alzheimer's drugs, have been subjected in this study for in silico and in vitro evaluation to check their potential against protein fibrillation, which is a causative factor for multiple diseases such as Alzheimer's disease, Parkinson's disease, Huntington disease, cardiac myopathy, type-II diabetes mellitus and many others. Molecular docking and thereafter molecular dynamics (MD) simulations revealed that diltiazem, rosiglitazone, and milnacipran interact with the binding residues such as Asp52, Glu35, Trp62, and Asp101, which lie within the fibrillating region of HEWL. The MM-GBSA analysis revealed -7.86, -5.05, and -10.29 kcal/mol as the binding energy of diltiazem, rosiglitazone, and milnacipran. The RMSD and RMSF calculations revealed significant stabilities of these ligands within the binding pocket of HEWL. While compared with two reported ligands inhibiting HEWL fibrillation, milnacipran depicted almost similar binding potential with one of the known ligands (Ligand binding affinity -10.66 kcal/mol) of HEWL. Furthermore, secondary structure analyses revealed notable inhibition of the secondary structural changes with our candidate ligand; especially regarding retention of the 3/10 α-helix both by DSSP simulation, Circular dichroism, and FESEM-based microscopic image analyses. Taking further into experimental validation, all three ligands inhibited fibrillation in HEWL in simulated conditions as revealed by blue shift in Congo red assay and later expressing percentage inhibition in ThioflavinT assay as well. However, dose-dependent kinetics revealed that the antifibrillatory effects of drugs are more pronounced at low protein concentrations.Communicated by Ramaswamy H. Sarma.

Finding structural requirements of structurally diverse α-glucosidase and α-amylase inhibitors through validated and predictive 2D-QSAR and 3D-QSAR analyses.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by hyperglycemic state. The α-glucosidase and α-amylase are considered two major targets for the management of Type 2 DM due to their ability of metabolizing carbohydrates into simpler sugars. In the current study, cheminformatics analyses were performed to develop validated and predictive models with a dataset of 187 α-glucosidase and α-amylase dual inhibitors. Separate linear, interpretable and statistically robust 2D-QSAR models were constructed with datasets containing the activities of α-glucosidase and α-amylase inhibitors with an aim to explain the crucial structural and physicochemical attributes responsible for higher activity towards these targets. Consequently, some descriptors of the models pointed out the importance of specific structural moieties responsible for the higher activities for these targets and on the other hand, properties such as ionization potential and mass of the compounds as well as number of hydrogen bond donors in molecules were found to be crucial in determining the binding potentials of the dataset compounds. Statistically significant 3D-QSAR models were developed with both α-glucosidase and α-amylase inhibition datapoints to estimate the importance of 3D electrostatic and steric fields for improved potentials towards these two targets. Molecular docking performed with selected compounds with homology model of α-glucosidase and X-ray crystal structure of α-amylase largely supported the interpretations obtained from the cheminformatic analyses. The current investigation should serve as important guidelines for the design of future α-glucosidase and α-amylase inhibitors. Besides, the current investigation is entirely performed by using non-commercial open-access tools to ensure easy accessibility and reproducibility of the investigation which may help researchers throughout the world to work more on drug design and discovery.

Critical Analysis and Optimization of Stoichiometric Ratio of Drug-Coformer on Cocrystal Design: Molecular Docking, In Vitro and In Vivo Assessment.

In this present research, an attempt has been made to address the influence of drug-coformer stoichiometric ratio on cocrystal design and its impact on improvement of solubility and dissolution, as well as bioavailability of poorly soluble telmisartan. The chemistry behind cocrystallization and the optimization of drug-coformer molar ratio were explored by the molecular docking approach, and theoretical were implemented practically to solve the solubility as well as bioavailability related issues of telmisartan. A new multicomponent solid form, i.e., cocrystal, was fabricated using different molar ratios of telmisartan and maleic acid, and characterized by SEM, DSC and XRD studies. The molecular docking study suggested that specific molar ratios of drug-coformer can successfully cluster with each other and form a specific geometry with favourable energy conformation to form cocrystals. Synthesized telmisartan-maleic acid cocrystals showed remarkable improvement in solubility and dissolution of telmisartan by 9.08-fold and 3.11-fold, respectively. A SEM study revealed the formation of cocrystals of telmisartan when treated with maleic acid. DSC and XRD studies also confirmed the conversion of crystalline telmisartan into its cocrystal state upon treating with maleic acid. Preclinical investigation revealed significant improvement in the efficacy of optimized cocrystals in terms of plasma drug concentration, indicating enhanced bioavailability through improved solubility as well as dissolution of telmisartan cocrystals. The present research concluded that molecular docking is an important path in selecting an appropriate stoichiometric ratio of telmisartan: maleic acid to form cocrystals and improve the solubility, dissolution, and bioavailability of poorly soluble telmisartan.

Neuropharmacological Alterations by a Rice Contaminant Stenotrophomonas maltophilia: a Detailed Bio-molecular and Mechanistic Landscape.

Contaminated rice is a major source of food poisoning in human communities where our earlier study showed Stenotrophomonas maltophilia, a Gram-negative bacillus, has been a major contaminant of the stored rice. In the present study, mono- and di-unsaturated fatty acids (UFAs) such as 18:1 ω 7 c, 16:1 ω 6 c, 16:1 ω 7 c, and 18:2 ω 6,9 c long-chain fatty acids have been found as the chief constituents of S. maltophilia boiled cell lysate. Throughout the study, both acute and chronic exposure of the cell lysate showed a decrease in the locomotor activity and a time-dependent increase of the depression (p < 0.001-0.0001, two-way ANOVA), supported by bioamine (dopamine, noradrenaline, adrenaline, serotonin, and GABA) depletion in rodents' brain possibly due to UFA-amino acid decarboxylase interaction favoring bioamine depletion as revealed by our study. Furthermore, the UFA-rich cell lysate revealed dose-dependent inhibition of murine brain microglial cell viability in vitro with concomitant increase of reactive oxygen species (ROS) inside the cell. Destruction of neuroprotective and neurotrophin releasing microglial cells, augmentation of brain ROS, and inflaming brain tissue resulting in infiltration of polymorphonuclear leucocytes also suggest to cause neurotoxicity by UFA derived from Stenotrophomonas maltophilia.

Recent Advancements in Green Synthesis of Nanoparticles for Improvement of Bioactivities: A Review.

Natural products have widely been used in applications ranging from antibacterial, antiviral, antifungal, and various other medicinal applications. The use of these natural products was recognized way before the establishment of basic chemistry behind the disease and the chemistry of plant metabo-lites. After the establishment of plant chemistry, various new horizons evolved, and the application of natural products breached the orthodox limitations. In one such interdisciplinary area, the use of plant materials in the synthesis of nanoparticles (NPs) has exponentially emerged. This advancement has offered various environment-friendly methods where hazardous chemicals are completely replaced by natural products in the sophisticated and hectic synthesis processes. This review is an attempt to under-stand the mechanism of metal nanoparticles synthesis using plant materials. It includes details on the role of the plant's secondary metabolites in the synthesis of nanoparticles including the mechanism of action. In addition, the use of these nanomaterials has widely been discussed along with the possible mechanism behind their antimicrobial and catalytic action.

Targeting mangiferin loaded N-succinyl chitosan-alginate grafted nanoparticles against atherosclerosis - A case study against diabetes mediated hyperlipidemia in rat.

Mangiferin (MGF), from Mangifera indica is well reported for its hypoglycemic activity and hypolipidemic activity. However, MGF suffers therapeutic limitation due to poor solubility causing disparaging bioavailability. Herein to address this problem, we have incorporated MGF in alginate grafted N-succinylated chitosan (NSC) nanomatrix. Characterization by molecular docking, FT-IR and 2D-NMR (COSY) has revealed that MGF could reinforce interaction with NSC. The OH and CH2OH groups of MGF may set interactions with pyranosic OH, CH2OH, NH2 (or NH-succinyl and COOH-succinyl) of NSC. The NSC-MGF nanoconjugate revealed a spherical particle geometry of 100 âˆ¼ 200 nm size. The encapsulated MGF showed 100% release in vitro. In vivo, NSC-MGF nanoconjugate revealed blood glucose lowering from 300 mg/dL to âˆ¼ 90 mg/dL as well as âˆ¼ 37% lowering of total plasma cholesterol. This is well comparative to the earlier reports which acknowledged only 1 âˆ¼ 36% lowering of plasma cholesterol with MGF. Furthermore, NSC-MGF lowered serum trigyceride to âˆ¼ 61%, while in earlier studies, only 10 âˆ¼ 40% serum triglycerides reduction was found with solitary MGF.

Assessment of Effects of Solvents on Cocrystallization by Computational Simulation Approach.

PURPOSE: The sole purpose of this study is to improve the solubility and dissolution of telmisartan by cocrystallization technique and apply a computational simulation approach to assess the nature of chemical interactions between telmisartan and coformer as well as the solvent contribution to the molecules for furnishing cocrystallization. METHODS: The effects of various concentrations of coformer i.e. oxalic acid on physicochemical parameters and drug release were investigated. RESULTS: Solubility studies suggested that cocrystallization technique with oxalic acid helps to increase the solubility of pure telmisartan of about 7 folds and drug release study revealed that telmisartan-oxalic acid cocrystals showed greater dissolution as compared to pure telmisartan. SEM study suggested that prepared telmisartan cocrystals showed rhomboid-shaped crystals with sharp edges and smooth surface. FTIR study revealed that shifting in the vibrational frequencies of C=O group of telmisartan in telmisartan- oxalic acid cocrystal indicates the formation of supra molecular hetero synthon of the cocrystal. DSC and XRD studies confirmed the formation of telmisartan-oxalic acid cocrystals. Computational simulation approach revealed that telmisartan and oxalic acid can interact with each other in the presence of methanol and water where oxalic acid can form interactions principally with the others. The interactions, thereof, may form several associations or bondings in between the drug and carrier modifying the planarity, bond energy, bond angles of both which subsequently lead to cocrystallization. CONCLUSION: So, the present research concluded that prepared telmisartan-oxalic acid cocrystal is a successful application of crystal engineering approach to improve the physicochemical properties as well as to enhance the solubility and dissolution of telmisartan.

Functionalized graphene oxide as a vehicle for targeted drug delivery and bioimaging applications.

Graphene oxide (GO) has attracted tremendous attention as a most promising nanomaterial among the carbon family since it emerged as a polynomial functional tool with rational applications in diverse fields such as biomedical engineering, electrocatalysis, biosensing, energy conversion, and storage devices. Despite having certain limitations due to its irreversible aggregation performance owing largely to the strong van der Waals interactions, efforts have been made to smartly engineer its surface chemistry for realistic multimodal applications. The use of such GO-based engineered devices has increased rapidly in the last few years, principally due to its excellent properties, such as huge surface area, honeycomb-like structure allowing vacant interstitial space to accommodate compounds, sp2 hybridized carbon, improved biocompatibility and cell surface penetration due to electronic interactions. Amongst multifaceted GO dynamics, in this review, attempts are made to discuss the advanced applications of GO or graphene-based materials (GBNs) in the biomedical field involving drug or therapeutic gene delivery, dual drug or drug-gene combination targeting, special delivery of drug cocktails to the brain, stimuli-responsive release of molecular payloads, and Janus-structured smart applications for polar-nonpolar combination drug loading followed by targeting together with smart bioimaging approaches. In addition, the advantages of duel-drug delivery systems are discussed in detail. We also discuss various electronic mechanisms, and detailed surface engineering to meet microcosmic criteria for its utilization, various novel implementations of engineered GO as mentioned above, together with discussions of its inevitable toxicity or disadvantages. We hope that the target audience, belonging to biomedical engineering, pharmaceutical or material science fields, may acquire relevant information from this review which may help them design future studies in this field.

Simultaneous Pharmacokinetics Estimation of Nateglinide and Pioglitazone by RP-HPLC: Computational Study to Unlock the Synergism.

Nateglinide (NAT) and Pioglitazone (PIO) are an antidiabetic drugs combination and currently under clinical trial in countries like Japan. In this study, an alternative, a simple, sensitive high-performance liquid chromatography method has been developed (limit of detection: 15 ng/mL and limit of quantification: 50 ng/mL) for simultaneous estimation of this drug combination in rat plasma. Most remarkably, bioavailability of NAT has been increased markedly on coadministration with PIO, than when it was administered alone. Thus, PIO is assumed to retard the catabolism of NAT by inhibiting metabolic liver-microsomal enzyme, especially CYP2C9. Using a Waters Nova-Pak C 18 column (150 × 3.9 mm, 4 µm) and a mobile phase of acetonitrile: 10 mM KH2PO4 (60: 40, V/V (volume by volume)) pH 3.5, the analysis was performed at 210 nm with a flow rate of 1.5 mL/min. In silico docking via molecular dynamics simulation revealed that NAT-CYP2C9 binding affinity may be reduced after PIO attachment, presumably due to the binding site overlapping of the two drugs. Thus, it has been proposed that NAT and PIO may be an efficient synergistic fixed dose combination against diabetes mellitus, and the above method can foster a simple but highly sensitive bioanalytical estimation for routine analysis.

Functionalized graphene oxide as a nanocarrier for dual drug delivery applications: The synergistic effect of quercetin and gefitinib against ovarian cancer cells.

Graphene Oxide (GO) has been extensively studied in the field of biomedical sciences as one of the most promising biomaterials due to its exceptional physiochemical properties. Experts have long favored anticancer drug cocktails over single drugs, given that the former may provide a more balanced molecular basis for novel chemotherapeutic strategies. Here, we investigated a combinatorial anticancer drug treatment involving the well-proven anticancer drugs quercetin and gefitinib and compared it with gefitinib and quercetin loaded separately onto polyvinylpyrrolidone (PVP)-functionalized graphene oxide (GO-PVP). The loading and cancer cell cytotoxicity of the individual drug systems and their combined loading onto GO-PVP nanovehicles were investigated in PA-1 ovarian cancer cells and compared to their effects on IOSE-364 ovarian epithelial cells. In this report, the combined drug system loaded on the GO-PVP nanovehicle was found to be significantly more toxic than the individual drug loaded systems, as well as the free drugs, toward PA-1 cells compared to the toxicity toward IOSE-364 cells. The combined drug system loaded on the GO-PVP nanovehicle is likely to be more successful than individual drug therapy, given the stronger impact of the combinatorial approach and the efficiency of chemotherapeutic delivery.

Discovery of nano-piperolactam A: A nonsteroidal contraceptive lead acting through down-regulation of interleukins.

Elevated serum interleukins (IL-6, IL-1ß) over baseline concentration help in blastocyst adhesion to the uterine endometrium in the early phase of pregnancy. A nano PLA (Piperolactam A)-HPBCD (2-hydroxy-propyl-ß-cyclodextrin) inclusion complex was developed as an interleukin down-regulator that exhibited 100% anti-implantation activity in rodents at a dose as low as 2.5-5.0 mg/kg. On metabolomics study, among major glyco-lipo-protein metabolites, only serum low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL) levels revealed alteration by the formulation. Administration of PLA-HPBCD did not cause changes in serum estrogen and progesterone levels. However, IL-6 and IL-1ß failed to increase post PLA-HPBCD administration; hence, it is assumed to be the mode of the drug's abortifacient action. In addition, absence of signs of either acute or chronic toxicity suggests the formulation was considerably non-toxic. Therefore, the nano-PLA conjugate promises as a non-steroidal contraceptive lead apart from ormeloxifene, the only non-steroidal anti-fertility agent currently available globally.

Genome mining, in silico validation and phase selection of a novel aldo-keto reductase from Candida glabrata for biotransformation.

Previously, we published cloning, overexpression, characterization and subsequent exploitation of a carbonyl reductase (cr) gene, belonging to general family aldo-keto reductase from Candida glabrata CBS138 to convert keto ester (COBE) to a chiral alcohol (ethyl-4-chloro-3-hydroxybutanoate or CHBE). Exploiting global transcription factor CRP, rDNA and transporter engineering, we have improved batch production of CHBE by trinomial bioengineering. Herein, we present the exploration of cr gene in Candida glabrata CBS138 through genome mining approach, in silico validation of its activity and selection of its biocatalytic phase. For exploration of the gene under investigation, 3 template genes were chosen namely Saccharomyces cerevisae YDR541c, YGL157w and YOL151w. The CR showed significant homology match, overlapping of substrate binding site and NADPH binding site with the template proteins. The binding affinity of COBE toward CR (-4.6 Kcal/ mol) was found higher than that of the template proteins (-3.5 to -4.5 Kcal/ mol). Biphasic biocatalysis with cofactor regeneration improved product titer 4∼5 times better than monophasic biotransformation. Currently we are working on DNA Shuffling as a next level of strain engineering and we demonstrate this approach herein as a future strategy of biochemical engineering.

Fabrication of ß-cyclodextrin-mediated single bimolecular inclusion complex: characterization, molecular docking, in-vitro release and bioavailability studies for gefitinib and simvastatin conjugate.

OBJECTIVES: Introduction of multiple molecules in a single inclusion complex, albeit cheaper, lacks conclusive attempts in earlier drug delivery reports. This manuscript emphasizes simultaneous incorporation of two anticancer drugs, gefitinib (G) and simvastatin (S), in a single molecule of ß-cyclodextrin for the first time to achieve effective drug delivery. METHODS: The inclusion complex (GSBCD) was prepared by cosolvent evaporation technique using ß-cyclodextrin (BCD) as carrier. Characterization of GSBDC was performed by Fourier transform infrared spectroscopy, COSY, differential scanning calorimetry, X-ray diffraction and dynamic light scattering analyses, which were ascribed to the complex formation inside BCD cavity, micronization of drugs and conversion to amorphous state. KEY FINDINGS: The complex revealed entrapment of G and S in 3 ± 0.48: 2 ± 0.19 molar ratio and showed more than 3.5 and 10 fold increase in drug release in in vitro and in vivo, respectively. Docking and COSY studies revealed molecular alignment into BCD central cavity that been achieved via hydrogen bonding between certain groups of the ligands (G and S) and the polar heads of BCD. Partial incorporation of the molecular backbone inside inclusion complex suggests superficial contact with the solvent indicating slow steady release kinetics. CONCLUSIONS: This approach of forming inclusion complex with multiple molecules within a single cavity can be a landmark for further studies in drug delivery.

Pharmacoinformatics study of Piperolactam A from Piper betle root as new lead for non steroidal anti fertility drug development.

Fertility control is a burning problem all over the world to regulate population overflow and maintain ecological balance. This study is an in-silico approach to explore a non-steroidal lead as contraceptive agent in order to avoid several contraindications generated by steroidal analogues. Piperolactam A, an aristolactam isolated from Piper betle Linn. showed binding affinity towards estrogen and progesterone receptor as -8.9 and -9.0Kcal/mol (inhibition constant Ki=0.294µM and 0.249µM) respectively which is even larger than that of reported antagonists such as Rohitukine and OrgC (binding affinity -8.7 and -8.4Kcal/mol; Ki 0.443µM and 0.685µM respectively). The binding site exploration displayed more hydrogen bonding of Piperolactam A (His 524, Leu 346, Thr 347) than Rohitukine and OrgC (Leu 718) with associated receptors which was further confirmed by molecular dynamics simulations. The drug-likeliness of the compound has been proved from its tally with Lipinsky's Rule of Five and lowered toxicity such as cardiac toxicity, liver toxicity, mutagenicity and ecological toxicity. Endocrine disruptome and later docking guided molecular simulations revealed that Piperolactam A has weaker binding affinity and/or lower probability of binding with nuclear receptors especially hERG and cytochrome P450. The high Caco-2 permeability suggested more bioavailability hence more therapeutic efficacy of the drug.

An experimental modeling of trinomial bioengineering- crp, rDNA, and transporter engineering within single cell factory for maximizing two-phase bioreduction.

A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363mM with highest turnover number (112.77±3.95s-1). Solitary recombineering of such catalytic cell reproduced CHBE 161.04g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E. coli host yielded CHBE 477.54g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88g/L/gDCW. The CHBE production has thus been upscaled to 8-12 times than those reported so far. qRT-PCR studies revealed that both membrane efflux channels such as acrAB as well as ROS scavenger genes such as ahpCF have been activated by engineering crp. Moreover, membrane protecting genes such as manXYZ together with solvent extrusion associated genes such as glpC have been upregulated inside mutant host. Although numerous proteins have been investigated to convert COBE to CHBE; this is the first approach to use engineering triad involving crp engineering, recombinant DNA engineering and transporter engineering together for improving cell performance during two-phase biocatalysis.

In Vitro susceptibilities of wild and drug resistant Leishmania donovani amastigotes to piperolactam A loaded hydroxypropyl-ß-cyclodextrin nanoparticles.

Leishmaniasis is an epidemic in various countries, and the parasite Leishmania donovani is developing resistance against available drugs. In the present study the antileishmanial action of piperolactam A (PL), isolated after bioactivity guided fractionation from root extracts of Piper betle was accentuated in detail. Activity potentiation was achieved via cyclodextrin complexation. Crude hydro-ethanolic extract (PB) and three fractions obtained from PB and fabricated PL-hydroxypropyl-ß-cyclodextrin (HPBCD) nanoparticles were evaluated for antileishmanial activity. Tests were performed against L. donovani wild-type, sodium stibogluconate, paromomycin and field isolated (GE1) resistant strains in axenic amastigote and amastigote in macrophage models. PL-HPBCD complex was characterized and FITC loaded HPBCD nanoparticles were assessed for macrophage internalization in confocal microscopic studies. Isolated and purified PL from most potent, alkaloid rich ethyl acetate fraction of PB showed high level of antileishmanial activities in wild-type (IC50=36 µM), sodium stibogluconate resistant (IC50=103 µM), paromomycin resistant (IC50=91 µM) and field isolated resistant (IC50=72 µM) strains together with cytotoxicity (CC50=900 µM) in mouse peritoneal macrophage cells. Inclusion of PL in HPBCD nanoparticles resulted in 10-fold and 4-10-fold increase in selectivity indexes (CC50/IC50) for wild-type and drug resistant strains, respectively. Drug-carrier interactions were clearly visualized in FT-IR studies. Complete incorporation of PL in HPBCD cavity was ascertained in DSC and XRD analyses. 180nm size stable nanospheres showed macrophage internalization within 1h of incubation. Piperolactam A (PL), a representative of the inchoate skeleton of aristolactam chassis might be the source of safe and affordable antileishmanial agents for the cure of deadly Leishmania infections.

Rewiring global regulator cAMP receptor protein (CRP) to improve E. coli tolerance towards low pH.

Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we tried to improve strain acidic tolerance from its transcription level, i.e. we adopted error-prone PCR method to engineer global regulator cAMP receptor protein (CRP) of Escherichia coli to improve its performance at low pH. The best mutant AcM1 was identified from random mutagenesis libraries based on its growth performance. AcM1 almost doubled (0.113h(-1)) the growth rate of the control (0.062h(-1)) at pH 4.24. It also demonstrated better thermotolerance than the control at 48°C, whose growth was completely inhibited at this temperature. Quantitative real time reverse transcription PCR results revealed a stress response overlap among low pH stress-, oxidative stress- and osmotic stress-related genes. The chief enzyme responsible for cell acid tolerance, glutamate decarboxylase, demonstrated over twofold activity in AcM1 compared to the control. Differential binding properties of AcM1 mutant CRP with Class-I, II, and III CRP-dependent promoters suggested that modifications to native CRP may lead to transcription profile changes. Hence, we believe that transcriptional engineering of global regulator CRP can provide a new strain engineering alternative for E. coli.

Enhancing E. coli tolerance towards oxidative stress via engineering its global regulator cAMP receptor protein (CRP).

Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP), which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3) were identified with improved tolerance via H(2)O(2) enrichment selection. The best mutant OM3 could grow in 12 mM H(2)O(2) with the growth rate of 0.6 h(-1), whereas the growth of wild type was completely inhibited at this H(2)O(2) concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS), which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H(2)O(2) treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS- and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay suggested that catalase (katE) could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.