Enhanced chaotrope tolerance and (S)-2-hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans.

Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacIQ /Ptrc system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2 O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L-1 upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL-1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g-1 benzaldehyde and 0.089 g 2-HPP g cell dry weight-1 h-1 , respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633.

Optimized enantioselective (S)-2-hydroxypropiophenone synthesis by free- and encapsulated-resting cells of Pseudomonas putida.

BACKGROUND: Aromatic α-hydroxy ketones, such as S-2-hydroxypropiophenone (2-HPP), are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of 2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida, previously grown in a culture medium containing ammonium mandelate, are a source of native benzoylformate decarboxylase (BFD) activity. BFD produced by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified enzyme preparations. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD-catalyzed enantioselective cross-coupling reaction. RESULTS: The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 mL of 200 mM phosphate buffer (pH 7) for 3 h. The optimal biomass concentration was assessed to be 0.006 g dry cell weight (DCW) mL- 1. 2-HPP titer, yield and productivity using the free cells were 1.2 g L- 1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP g- 1 DCW h- 1, respectively, under optimized biotransformation conditions (30 °C, 200 rpm). Calcium alginate (CA)-polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable beads degradation. Moreover, there was no production of benzyl alcohol as an unwanted by-product. CONCLUSIONS: Bioconversion by whole P. putida resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.

Nicotinamide adenine dinucleotide hydrogen regeneration in a microbial electrosynthesis system by Enterobacter aerogenes.

Costly cofactors such as nicotinamide adenine dinucleotide hydrogen (NADH) are essential to have high activity in many redox enzymatic processes. Cofactor regeneration methods have been suggested to improve the economic aspects of the system. Here, we introduce a microbial electrosynthesis process to regenerate NADH in a two-chamber set-up with Enterobacter aerogenes biofilm as the bio-cathode. The effects of several important factors on the regeneration efficiency were studied and the highest NADH regeneration yield was achieved equal to 65 % at the potential of -1.5 V and the initial NAD+ concentration of 1 mM after 8 h of operation. The regenerated cofactor was highly enzymatically active (93 ± 4 %) which was a great merit of the process. Studying the kinetics of regeneration revealed that the electron transfer rate to the biofilm was the limiting factor. We tried to remove the limitation through co-culturing Pseudomonas aeruginosa and producing more electrochemical active compounds in the biofilm. Although, this modification was not effective for the regeneration yield, it showed that the external potential implicitly influenced the regeneration process by changing the internal microbial cell metabolic fluxes. Finally, it can be concluded that the microbial electrosynthesis is a promising green process for NADH regeneration.

Tailored lipopeptide surfactants as potentially effective drugs to treat SARS-CoV-2 infection.

Finding effective drugs to treat SARS-CoV-2 infection as a complementary step to the extensive vaccination is of the great importance to overcome the current pandemic situation. It has been shown that some bio-active unsaturated fatty acids such as Arachidonic Acid (AA) can reduce the infection severity and even destroy the virus by disintegration of the virus lipid envelope. On the other hand, it has been reported that several designed peptides with an activity similar to the angiotensin converting enzyme 2 (ACE-2), which has a high affinity towards the novel corona virus spike protein, can inhibit the viral infection through concealing the spike proteins from the cell surfaces ACE-2. Binding the mentioned peptides to the bio-active lipids like AA will result in a lipopeptide surfactant molecule with the synergistic effect of both the active moieties in its structure to treat the novel corona infection. In addition, the peptide segment increases the aqueous solubility of the lipid segment and enables the targeted delivery of the surfactant molecule to the virus. The resultant lipopeptide would be a potentially effective drug for SARS-CoV-2 infection treatment with the minimum side effects.

The promiscuous potential of cellulase in degradation of polylactic acid and its jute composite.

It has been suggested that cellulolytic enzymes can be effective on the degradation of PLA samples. The idea was investigated by examining the impact of cellulase on degradation of PLA and PLA-jute (64/36) composite in an aqueous medium. The obtained results demonstrated 55% and 61% thickness reduction in PLA and PLA-jute specimens after four months of treatment, respectively. Gel permeation chromatography (GPC) showed significant decline in the number average molecular weight (Mn) approximately equal to 85% and 80% for PLA and PLA-jute in comparison with their control. The poly dispersity index (PDI) of PLA and PLA-jute declined 41% and 49% that disclosed more homogenous distribution in molecular weight of the polymer after treatment with cellulase. The cellulase promiscuity effect on PLA degradation was further revealed by Fourier-transform infrared spectroscopy (FT-IR) analysis where substantial decrease in the peak intensities of the polymer related functional groups were observed. In addition, PLA biodegradation was studied in more detail by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) of control and cellulase treated specimens. The obtained results confirmed the promiscuous function of cellulase in the presence or the absence of jute as the specific substrate of cellulase. This can be considered as a major breakthrough to develop effective biodegradation processes for PLA products at the end of their life cycle.

Improving l-phenylacetylcarbinol production in Saccharomyces cerevisiae by in silico aided metabolic engineering.

L-Phenylacetylcarbinol (L-PAC) which is used as a precursor for the production of ephedrine and pseudoephedrine is the first reported biologically produced α-hydroxy ketone compound. l-PAC is commercially produced by the yeast Saccharomyces cerevisiae. Yeast cells transform exogenously added benzaldehyde into l-PAC by using the action of pyruvate decarboxylase (PDC) enzyme. In this work, genome-scale model and flux balance analysis were used to identify novel target genes for the enhancement of l-PAC production in yeast. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene and minimization of metabolic adjustments. Six single gene deletion strains, namely Δrpe1, Δpda1, Δadh3, Δadh1, Δzwf1 and Δpdc1, were predicted in silico and further tested in vivo by using knock-out strains cultivated semi-anaerobically on glucose and benzaldehyde as substrates. Δzwf1 mutant exhibited the highest l-PAC formation (2.48 g/L) by using 2 g/L of benzaldehyde which is equivalent to 88 % of the theoretical yield.

Investigation of the Role of Glucose Decorated Chitosan and PLGA Nanoparticles as Blocking Agents to Glucose Transporters of Tumor Cells.

PURPOSE: Glucose decorated PLGA and chitosan nanoparticles (GPNPs and GCNPs) have been developed to examine the possibility of preventing the facilitated glucose transport to the cells through blocking the glucose transporters (Gluts) overexpressed by tumor cells. METHODS: The MTT assay was used to assess the cytotoxicity towards human colon tumor (HT-29) cells in 72 hrs. Fluorescence microscopy was employed to confirm the attachment of GPNPs to the cells. Moreover, the GPNPs effects on the apoptotic rate of HT-29 cells were analyzed. Finally, the expression levels of GLUT-1 and GLUT-4 by real-time polymerase chain reaction (RT-PCR) were assayed to investigate the response of HT-29 cells to blocking their Gluts by GPNPs. RESULTS: The stability studies showed that the synthesized complexes were mostly stable (more than 80%) at various temperatures (4 to 40ºC) and pH (5.4 to 7.4) conditions. Results indicated that the survival rate of the cells was decreased to 43% and 46% after treatment with GCNPs and GPNPs, respectively. Also, the apoptosis assay results showed that the percentage of viable cells reduced to 47% after GPNPs treatment. These observations were justified by the specific interactions between the glucose terminals and the cells Gluts which resulted in blocking the entries of nutrients to the cells. It was revealed that the GLUT-1 mRNA expression after the first 24 h of treatment by GPNPs was upregulated to more than 145%, while the direction was reversed after 72 h (expression less than 45%), which coincided with the cells death. In the first 24 h, the glucose deprivation stimulated the expression of Glut-1 while the apoptotic enzymes expression was dominant at the end of 72 h treatment time. CONCLUSION: Finally, it can be concluded that the glucose-nanoparticle complexes could be considered as promising agents in cancer therapy.

Investigation of platinum recovery from a spent refinery catalyst with a hybrid of oxalic acid produced by Aspergillus niger and mineral acids.

The capability of oxalic acid produced by Aspergillus niger was investigated for bioleaching of platinum from a refinery reforming catalyst. The spent medium mode was selected for bioleaching because of its higher efficiency at favorable pH and temperature conditions. The effects of several important factors such as the pulp density, pH and temperature on platinum recovery were optimized using Box-Behnken design of response surface methodology. The results indicated that pH adjustment during the bioleaching process increases the final platinum recovery significantly. The obtained optimum conditions were 1% for the pulp density, 0.5 for the medium pH, and 70 °C for the temperature which led to 37% platinum recovery. The significance of oxalic acid as the leaching agent in platinum bioleaching was highlighted by investigating the recovery of a blank medium without oxalic acid at the optimum conditions which was just about 13%. The presented method can be utilized in an environmentally friendly process to recover platinum from industrial catalysts.

Production of xanthan gum by free and immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii.

Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan production.

Optimizing conditions for production of high levels of soluble recombinant human growth hormone using Taguchi method.

Human growth hormone (hGH) is synthesized and stored by somatotroph cells of the anterior pituitary gland and can effect on body metabolism. This protein can be used to treat hGH deficiency, Prader-Willi syndrome and Turner syndrome. The limitations in current technology for soluble recombinant protein production, such as inclusion body formation, decrease its usage for therapeutic purposes. To achieve high levels of soluble form of recombinant human growth hormone (rhGH) we used suitable host strain, appropriate induction temperature, induction time and culture media composition. For this purpose, 32 experiments were designed using Taguchi method and the levels of produced proteins in all 32 experiments were evaluated primarily by ELISA and dot blotting and finally the purified rhGH protein products assessed by SDS-PAGE and Western blotting techniques. Our results indicate that media, bacterial strains, temperature and induction time have significant effects on the production of rhGH. The low cultivation temperature of 25°C, TB media (with 3% ethanol and 0.6M glycerol), Origami strain and a 10-h induction time increased the solubility of human growth hormone.

Classification of DNA minor and major grooves binding proteins according to the NLSs by data analysis methods.

High-mobility group proteins are a superfamily of DNA-binding proteins that bind to the DNA minor groove and bend it, whereas most of the transcription factors such as centromere protein B (CENP-B), octamer (Oct)-1, growth factor independence 1 (Gfi-1), and WRKY bind to the major groove of DNA. Classification of proteins using their DNA-binding features is the aim of this study. Nuclear localization signals play more important roles in entering DNA-binding proteins to nucleus and doing their functions; therefore, they have been considered as a feature which is important for DNA-binding manner in proteins. Nuclear localization signals (NLSs) were predicted by two prediction web servers, and then, their sequence ordered features were extracted by Chou's pseudo amino acid composition (PseAAC) and ProtParam. Multilayer perceptron was used as an artificial neural network for analyzing the features by calculating the correlation coefficient and 30-fold cross-validation. Another used data-analyzing program was principal component analysis of the Minitab software. By calculating the eigenvalues and considering five principal components, the sequence length of NLSs was known as the best feature for classifying DNA-binding proteins. Minimum mean squared error (MSE) (0.1098) and the highest R (2) (0.963) mean that there is a significant difference between the NLS length of the DNA major groove and minor groove binder proteins. Results showed that it is possible to classify DNA major groove and minor groove binder proteins by their NLS sequences as a feature.