Multisite Mutation of the Escherichia coli cAMP Receptor Protein: Enhancing Xylitol Biosynthesis by Activating Xylose Catabolism and Improving Strain Tolerance.

The bioproduction of xylitol from hemicellulose hydrolysate has good potential for industrial development. However, xylitol productivity has always been limited due to corncob hydrolysate toxicity and glucose catabolic repression. To address these challenges, this work selected the S83 and S128 amino acid residues of the cyclic AMP receptor protein (CRP) as the modification target. By introducing multisite mutation in CRP, this approach successfully enhanced xylose catabolism and improved the strain's tolerance to corncob hydrolysate. The resulting mutant strain, designated as CPH (CRP S83H-S128P), underwent fermentation in a 20 L bioreactor with semicontinuous feeding of corncob hydrolysate. Remarkably, xylitol yield and xylitol productivity for 41 h fermentation were 175 and 4.32 g/L/h, respectively. Therefore, multisite CRP mutation was demonstrated as an efficient global regulatory strategy to effectively improve xylitol productivity from lime-pretreated corncob hydrolysates.

Production of red yeast rice rich in monacolin K by variable temperature solid fermentation of Monascus purpureus.

Hypercholesterolemia represents a serious public health problem as it significantly increases the risk of developing cardiovascular diseases. Monacolin K (MK) in red yeast rice is an active compound that can effectively lower plasma cholesterol. To enhance the yield of MK in solid state fermentation of Monascus purpureus HNU01, the effects of different variables were systematically examined in single-factor experiments. The optimal conditions for the production of red yeast rice rich in MK were as follows: initial pH value 5.5, initial moisture content 40% w/w, glucose 50 g L-1, peptone 20 g L-1, MgSO4 0.5 g L-1, KH2PO4 1 g L-1, variable temperature fermentation (30 °C for the first 3 days and then 24 °C for 15 days), total fermentation time of 18 days, and additional water added at day 4 at 10% w/w. Under the above optimized conditions, the MK content of red yeast rice produced by fermentation was 9.5 mg g-1. No citrinin was detected in any of the batches of fermentation products. The results will be useful for the large-scale production of high-quality red yeast rice with health benefits for consumers.

Engineered Biosynthesis through the Adenylation Domains from Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases, consisted of multiple catalytic domains, are involved in the biosynthesis of an important family of bioactive natural products in a coordinated manner. Among the functional domains, adenylation domains are specifically responsible for recognizing carboxylic acid building blocks and synthesizing aminoacyl adenylates. Given their critical roles in the biosynthesis of the growing peptide, A-domains are also referred to as the "gatekeeper". In this review, very recent developments on the A-domains from NRPSs are reviewed to expand the fundamental knowledge of the A domain, including knowledge on the structures, functions, and molecular interactions. Several recent examples were also discussed to highlight the great potential of A-domain engineering. This study should provide a framework for the combinatorial biosynthesis or synthetic biology-driven microbial production of novel nonribosomal peptides.

Genetically Engineering Escherichia coli to Produce Xylitol from Corncob Hydrolysate without Lime Detoxification.

Before fermentation with hemicellulosic hydrolysate as a substrate, it is generally necessary to detoxify the toxic substances that are harmful to microorganism growth. Cyclic AMP receptor protein (CRP) is a global regulator, and mutation of its key sites may have an important impact on E. coli virulence tolerance. Using corncob hydrolysate without ion-exchange or lime detoxification as the substrate, shake flask fermentation experiments showed that CRP mutant IS5-dG (I112L, T127G, A144T) produced 18.4 g/L of xylitol within 34 h, and the OD600 was 9.7 at 24 h; these values were 41.5% and 21.3% higher than those of the starting strain, IS5-d, respectively. This mutant produced 82 g/L of xylitol from corncob hydrolysate without ion-exchange or lime detoxification during fed-batch fermentation in a 15-L bioreactor, with a productivity of 1.04 g/L/h; these values were 173% and 174% higher than the starting strain, respectively. To our knowledge, this is the highest xylitol concentration and productivity produced by microbial fermentation using completely non-detoxified hemicellulosic hydrolysate as the substrate to date. This study also showed that alkali neutralization, high temperature sterilization, and fermentation of the hydrolysate had important effects on the xylose loss rate and xylitol production.

Advances in Research at Synthesis Process Optimization and Quality Standard Improvement of O-desmethylvenlafaxine Succinate.

We herein describe an optimal approach for the efficient synthesis of O-desmethylvenlafaxine succinate monohydrate (DVS) with high yield and high purity through 5-step reactions, including benzyl protection of the phenolic hydroxyl group, cyclohexanone condensation, deprotection, cyano reduction, dimethylation, and succinic acid salt formation from p-hydroxybenzene acetonitrile as a starting material. 4-Benzyloxyphenylacetonitrile (Intermediate I) was prepared by the hydroxyl protection of the bromide benzyl-p-hydroxyphenylacetonitrile catalyzed by potassium carbonate with 99.83% purity and 98.92% yields. The 1, 2-nucleophilic addition of intermediate I to cyclohexanone promoted by sodium hydroxide with the homogeneous catalyst (n-Bu)4N+Br- to the preparation of 1-[Cyano(4-benzyloxyphenyl)methyl]cyclohexanol (Intermediate II) was obtained by 99.13% purity and 99.71% yields. Cyclohexanone residues and benzyl bromide residues were trace, and tetrabutylammonium bromide residues were UNDER 0.7 ppm, which further improves the residual standards for genotoxic impurities (GIs). 1-[2-amino-1-(4-hydroxyphenyl)ethyl]cyclohexanol hydrochloride (Intermediate III) was prepared by 10% palladium-carbon under 2.0 MPa up to 98.32% purity and 94.20% yields. O-desmethylvenlafaxine (ODV) was synthesized by dimethylation of intermediate III with 37% formaldehyde solution and 85% formic acid solution. The highest purity was up to 99.20% and the yield was up to 84.77%. O-desmethylvenlafaxine succinate monohydrate (DVS) was formed from succinic acid and O-desmethylvenlafaxine (ODV) and crystallized in a mixed solvent of acetone and water (3:1) to obtain 99.92% purity and 90.27% yields. The 5-step total yields of desvenlafaxine succinate monohydrate is 71.09%, and its crystal form has characteristic peaks at 5, 10, 21, and 26 min by XRD powder diffraction, which is consistent with the crystalline form I. Compared with conventional synthesis strategy, we revealed a novel and green process with a high total yield, high atomic economy, low environmental pollution, high operational safety, and high residual standards for genotoxic impurities (GIs), which improves drug safety.

Metabolic Behaviors of Aconitum Alkaloids in Different Concentrations of Aconiti Lateralis Radix Praeparata and Effects of Aconitine in Healthy Human and Long QT Syndrome Cardiomyocytes.

Aconiti Lateralis Radix Praeparata (Fu Zi) is the processed lateral root of Aconitum carmichaelii Debx, which is widely used in emergency clinics. Poisoning incidents and adverse reactions occur with the improper intake of Fu Zi. Metabolic characteristics of aconitum alkaloids of Fu Zi may vary, and the effects of Fu Zi in healthy and Long QT syndrome (LQTS) patients is unknown. In this experiment, 24 Sprague Dawley rats were randomly divided into three groups: 2.0, 1.0, and 0.5 g/kg dose groups, and blood samples were collected after the oral administration of Fu Zi extract. We used an ultra-high performance liquid chromatography-tandem mass spectrometry system to detect the concentrations of six aconitum alkaloids. Cell toxicity, calcium imaging, and patch-clamp recordings of human induced pluripotent stem cells-cardiomyocytes (hiPSC-CMs) of aconitine in healthy and LQTS were observed. We found that the AUC(0-48h), Cmax, and t1/2 of the six compounds increased with the multiplicative dosages; those in the high group were significantly higher than those in the low group. Aconitine concentration-dependently decreased the amplitude, which has no significant effect on the cell index of normal hiPSC-CMs. Aconitine at 5.0 µM decreased the cell index between 5-30 min for LQTS hiPSC-CMs. Meanwhile, aconitine significantly increased the frequency of calcium transients in LQTS at 5 µM. Aconitine significantly shortened the action potential duration of human cardiomyocytes in both normal and LQTS groups. These results show metabolic behaviors of aconitum alkaloids in different concentrations of Fu Zi and effects of aconitine in healthy and LQTS patients.

Photo-Responsive Micelles with Controllable and Co-Release of Carbon Monoxide, Formaldehyde and Doxorubicin.

Endogenous gases have attracted much attention due to their potent applications in disease therapies. The combined therapy, including gaseous molecules and other medicines that can create synergistic effects, is a new way for future treatment. However, due to the gaseous state, gas utilization in medical service is still limited. To pave the way for future usage, in this work, an amphiphilic block copolymer containing nitrobenzyl ether, 3-hydroxyflavone (3-HF) derivatives and ether linker was constructed. The nitrobenzyl ether group endows the polymer with a photo-responsive character. Upon light illumination, 3-HF derivatives can be triggered for carbon monoxide (CO) release. The ether linker can also be released emitting formaldehyde (FA). The self-assembly induced micelle can encompass medicine, e.g., doxorubicin (DOX), into it and a controlled release of DOX can be realized upon light illumination. As far as we know, there is no report on the combination donor of CO and DOX and this is the first attempt on the co-release of CO, FA and DOX.

Preparation of calcium carbonate microrods from the gypsum scale layer of evaporation equipment.

The difficult-to-remove CaSO4 scale layer attached to an evaporator wall is a major problem in related industries. How to efficiently remove the CaSO4 scale layer and convert it into fine chemicals with high added value, so as to turn waste into treasure, is a current research hotspot. In this study, a CaSO4 scale layer was removed by 15 min rotary washing via a phase transfer route. Further, using the eluted calcium gluconate solution as a raw material and polyethylene glycol as the crystal control agent, CaCO3 was prepared by a CO2 carbonization method. The preparation conditions of CaCO3 were optimized by single factor experiments, and the phase and morphology of the prepared samples were characterized by XRD and FESEM. The results show that the optimized conditions are as follows: reaction temperature 80 °C, reaction time 1 h, polyethylene glycol addition 3%, and a stirring rate of 400 rpm. The samples prepared under these conditions are pure-phase calcite-type CaCO3 microrods with lengths of 1-2 µm and diameters of 300-500 nm.

Increasing NADPH Availability for Xylitol Production via Pentose-Phosphate-Pathway Gene Overexpression and Embden-Meyerhof-Parnas-Pathway Gene Deletion in Escherichia coli.

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA, pfkB, and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.

Combination of the CRP mutation and ptsG deletion in Escherichia coli to efficiently synthesize xylitol from corncob hydrolysates.

The biotechnology-based production of xylitol has received widespread attention because it can use cheap and renewable lignocellulose as a raw material, thereby decreasing costs and pollution. The simultaneous use of various sugars in lignocellulose hydrolysates is a primary prerequisite for efficient xylitol production. In this study, a ΔptsG and crp* combinatorial strategy was used to generate Escherichia coli W3110 strain IS5-dI, which completely eliminated glucose repression and simultaneously used glucose and xylose. This strain produced 164 g/L xylitol from detoxified corncob hydrolysates during a fed-batch fermentation in a 15-L bioreactor, which was 14.7% higher than the xylitol produced by the starting strain, IS5-d (143 g/L), and the xylitol productivity was 3.04 g/L/h. These results represent the highest xylitol concentration and productivity reported to date for bacteria and hemicellulosic sugars. Additionally, strain IS5-dG, which differs from IS5-dI at CRP amino acid residue 127 (I127G), was tolerant to the toxins in corncob hydrolysates. In a fed-batch fermentation experiment involving a 15-L bioreactor, IS5-dG produced 137 g/L xylitol from non-detoxified corncob hydrolysates, with a productivity of 1.76 g/L/h. On the basis of these results, we believe that IS5-dI and IS5-dG may be useful host strains for the industrial-scale production of xylitol from detoxified or non-detoxified corncob hydrolysates.

Efficient production of xylitol by the integration of multiple copies of xylose reductase gene and the deletion of Embden-Meyerhof-Parnas pathway-associated genes to enhance NADPH regeneration in Escherichia coli.

Cofactor supply is a rate-limiting step in the bioconversion of xylose to xylitol. Strain WZ04 was first constructed by a novel simultaneous deletion-insertion strategy, replacing ptsG, xylAB and ptsF in wild-type Escherichia coli W3110 with three mutated xylose reductase genes (xr) from Neurospora crassa. Then, the pfkA, pfkB, pgi and/or sthA genes were deleted and replaced by xr to investigate the influence of carbon flux toward the pentose phosphate pathway and/or transhydrogenase activity on NADPH generation. The deletion of pfkA/pfkB significantly improved NADPH supply, but minimally influenced cell growth. The effects of insertion position and copy number of xr were examined by a quantitative real-time PCR and a shake-flask fermentation experiment. In a fed-batch fermentation experiment with a 15-L bioreactor, strain WZ51 produced 131.6 g L-1 xylitol from hemicellulosic hydrolysate (xylitol productivity: 2.09 g L-1 h-1). This study provided a potential approach for industrial-scale production of xylitol from hemicellulosic hydrolysate.

The advancement of multidimensional QSAR for novel drug discovery - where are we headed?

INTRODUCTION: The Multidimensional quantitative structure-activity relationship (multidimensional-QSAR) method is one of the most popular computational methods employed to predict interesting biochemical properties of existing or hypothetical molecules. With continuous progress, the QSAR method has made remarkable success in various fields, such as medicinal chemistry, material science and predictive toxicology. Areas covered: In this review, the authors cover the basic elements of multidimensional -QSAR including model construction, validation and application. It includes and emphasizes the very recent developments of multidimensional -QSAR such as: HQSAR, G-QSAR, MIA-QSAR, multi-target QSAR. The advantages and disadvantages of each method are also discussed and typical examples of their application are detailed. Expert opinion: Although there are defects in multidimensional-QSAR modeling, it is still of enormous help to chemists, biologists and other researchers in various fields. In the authors' opinion, the latest more precise and feasible QSAR models should be further developed by integrating new descriptors, algorithms and other relevant computational techniques. Apart from being applied in traditional fields (e.g. lead optimization and predictive risk assessment), QSAR should be used more widely as a routine method in other emerging research fields including the modeling of nanoparticles(NPs), mixture toxicity and peptides.