Analysis of the complete genome sequence of Paenibacillus sp. lzh-N1 reveals its antagonistic ability.

BACKGROUND: Plant diseases caused by pathogenic fungi are devastating. However, commonly used fungicides are harmful to the environment, and some are becoming ineffective due to fungal resistance. Therefore, eco-friendly biological methods to control pathogenic fungi are urgently needed. RESULTS: In this study, a strain, Paenibacillus sp. lzh-N1, that could inhibit the growth of the pathogenic fungus Mycosphaerella sentina (Fr) Schrorter was isolated from the rhizosphere soil of pear trees, and the complete genome sequence of the strain was obtained, annotated, and analyzed to reveal the genetic foundation of its antagonistic ability. The entire genome of this strain contained a circular chromosome of 5,641,488 bp with a GC content of 45.50%. The results of species identification show that the strain belongs to the same species as P. polymyxa Sb3-1 and P. polymyxa CJX518. Sixteen secondary metabolic biosynthetic gene clusters were predicted by antiSMASH, including those of the antifungal peptides fusaricidin B and paenilarvins. In addition, biofilm formation-related genes containing two potential gene clusters for cyclic lactone autoinducer, a gene encoding S-ribosylhomocysteine lyase (LuxS), and three genes encoding exopolysaccharide biosynthesis protein were identified. CONCLUSIONS: Antifungal peptides and glucanase biosynthesized by Paenibacillus sp. lzh-N1 may be responsible for its antagonistic effect. Moreover, quorum sensing systems may influence the biocontrol activity of this strain directly or indirectly.

Progress in research and application development of surface display technology using Bacillus subtilis spores.

Bacillus subtilis is a widely distributed aerobic Gram-positive species of bacteria. As a tool in the lab, it has the advantages of nonpathogenicity and limited likelihood of becoming drug resistant. It is a probiotic strain that can be directly used in humans and animals. It can be induced to produce spores under nutrient deficiency or other adverse conditions. B. subtilis spores have unique physical, chemical, and biochemical characteristics. Expression of heterologous antigens or proteins on the surface of B. subtilis spores has been successfully performed for over a decade. As an update and supplement to previously published research, this paper reviews the latest research on spore surface display technology using B. subtilis. We have mainly focused on the regulation of spore coat protein expression, display and application of exogenous proteins, and identification of developing research areas of spore surface display technology.

Biocatalytic production of 2,5-furandicarboxylic acid: recent advances and future perspectives.

2,5-Furandicarboxylic acid (FDCA) is attracting increasing attention because of its potential applications as a sustainable substitute to petroleum-derived terephthalic acid for the production of bio-based polymers, such as poly(ethylene 2,5-furandicarboxylate) (PEF). Many catalytic methods have been developed for the synthesis of FDCA, including chemocatalysis, biocatalysis, photocatalysis, and electrocatalysis. Biocatalysis is a promising approach with advantages that include mild reaction condition, lower cost, higher selectivity, and environment amity. However, the biocatalytic production of FDCA has hardly been reviewed. To fully understand the current research developments, this review comprehensively considers the research progress on toxic effects and biodegradation of furan aldehydes, and then summarizes the latest achievements concerning the synthesis of FDCA from 5-hydroxymethylfurfural and other chemicals, such as 2-furoic acid and 5-methoxymethylfurfural. Our primary focus is on biocatalytic methods, including enzymatic catalysis (in vitro) and whole-cell catalysis (in vivo). Furthermore, future research directions and general developmental trends for more efficient biocatalytic production of FDCA are also proposed.

Production of trans-2,3-dihydro-3-hydroxyanthranilic acid by engineered Pseudomonas chlororaphis GP72.

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic ß-amino acid that can be used for the synthesis of chiral materials and nonnatural peptides. The aim of this study was to accumulate DHHA by engineering Pseudomonas chlororaphis GP72, a nonpathogenic strain that produces phenazine-1-carboxylic acid and 2-hydroxyphenazine. First, the phzF deletion mutant DA1 was constructed, which produced 1.91 g/L DHHA. Moreover, rpeA and pykF were disrupted and then ppsA and tktA were co-expressed in strain DA1. The resulting strain DA4 increased DHHA concentration to 4.98 g/L, which is 2.6-fold than that of DA1. The effects of the addition of glucose, glycerol, L-tryptophan, and Fe3+on DHHA production were also investigated. Strain DA4 produced 7.48 g/L of DHHA in the culture medium in the presence of 12 g/L glucose and 3 mM Fe3+, which was 1.5-fold higher than the strain in the original fermentation conditions. These results indicate the potential of P. chlororaphis GP72 as a DHHA producer.

Genetic engineering of Pseudomonas chlororaphis GP72 for the enhanced production of 2-Hydroxyphenazine.

BACKGROUND: The biocontrol strain Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere synthesizes three antifungal phenazine compounds, 2-Hydroxyphenazine (2-OH-PHZ), 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) and phenazine-1-carboxylic acid (PCA). PCA has been a commercialized antifungal pesticide registered as "Shenqinmycin" in China since 2011. It is found that 2-OH-PHZ shows stronger fungistatic and bacteriostatic activity to some pathogens than PCA. 2-OH-PHZ could be developed as a potential antifungal pesticide. But the yield of 2-OH-PHZ generally is quite low, such as P. chlororaphis GP72, the production of 2-OH-PHZ by the wide-type strain is only 4.5 mg/L, it is necessary to enhance the yield of 2-OH-PHZ for its application in agriculture. RESULTS: Different strategies were used to improve the yield of 2-OH-PHZ: knocking out the negative regulatory genes, enhancing the shikimate pathway, deleting the competing pathways of 2-OH-PHZ synthesis based on chorismate, and improving the activity of PhzO which catalyzes the conversion of PCA to 2-OH-PHZ, although the last two strategies did not give us satisfactory results. In this study, four negative regulatory genes (pykF, rpeA, rsmE and lon) were firstly knocked out of the strain GP72 genome stepwise. The yield of 2-OH-PHZ improved more than 60 folds and increased from 4.5 to about 300 mg/L. Then six key genes (ppsA, tktA, phzC, aroB, aroD and aroE) selected from the gluconeogenesis, pentose phosphate and shikimate pathways which used to enhance the shikimate pathway were overexpressed to improve the production of 2-OH-PHZ. At last a genetically engineered strain that increased the 2-OH-PHZ production by 99-fold to 450.4 mg/L was obtained. CONCLUSIONS: The 2-OH-PHZ production of P. chlororaphis GP72 was greatly improved through disruption of four negative regulatory genes and overexpression of six key genes, and it is shown that P. chlororaphis GP72 could be modified as a potential cell factory to produce 2-OH-PHZ and other phenazine biopesticides by genetic and metabolic engineering.

Development of Raw Materials and Technology for Pulping-A Brief Review.

Paper is one of the most significant inventions in human civilization, which considerably advanced global cultural development. Pulping is a key step in the conversion of fiber raw materials into paper. Since its inception, pulping has rapidly evolved, continually adapting to technological advancements. Researchers are constantly investigating various types of raw materials for pulping. In this review, some of the materials employed in pulping are outlined, and the fiber content, pulping method, as well as the strength of wood and non-wood crop straw as pulping raw materials are analyzed and discussed. In addition, this review explores the effects of different materials under various pulping conditions and assesses the future trends in raw material selection for pulping while considering the current global environmental pressures.

Biosynthesis and genetic engineering of phenazine-1-carboxylic acid in Pseudomonas chlororaphis Lzh-T5.

Phenazine-1-carboxylic acid (PCA) is a biologically active substance with the ability to prevent and control crop diseases. It was certified as a pesticide by the Ministry of Agriculture of China in 2011 and was named "Shenzimycin." Lzh-T5 is a Pseudomonas chlororaphis strain found in the rhizosphere of tomatoes. This strain can produce only 230 mg/L of PCA. We used LDA-4, which produces the phenazine synthetic intermediate trans-2,3-dihydro-3-hydroxyanthranilic acid in high amounts, as the starting strain. By restoring phzF and knocking out phzO, we achieved PCA accumulation. Moreover, PCA production was enhanced after knocking out negative regulators, enhancing the shikimate pathway, and performing fed-batch fermentation, thus resulting in the production of 10,653 mg/L of PCA. It suggested that P. chlororaphis Lzh-T5 has the potential to become an efficiency cell factory of biologically active substances.

Construction and Characterization of Fitting Equations for a New Wheat Straw Pulping Method.

The pretreatment of pulp with enzymes has been extensively studied in the laboratory. However, due to cost constraints, the application of enzymes in the pulp and paper industry is very limited. In this paper, an environment-friendly and efficient pulping method is proposed as an alternative to traditional pulping and papermaking methods. This new method overcomes the low efficiency and extreme pollution problems associated with traditional pulping methods. In addition, fitting equations for the new pulping method are constructed using data on enzyme treatments, which reflect the effect of enzymes and enable the realization of real-time control of the pulping process. The experimental results show that the efficiency of the pulping and papermaking process can be improved using biological enzymes, and the separation of cellulose can be facilitated using mixed enzymes, which have a better effect than single enzymes.

The synthesis and highly effective antibacterial properties of Cu-3, 5-dimethy l-1, 2, 4-triazole metal organic frameworks.

The influence of metal ions, the state of metal salt, and ligands on the sterilization ability of (Metalorganic frameworks) MOFs to effectively achieve sterilization has been investigated in this study. Initially, the MOFs were synthesized by elements of Zn, Ag, and Cd for the same periodic and main group of Cu. This illustrated that the atomic structure of Cu was more beneficial for coordinating with ligands. To further induce the maximum amount of Cu2+ ions in the Cu-MOFs to achieve the highest sterilization, various Cu-MOFs synthesized by the different valences of Cu, various states of copper salts, and organic ligands were performed, respectively. The results demonstrated that Cu-MOFs synthesized by 3, 5-dimethyl-1, 2, 4-triazole and tetrakis (acetonitrile) copper(I) tetrafluoroborate presented the largest inhibition-zone diameter of 40.17 mm towards Staphylococcus Aureus (S. aureus) under dark conditions. The proposed mechanism of Cu (Ⅱ) in MOFs could significantly cause multiple toxic effects, such as the generation of reactive oxygen species, and lipid peroxidation in S. aureus cells, when the bacteria was anchored by the Cu-MOFs via electrostatic interaction. Finally, the broad antimicrobial properties of Cu-MOFs against Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), and S. aureus were demonstrated. In conclusion, the Cu-3, 5-dimethyl-1, 2, 4-triazole MOFs appeared to be potential antibacterial catalysts in the antimicrobial field.

Gasless single-incision laparoscopic appendectomy.

BACKGROUND: Single-incision laparoscopic surgery has received increasing attention recently. This report describes a novel technique for single-incision laparoscopic appendectomy. METHODS: From August 2008 to October 2009, 69 consecutive patients with acute appendicitis underwent gasless single-incision laparoscopic appendectomy under epidural anesthesia at our department. An approximately 1.8-cm-long incision was made at McBurney's point. Once the abdominal wall was entered, an abdominal wall lifting device was employed in the right lower quadrant to lift up the abdominal wall to establish the operating space. Appendectomies were accomplished by use of three techniques: extracorporeally, combined extra- and intracorporeally, and intracorporeally. A drainage tube was inserted in right iliac fossa through the small incision when severe inflammation at the base of appendix or local peritonitis was present at time of operation. RESULTS: Of the 69 patients, 66 underwent successful gasless single-incision laparoscopic appendectomy. In the remaining three patients the procedure was converted to open by extending the length of the incision at McBurney's point. Average operative time was 51 min (range 38-72 min). Abdominal cavity was drained in four patients. Patients resumed mobility within 24 h postoperatively. No complications occurred intraoperatively. Postoperative complication occurred in two patients: localized abscess in one, and wound infection in another; both were treated successfully with conservative management. CONCLUSIONS: Appendectomy can be safely performed through gasless single-incision laparoscopic surgery. Further study is required to investigate any potential advantage of this method over conventional laparoscopic techniques.

A Valuable Product of Microbial Cell Factories: Microbial Lipase.

As a powerful factory, microbial cells produce a variety of enzymes, such as lipase. Lipase has a wide range of actions and participates in multiple reactions, and they can catalyze the hydrolysis of triacylglycerol into its component free fatty acids and glycerol backbone. Lipase exists widely in nature, most prominently in plants, animals and microorganisms, among which microorganisms are the most important source of lipase. Microbial lipases have been adapted for numerous industrial applications due to their substrate specificity, heterogeneous patterns of expression and versatility (i.e., capacity to catalyze reactions at the extremes of pH and temperature as well as in the presence of metal ions and organic solvents). Now they have been introduced into applications involving the production and processing of food, pharmaceutics, paper making, detergents, biodiesel fuels, and so on. In this mini-review, we will focus on the most up-to-date research on microbial lipases and their commercial and industrial applications. We will also discuss and predict future applications of these important technologies.

Genetic engineering of Pseudomonas chlororaphis Lzh-T5 to enhance production of trans-2,3-dihydro-3-hydroxyanthranilic acid.

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic ß-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in Pseudomonas spp. Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes (psrA, pykF, and rpeA) were disrupted in the genome of P. chlororaphis Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe3+ was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result, P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.

Biosynthesis and Characterization of Medium-Chain-Length Polyhydroxyalkanoate with an Enriched 3-Hydroxydodecanoate Monomer from a Pseudomonas chlororaphis Cell Factory.

Polyhydroxyalkanoates (PHAs) have been reported with agricultural and medical applications in virtue of their biodegradable and biocompatible properties. Here, we systematically engineered three modules for the enhanced biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA) in Pseudomonas chlororaphis HT66. The phzE, fadA, and fadB genes were deleted to block the native phenazine pathway and weaken the fatty acid ß-oxidation pathway. Additionally, a PHA depolymerase gene phaZ was knocked out to prevent the degradation of mcl-PHA. Three genes involved in the mcl-PHA biosynthesis pathway were co-overexpressed to increase carbon flux. The engineered strain HT4Δ::C1C2J exhibited an 18.2 g/L cell dry weight with 84.9 wt % of mcl-PHA in a shake-flask culture, and the 3-hydroxydodecanoate (3HDD) monomer was increased to 71.6 mol %. Thermophysical and mechanical properties of mcl-PHA were improved with an enriched ratio of 3HDD. This study demonstrated a rational metabolic engineering approach to enhance the production of mcl-PHA with the enriched dominant monomer and improved material properties.

The LuxS/AI-2 Quorum-Sensing System Regulates the Algicidal Activity of Shewanella xiamenensis Lzh-2.

Cyanobacterial blooming is an increasing environmental issue all over the world. Algicidal bacteria are potential tools for the control of algal blooms. The algicidal activity in many bacteria exhibits quorum-sensing (QS) dynamics and the regulatory mechanism of this activity in these bacteria is unclear. In this study, combining genomic sequencing and genome editing, we have identified that the primary quorum-sensing system in the isolated algicidal strain Shewanella xiamenensis Lzh-2 is the LuxS/AI-2 signaling pathway. Disruption of the QS system through recombination deletion of the LuxS gene led to a loss of algicides production and algicidal activity. Restoration of the LuxS gene in the deletion mutant compensated the QS system and recovered the algicidal activity. Consequently, we proved that Lzh-2 regulates the algicidal activity through LuxS/AI-2 quorum-sensing system.

Conductive Hydrogels-A Novel Material: Recent Advances and Future Perspectives.

A conductive hydrogel is a kind of polymer material having substantial potential applications with various properties, including high toughness, self-recoverability, electrical conductivity, transparency, freezing resistance, stimuli responsiveness, stretchability, self-healing, and strain sensitivity. Herein, according to the current research status of conductive hydrogels, properties of conductive hydrogels, preparation methods of different conductive hydrogels, and their application in different fields, such as sensor and actuator fabrication, biomedicine, and soft electronics, are introduced. Furthermore, the development direction and application prospects of conductive hydrogels are proposed.

Engineering a Synthetic Pathway for Gentisate in Pseudomonas Chlororaphis P3.

Pseudomonas chlororaphis P3 has been well-engineered as a platform organism for biologicals production due to enhanced shikimate pathway and excellent physiological and genetic characteristics. Gentisate displays high antiradical and antioxidant activities and is an important intermediate that can be used as a precursor for drugs. Herein, a plasmid-free biosynthetic pathway of gentisate was constructed by connecting the endogenous degradation pathway from 3-hydroxybenzoate in Pseudomonas for the first time. As a result, the production of gentisate reached 365 mg/L from 3-HBA via blocking gentisate conversion and enhancing the gentisate precursors supply through the overexpression of the rate-limiting step. With a close-up at the future perspectives, a series of bioactive compounds could be achieved by constructing synthetic pathways in conventional Pseudomonas to establish a cell factory.

Metabolic Engineering of Pseudomonas chlororaphis Qlu-1 for the Enhanced Production of Phenazine-1-carboxamide.

Phenazine-1-carboxylic acid (PCA), the primary active ingredient of Shenqinmycin, was awarded the China Pesticide Certificate in 2011 due to its excellent antibacterial action. Phenazine-1-carboxamide (PCN) is a derivative of PCA, which is modified by the phzH gene, and its anti-bacterial effect is better than that of PCA. At present, PCN can be produced via Pseudomonas fermentation using an opportunistic pathogen, Pseudomonas aeruginosa. Qlu-1 is an environmentally friendly strain of Pseudomonas chlororaphis that can produce phenazine derivatives. We replaced the phzO gene with the phzH gene from P. aeruginosa to achieve PCN accumulation. Different strategies were used to enhance PCN production: knocking out of negative regulatory factors, enhancing the shikimate pathway by gene overexpression and gene knocking, and using fed-batch fermentation. Finally, an engineered strain of P. chlororaphis was produced, which produced 11.45 g/L PCN. This achievement indicates that Qlu-1 could be modified as a potential microbial cell factory for PCN production by metabolic engineering.

Trehalose Production Using Recombinant Trehalose Synthase in Bacillus subtilis by Integrating Fermentation and Biocatalysis.

Trehalose, a stable nonreducing disaccharide, protects biomolecules against environmental stress. However, trehalose production using secretory trehalose synthase (TreS) by Bacillus subtilis has not been well studied. In this study, a mutant TreS was successfully secreted and expressed in B. subtilis WB800N. The extracellular enzyme activity of TreS regulated by the P43 promoter and SPPhoD signal peptide in recombinant B. subtilis WB800N reached 23080.6 ± 1119.4 U/L in a 5-L fermenter after optimizing the culture medium, while xpF, skfA, lytC, and sdpC were knocked out. To reduce maltose consumption, malP and amyE corresponding to maltose transporters were further deleted. To simplify the trehalose production process, we invented a fermentation-coupling biocatalysis process involving recombinant bacteria fermentation to secrete TreS and simultaneous conversion of maltose to trehalose by TreS and found that the conversion rate of maltose to trehalose reached 75.5%, suggesting that this is an efficient strategy for large-scale trehalose production using recombinant B. subtilis.

Application of a proton quantitative nuclear magnetic resonance spectroscopy method for the determination of actinodaphnine in Illigera aromatica and Illigera henryi.

Illigera aromatica S. Z. Huang et S. L. Mo and Illigera henryi W. W. Sm., belonging to the genus Illigera (Hernandiaceae), are used as herbal medicines for promoting blood circulation and treating tuberculosis. Actinodaphnine, the major bioactive alkaloid, plays an important role in the quality controls of the herbs. In the present study, a rapid, simple, accurate, and precise proton quantitative nuclear magnetic resonance (1H-qNMR) method was developed to determine the content of actinodaphnine in I. aromatica and I. henryi. DMSO-d6 enabled satisfactory separation of the signals to be integrated in 1H NMR spectrum. 1,4-Dinitrobenzene was selected as an internal standard. The limits of determination and quantitation were 0.005 and 0.038 mg/mL, respectively. This work implied that 1H-qNMR represents a feasible alternative to HPLC-based methods for quantitation of actinodaphnine in I. aromatica and I. henryi and is suitable for the quality control of I. aromatica and I. henryi.

Improving the acetylcholinesterase inhibitory effect of Illigera aromatica by fermentation with Clonostachys rogersoniana.

Illigera aromatica was fermented by Clonostachys rogersoniana. The acetylcholinesterase (AChE) inhibitory effects of unfermented and fermented I. aromatica revealed that C. rogersoniana-fermented I. aromatica (CFIA) induced significantly more AChE inhibitory activity (IC50: 35.4 ± 2.1 µg/mL). The biotransformation of actinodaphnine (1) into (4R,6aS)-4-hydroxyactinodaphnine (2) was found during the fermentation, which played an important role in the improvement of the AChE inhibitory activity of I. aromatica. Subsequently, the fermentation conditions-including the solid-liquid ratio, fermentation temperature, and fermentation time-were optimized. I. aromatica immersed in 100-200% water and fermented with C. rogersoniana at ambient temperature for 30 days was conducive to the biotransformation of actinodaphnine (1) and improved the AChE inhibitory activity of I. aromatica. The present study provides a novel approach for improving the pharmacological effect of I. aromatica and suggests that CFIA may be used as an alternative AChE inhibitor.