Complete mitochondrial genome of the mulberry psyllid Paurocephala sauteri Enderlein (Hemiptera: Psyllidae) and phylogenetic analysis.

Paurocephala sauteri (Enderlein, 1914) (Hemiptera: Psyllidae) is a species of a psyllid distributed in Asia. Mulberry is the only known host for P. sauteri until now. The complete mitogenome of P. sauteri (accession number: MT759765) 14,963 bp in size, including 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs genes. The base composition of the whole P. sauteri mitogenome is 40.26% for A, 7.86% for G, 34.07% for T, and 11.81% for C, with a high AT bias of 80.33%. The mitochondrial genome of P. sauteri was sequenced and annotated as the first representative of family Paurocephalidae. The present data could contribute to a detailed phylogeographic analysis of this valuable economic insect for further study in differentiating closely related species.

[Biosynthesis analysis of hederagenin pathway and its construction in yeast cells].

Hederagenin is an effective constituent of many medical plants, such as Clematidis Radix, and has a wide range of applications in anti-tumor, anti-inflammatory, antidepressant, hepatoprotective antibacterial, et al. In order to obtain the efficient production of yeast cells for hederagenin,we successfully cloned and screened out a P450 gene MdMA02 from Malus×domestica which can catalyze oleanolic acid C-23 oxidation with our developed plug and play platform. Its amino acid homology is only 32% as compared to characterized CYP72A68v2. By transforming MdMA02 to the oleanolic acid-producing strain BY-OA, a hederagenin-producing strain was constructed and hederagenin's titer could achieve 101 mg·L⁻¹ using high cell density fermentation, which was 337 times higher than in shake flasks culturing. This study provides a basis for further research on promoting the creation of oleanane-type pentacyclic triterpenoids biosynthetic pathway analysis and relative cell factories construction.

Comparative study on fermentation performance in the genome shuffled Candida versatilis and wild-type salt tolerant yeast strain.

BACKGROUND: The fermentation performance of a genome-shuffled strain of Candida versatilis S3-5, isolated for improved tolerance to salt, and wild-type (WT) strain were analysed. The fermentation parameters, such as growth, reducing sugar, ethanol, organic acids and volatile compounds, were detected during soy sauce fermentation process. RESULTS: The results showed that ethanol produced by the genome shuffled strain S3-5 was increasing at a faster rate and to a greater extent than WT. At the end of the fermentation, malic acid, citric acid and succinic acid formed in tricarboxylic acid cycle after S3-5 treatment elevated by 39.20%, 6.85% and 17.09% compared to WT, respectively. Moreover, flavour compounds such as phenethyl acetate, ethyl vanillate, ethyl acetate, isoamyl acetate, ethyl myristate, ethyl pentadecanoate, ethyl palmitate and phenylacetaldehyde produced by S3-5 were 2.26, 2.12, 2.87, 34.41, 6.32, 13.64, 2.23 and 78.85 times as compared to WT. CONCLUSIONS: S3-5 exhibited enhanced metabolic ability as compared to the wild-type strain, improved conversion of sugars to ethanol, metabolism of organic acid and formation of volatile compounds, especially esters, Moreover, S3-5 might be an ester-flavour type salt-tolerant yeast. © 2016 Society of Chemical Industry.

Direct cloning, expression of a thermostable xylanase gene from the metagenomic DNA of cow dung compost and enzymatic production of xylooligosaccharides from corncob.

OBJECTIVES: To acquire a thermostable xylanase, that is suitable for xylooligosaccharide production from pretreated corncobs, the metagenomic method was used to obtain the gene from an uncultured environmental microorganism. RESULTS: A thermostable xylanase-encoding gene (xyn10CD18) was cloned directly from the metagenomic DNA of cow dung compost. When xyn10CD18 was expressed in Bacillus megaterium MS941, extracellular xylansae activity at 106 IU/ml was achieved. The purified recombinant Xyn10CD18 was optimally active at pH 7 and 75 °C as measured over 10 min. It retained over 55% of its initial activity at 70 °C and pH 7 after 24 h. Its action on birchwood xylan for 18 h liberated xylooligosaccharides with 2°-4° of polymerization, with xylobiose and xylotetraose as the main products. When pretreated corncobs were hydrolyzed by Xyn10CD18 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80% and the xylose was just increased by 3%. CONCLUSION: Xyn10CD18 is a thermostable endoxylanase and is a promising candidate for biomass conversion and xylooligosaccharide production.

Purification and characterization of a thermostable xylanase from Paenibacillus sp. NF1 and its application in xylooligosaccharides production.

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by Ca(2+), Ba(2+), DTT, and ß-mercaptoethanol, but was inhibited by Fe(3+), Zn(2+), Fe(2+), Cu(2+), SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at 60°C and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature (70°C-80°C) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The end products of high efficient oat spelt xylan hydrolysis by XynNF (an endoxylanase) containing 95.8% xylooligosaccharides of 2-4 degree of polymerization (DP2-4) with the enrichment of xylobiose (61.5%) indicated that XynNF is a promising candidate for xylooligosaccharides production.

[Construction of Saccharomyces cerevisiae cell factories for lycopene production].

For microbial production of lycopene, the lycopene synthetic genes from Pantoea agglomerans were integrated into Saccharomyces cerevisiae strain BY4742, to obtain strain ZD-L-000 for production of 0.17 mg · L(-1) lycopene. Improving supplies of isoprenoid precursors was then investigated for increasing lycopene production. Four key genes were chosen to be overexpressed, inclu- ding truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMG1), which is the major rate-limiting enzyme in the mevalonate (MVA) pathway, a mutated global regulatory factor gene (upc2.1), a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1), which is a key enzyme in the diterpenoid synthetic pathway, and GGPP synthase gene (SaGGPS) from Sulfolobus acidocaldarius. Over-expression of upc2.1 could not improve lycopene production, while over-expression of tHMGI , BTS1-ERG20 and SaGGPS genes led to 2-, 16. 9- and20. 5-fold increase of lycopene production, respectively. In addition, three effective genes, tHMG1, BTS1-ERG20 and SaGGPS, were integrated into rDNA sites of ZD-L-000, resulting in strain ZD-L-201 for production of 13.23 mg · L(-1) lycopene, which was 77-fold higher than that of the parent strain. Finally, two-phase extractive fermentation was performed. The titer of lycopene increased 10-fold to 135.21 mg · L(-1). The engineered yeast strains obtained in this work provided the basis for fermentative production of lycopene.

Construction of Escherichia coli strains producing L-serine from glucose.

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.

Optimization of nitrogen source for enhanced production of squalene from thraustochytrid Aurantiochytrium sp.

Nitrogen (N) sources, the critical medium component, were optimized for squalene production by microalga Aurantiochytrium sp. in heterotrophic cultures. In screening experiments monosodium glutamate, yeast extract and tryptone were found to enhance cell growth and squalene production. The optimal levels of the three nitrogen sources were further determined through central composite experimental design. The squalene content and yield were both influenced not only by monosodium glutamate, tryptone and yeast extract, but also by their interactions. The squalene content and squalene yield were described by the second-order polynomial equations with high confidence levels (>99%). The optimal concentrations of monosodium glutamate, yeast extract and tryptone were predicted to be 6.61 g/L, 6.13 g/L and 4.50 g/L for squalene content and 6.94 g/L, 6.22 g/L and 4.40 g/L for squalene yield, respectively. In the verification experiment, the squalene content and squalene yield reached 0.72 mg/g and 5.90 mg/L, respectively, which were much higher than those obtained in previous studies.

Screening and characterization of squalene-producing thraustochytrids from Hong Kong mangroves.

Eighteen strains of thraustochytrids were newly isolated from Hong Kong mangroves, and their fatty acid and squalene contents were analyzed. All strains could grow well heterotrophically with glucose as the sole carbon source. All of them had the typical fatty acid profile of thraustchytrids and could produce a large amount of docosahexaenoic acid. The cell dry weight ranged from 5.49 to 15.62 g/L and squalene content from 0.02 to 0.18 mg/g at 72 h. The highest squalene-producing strain, BR-MP4-A1 was identified as a new strain of Aurantiochytrium species through sequence comparison of the 18S rRNA gene. The highest biomass of Aurantiochytrium sp. BR-MP4-A1 was achieved at 72 h, whereas its squalene content reached the maximum of 0.567 mg/g at 36 h but decreased rapidly thereafter. The production of squalene by thraustochytrids might be highly influenced by culture conditions.

Expression, purification, and characterization of phosphatidylserine synthase from Escherichia coli K12 in Bacillus subtilis.

Although phosphatidylserine synthase (PSS) from Escherichia coli is an ideal enzyme for phospholipid production, its application in the food industry has been limited because of the low PSS yield. In this study, the pss gene was cloned from E. coli K(12) and expressed in Bacillus subtilis DB104, and the recombinant PSS was characterized subsequently. PSS was purified to 39.59-fold, and the highest activity was detected as 13.62 U/mg. The enzyme was found to be stable in a pH range of 6.5-9.5, with optimal pH values of 8.0 for hydrolysis and 7.0 for transphosphatidylation, respectively. The optimal temperature for PSS activity was 35 degrees C. The enzyme activity could be detected after 1 h of heating at 65 degrees C. Among the detected detergents and metal ions, Triton X-100, Ca(2+), Mn(2+), and Co(2+) could improve PSS activity. The transformation of phosphatidylcholine to phosphatidylserine under PSS catalyzation was carried out in a biphasic system, which confirmed the actual catalyzing ability of the recombinant protein.

[Cloning, characterization and application of the promoter region of the alkaline protease gene in Bacillus alcalophillus PB92].

Promoter function fragment of alkaline protease gene (GenBank accession number: EU130686) was cloned from Bacillus alcalophillus PB92 genome by TAIL-PCR. Sequenced and analyzed revealed that it contains several typical promoter characterized regions. Two reverse translation frames were located in -538~-370 bp and-275~-128 bp. Deletion analysis of the sequence demonstrated that 414 bp to 619 bp upstream of the TSS showed predominant promoter activity, and a 105 bp length sequence can serve as this function. Additionally, a representative Sec-type signal peptide structure was detected in PB92 AprE signal peptide. By cloning the PaprE and alkaline protease signal peptide gene into pBE2, an expression vector pBEAC was constructed, and a plant sweet protein monellin gene was highly expressed in B. subtilis 1A751.

Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations.

This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k(cat)/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k(cat)/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.

Natamycin production by Streptomyces gilvosporeus based on statistical optimization.

Natamycin has been widely used as a natural preservative to prevent mold contamination in food. In this study, statistically based experimental designs were employed for the optimization of medium components for natamycin production by Streptomyces gilvosporeus. After glucose, yeast extract, and soy peptone were screened as suitable carbon and nitrogen sources, a full factorial design was used to evaluate the effects of various factors on natamycin production. Glucose and pH were identified as having significant effects (with confidence level >90%). Glucose concentration and initial pH were subsequently optimized by use of a central composite design. The result indicated that glucose and pH had a significant interactive effect on natamycin production. The optimal glucose concentration and initial pH value were 38.2 g/L and 7.8, respectively. This optimization strategy led to a natamycin yield of 2.45 g/L, which was nearly 90% higher than that in the original medium.

Characterisation of mutagenised acid-resistant alpha-amylase expressed in Bacillus subtilis WB600.

Based on the original thermostable alpha-amylase gene from Bacillus licheniformis, two amino acids were site-directed mutagenised by polymerase chain reaction to obtain a new gene. This gene, with Leu134-->Arg and Ser320-->Ala, was substituted for acid-resistant capability previously. To favor purification of the product, high-level expression and secretion of mature, authentic and stable recombinant mutagenised alpha-amylase were achieved with protease-deficient strain Bacillus subtilis WB600 as the host. The recombinant mutagenised alpha-amylase with the activity of 4,700 U/mL was then purified by ammonium sulphate fractionation, anion exchange and gel filtration, consecutively. By multi-step purification, the specific activity of the recombinant protein was up to 916.7 U/mg with a 187.1-fold purification. The mutagenised protein was found to be more acid resistant than the native protein. The optimum pH and stable range of pH with the mutagenised protein was 4.5 and 4.0 to 6.5, respectively, compared with pH 6.5 and 5.5 to 7.0 as the favorite pH and pH stability range of the native protein.

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate.

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH4)2SO4 (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH2PO4. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.

Statistical optimization of medium components for avilamycin production by Streptomyces viridochromogenes Tü57-1 using response surface methodology.

A fermentation medium for avilamycin production by Streptomyces viridochromogenes Tü57-1 has been optimized. Important components and their concentrations were investigated using fractional factorial design and Box-Behnken Design. The results showed that soybean flour, soluble starch, MgSO4.7H2O and CaCl2.2H2O are important for avilamycin production. A polynomial model related to medium components and avilamycin yield had been established. A high coefficient of determination (R2 = 0.92) was obtained that indicated good agreement between the experimental and predicted values of avilamycin yield. Student's T-test of each coefficient showed that all the linear and quadratic terms had significant effect (P > |T| < 0.05) on avilamycin yield. The significance of tested components was related to MgSO4.7H2O (0.37 g/L), CaCl2.2H2O (0.39 g/L), soybean flour (21.97 g/L) and soluble starch (37.22 g/L). The yield of avilamycin reached 88.33 +/- 0.94 mg/L (p < 0.05) that was 2.8-fold the initial yield.

[Overexpression of a sweet protein monellin in Escherichia coli].

According to the amino acid sequence of monellin, a single chain 294bp monellin gene was synthesized and inserted into vector pET-22b to yield the recombinant secretion plasmid pETMO. The single-chain monellin gene was designed based on the biased codons of E. coli so that its expression would be then optimized. Under the expressing conditions, monellin was produced accounting for 44.8% of total soluble proteins. The E. coli-expressed single-chain monellin is 3000 times sweeter than sucrose. The thermal-stability and acid-resistance of the protein are higher than the natural monellin.

[Characteristics of a new fibrinolytic enzyme produced from Rhizopus chinensis 12#].

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gamma-chains of fibrinogen simultaneously, and it also activated plasminogen to plasmin. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and Km was 0.23 mmol/L and Kcat 16.36 s(-1). The optimal temperature of the enzyme for hydrolying fibrin was 45 degrees C, and the optimal pH range of 6.8 - 8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5 +/- 0.1. The enzyme was a glycoprotein. EDTA, PCMB, PMSF inhibited the activety of the enzyme, and SBTI, Lys, TPCK, Aprotinine had none obvious inhibition, which suggested that the activity centre of the enzyme had hydrosulfuryl, metal and serine. The first 12 amino acids of the N-termimal sequence of the enzyme were NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly, and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeutic agent in thrombosis.

[Differential acute mortality among the allozyme genotypes of Oxya chinensis by pesticide avermectin].

The rice grasshopper Oxya chinensis exhibits polymorphic loci at Ldh, Gpi, Pgm and Me. The data of the mean number of alleles per locus (A = 2.8), percentage of polymorphic loci (P = 80.0%), the observed mean heterozygosities (Ho = 0.271 approximately 0.279) and the expected mean heterozygosities (He = 0.305 approximately 0.316) of the species suggest that O. chinensis possesses sufficient genetic diversity. It was hypothesized that the high polymorphisms at Ldh, Gpi, Pgm and Me might make it possible for pesticide avermectin to act as a selective agent through differential lethality among the insect individuals with different genotypes. In this study a total of 855 grasshoppers were injected with avermectin (1.3 x 10(-2) g/g) to obtain a mortality of 54% after 24 hours. The allozyme analysis was then employed to determine the genotypes of Ldh, Gpi, Pgm and Me for both dead and surviving individuals. Contingency table chi2 tests showed that avermectin displayed random lethal effects on the genotypes at the loci of Ldh, Pgm and Me, without correlation between the genotype and mortality. In contrast, at Gpi locus, the grasshopper demonstrated a mortality cline of Gpi-AA (38%), Gpi-AB (51%), Gpi-BB (58%) and Gpi-BC (74%). The significant mortality differences were found among the following genotype pairs: Gpi-AA vs. Gpi-BB, Gpi-AA vs. Gpi-BC and Gpi-AB vs. Gpi-BC. These data implied the Gpi-AA genotype was likely related to the specie's resistance to the pesticide avermectin. It was also noted that the Gpi-A allele was present in the genotypes with low morality,while Gpi-B was present in the genotypes with moderate mortality, and the individuals with Gpi-C allele exhibited the highest mortality. The data obtained in this study suggested that the increasing proportion of Gpi-AA genotype and perhaps Gpi-A allele in a population may be useful as a potential resistant biomarker of O. chinensis to pesticide avermectin.

[Impacts of malathion on population genetic structure of Oxya chinensis].

Allozyme electrophoresis was employed to compare the difference in mortality among the genotypes at two polymorphic loci of Pgm and Me of grasshopper Oxya chinensis individuals acutely exposed to 1.5g/L malathion which resulted in 56% mortality in 24 hours. The selective lethal effects were observed among the genotypes at Pgm locus but not at Me locus. It is noted that the genotype Pgm-ab experienced the highest mortality (80%), whereas Pgm-bb and Pgm-bc were 49%, lower than the average. The chi(2) tests showed significant difference in morality between Pgm-bb and Pgm-cc. After exposure the allele frequency of Pgm-b showed a notable increase among surviving individuals. The cluster analysis based on Roger's genetic distance indicated that the acute exposure to malathion can cause differentiation in genetic composition at population level in Oxya chinensis. Because malathion is commonly used as the insecticide for grasshopper control, the data obtained in this study suggest that the similar genotype-mortality effects may occur in crop fields.