Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host / 生物工程学报

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.

Endophytic Bacillus subtilis P10 from Prunus cerasifera as a biocontrol agent against tomato Verticillium wilt / Bacillus subtilis P10 endofítico de Prunus cerasifera como agente de biocontrole contra murcha de Verticillium de tomate

Endophytic bacteria serve key roles in the maintenance of plant health and growth. Few studies to date, however, have explored the antagonistic and plant growth-promoting (PGP) properties of Prunus cerasifera endophytes. To that end, we isolated endophytic bacteria from P. cerasifera tissue samples and used a dual culture plate assay to screen these microbes for antagonistic activity against Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum, and F. moniliforme. Of the 36 strains of isolated bacteria, four (strains P1, P10, P16, and P20) exhibited antagonistic effects against all five model pathogens, and the P10 strain exhibited the strongest antagonistic to five pathogens. This P10 strain was then characterized in-depth via phenotypic assessments, physiological analyses, and 16s rDNA sequencing, revealing it to be a strain of Bacillus subtilis. Application of a P10 cell suspension (1×108 CFU/mL) significantly enhanced the seed germination and seedling growth of tomato in a greenhouse setting. This P10 strain further significantly suppressed tomato Verticillium wilt with much lower disease incidence and disease index scores being observed following P10 treatment relative to untreated plants in pot-based experiments. Tomato plants that had been treated with strain P10 also enhanced defense-related enzymes, peroxidase, superoxide dismutase, and catalase activity upon V. dahliae challenge relative to plants that had not been treated with this endophytic bacterium. The results revealed that the P10 bacterial strain has potential value as a biocontrol agent for use in the prevention of tomato Verticillium wilt.

As bactérias endofíticas desempenham papel fundamental na manutenção da saúde e do crescimento das plantas. Poucos estudos até o momento, no entanto, exploraram as propriedades antagônicas e promotoras de crescimento de plantas (PGP) de endófitos de Prunus cerasifera. Para esse fim, isolamos bactérias endofíticas de amostras de tecido de P. cerasifera e usamos um ensaio de placa de cultura dupla para rastrear esses micróbios quanto à atividade antagonista contra Verticillium dahliae, Botryosphaeria dothidea, Fusarium oxysporum, F. graminearum e F. moniliforme. Das 36 cepas de bactérias isoladas, quatro (cepas P1, P10, P16 e P20) exibiram efeitos antagônicos contra todos os cinco patógenos modelo, e a cepa P10 exibiu o antagonista mais forte para cinco patógenos. Essa cepa P10 foi então caracterizada em profundidade por meio de avaliações fenotípicas, análises fisiológicas e sequenciamento de rDNA 16s, revelando ser uma cepa de Bacillus subtilis. A aplicação de uma suspensão de células P10 (1 × 108 UFC / mL) aumentou significativamente a germinação das sementes e o crescimento das mudas de tomate em casa de vegetação. Essa cepa P10 suprimiu ainda mais a murcha de Verticillium do tomate com incidência de doença muito menor e pontuações de índice de doença sendo observadas após o tratamento com P10 em relação a plantas não tratadas em experimentos baseados em vasos. As plantas de tomate que foram tratadas com a cepa P10 também aumentaram as enzimas relacionadas à defesa, peroxidase, superóxido dismutase e atividade da catalase após o desafio de V. dahliae em relação às plantas que não foram tratadas com essa bactéria endofítica. Os resultados revelaram que a cepa bacteriana P10 tem valor potencial como agente de biocontrole para uso na prevenção da murcha de Verticillium em tomate.

Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

Strategies and tools for metabolic engineering in Bacillus subtilis / 生物工程学报

As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.

Research progress and industrial application of Bacillus subtilis in systematic and synthetic biotechnology / 生物工程学报

Bacillus subtilis is a model strain for studying the physiological and biochemical mechanisms of microorganism, and is also a good chassis cell for industrial application to produce biological agents such as small molecule compounds, bulk chemicals, industrial enzymes, precursors of drugs and health product. In recent years, studies on metabolic engineering methods and strategies of B. subtilis have been increasingly reported, providing good tools and theoretical references for using it as chassis cells to produce biological agents. This review provides information on systematically optimizing the Bacillus subtilis chassis cell by regulating global regulatory factors, simplifying and optimizing the genome, multi-site and multi-dimensional regulating, dynamic regulating through biosensors, membrane protein engineering. For producing the protein reagent, the strain is optimized by optimizing the promoters, signal peptides, secretion components and building the expression system without chemical inducers. In addition, this review also prospects the important issues and directions that need to be focused on in the further optimization of B. subtilis in industrial production.

Effects of different application methods of Bacillus subtilis agent on soil microbial diversity and growth of muskmelon / 生物工程学报

Continuous planting of muskmelon and excessive application of chemical fertilizers have caused a series of problems, such as imbalance of the soil micro-ecological environment, serious soil-borne diseases and yield loss. Application of Bacillus subtilis agent is an important way to improve soil micro-ecological environment, prevent soil-borne diseases, and promote plant growth. In this study, B. subtilis was used as experimental agent to analyze the effects of different application methods on the soil microbial diversity and growth of muskmelon in greenhouse. The number of culturable microorganisms in soil was measured by dilution-plate method. The diversity of soil uncultivated microorganisms was determined by Illumina Miseq sequencing technology. The yield of muskmelon was measured by weighing method. The number of culturable bacteria in the root irrigation, hole application and dipping root application groups was higher than that of the control in different muskmelon growth stages, but there was no significant difference among the three different application methods. The number of soil fungi from B. subtilis agent treatment groups in flowering stage was significantly lower in comparison to the control group. However, B. subtilis agent treatment did not cause significant difference on soil fungi number at the fruiting and pulling stage. Diversity analysis of uncultured microorganisms showed that the Shannon index values of bacteria were higher and Simpson index values were lower respectively in the three B. subtilis treatment groups than that in the control. Moreover, the dipping root treatment produced the lowest Shannon index value and the highest Simpson index value of fungi. NMDS and cluster analysis showed that B. subtilis agents dipping root treatment significantly affected the bacterial and fungal flora, both of which were clustered into one independent branch. The application of B. subtilis agents, especially dipping root treatment, significantly decreased the abundance of Bacteroidetes, increased the abundance of Actinobacteria and Acidobacteria. The B. subtilis agent treatment didn't produce significant effect on the diversity of fungal flora except Chytridiomycota. The height, stem diameter and leaf area of muskmelon increased by applying B. subtilis agents, and dipping root treatment produced the most significant effect. As a new type of environmental protection fertilizer, B. subtilis agent can increase the number of soil culturable microorganisms, improve soil microbial diversity, and promote growth and yield. This study would provide a scientific basis for the rational application of B. subtilis.

A novel expression vector for the secretion of abaecin in Bacillus subtilis

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.

Lipopeptide production by Bacillus subtilis R1 and its possible applications

Abstract The possible application of a bacterial strain - Bacillus subtilis R1, isolated from an oil contaminated desert site in India, as biocontrol agent and its biosurfactant in microbial enhanced oil recovery are discussed. The biosurfactant production in minimal medium was carried out at different temperatures and salt concentrations, where it produced an efficient biosurfactant at 30-45 °C and in presence of up to 7% salt. It significantly reduced the surface tension from 66 ± 1.25 mN/m to 29 ± 0.85 mN/m within 24 h. In order to enhance the biosurfactant production, random mutagenesis of B. subtilis R1 was performed using chemical mutagen - ethyl methanesulfonate. Majority of the isolated 42 mutants showed biosurfactant production, but the difference was statistically insignificant as compared with parent strain R1. Therefore none of the mutants were selected for further study, and only parent strain R1 was studied. The biosurfactant was quite stable under harsh conditions for up to 10 days. The biosurfactant was extracted and characterized as similar to the lipopeptide group - surfactins and fengycin. The crude oil displacement experiments using biosurfactant broth in sand pack glass columns showed 33 ± 1.25% additional oil recovery. The strain also showed inhibition of various plant pathogenic fungi on potato dextrose agar medium.

Indole acetic acid and ACC deaminase from endophytic bacteria improves the growth of Solarium lycopersicum

Background: Endophytic bacteria are ubiquitous in all plant species contributing in host plant's nutrient uptake and helping the host to improve its growth. Moringa peregrina which is a medicinal plant, growing in arid region of Arabia, was assessed for the presence of endophytic bacterial strains. Results: PCR amplification and sequencing of 16S rRNA of bacterial endophytes revealed the 5 endophytic bacteria, in which 2 strains were from Sphingomonas sp.; 2 strains from Bacillus sp. and 1 from Methylobacterium genus. Among the endophytic bacterial strains, a strain of Bacillus subtilis LK14 has shown significant prospects in phosphate solubilization (clearing zone of 56.71 mm after 5 d), ACC deaminase (448.3 ± 2.91 nM α-ketobutyrate mg-1 h-1) and acid phosphatase activity (8.4 ± 1.2 nM mg-1 min-1). The endophytic bacteria were also assessed for their potential to produce indole-3-acetic acid (IAA). Among isolated strains, the initial spectrophotometry analysis showed significantly higher IAA production by Bacillus subtilis LK14. The diurnal production of IAA was quantified using multiple reactions monitoring method in UPLC/MS-MS. The analysis showed that LK14 produced the highest (8.7 uM) IAA on 14th d of growth. Looking at LK14 potentials, it was applied to Solanum lycopersicum, where it significantly increased the shoot and root biomass and chlorophyll (a and b) contents as compared to control plants. Conclusion: The study concludes that using endophytic bacterial strains can be bio-prospective for plant growth promotion, which might be an ideal strategy for improving growth of crops in marginal lands.

Tutorial for writing systematic reviews for the Brazilian Journal of Physical Therapy (BJPT) / Tutorial para elaboração de revisões sistemáticas para o Brazilian Journal of Physical Therapy (BJPT)

Systematic reviews aim to summarize all evidence using very rigorous methods in order to address a specific research question with less bias as possible. Systematic reviews are widely used in the field of physical therapy, however not all reviews have good quality. This tutorial aims to guide authors of the Brazilian Journal of Physical Therapy on how systematic reviews should be conducted and reported in order to be accepted for publication. It is expected that this tutorial will help authors of systematic reviews as well as journal editors and reviewers on how to conduct, report, critically appraise and interpret this type of study design. .

Revisões sistemáticas têm como objetivo sumarizar toda a evidência disponível, através de métodos rigorosos, para responder a uma pergunta de pesquisa específica com o mínimo de viés possível. Revisões sistemáticas são amplamente utilizadas na fisioterapia, porém nem todas as revisões possuem boa qualidade. Esse tutorial tem como objetivo guiar os autores do Brazilian Journal of Physical Therapy sobre como revisões sistemáticas deveriam ser conduzidas e descritas para que sejam aceitas para publicação. Espera-se que esse tutorial irá auxiliar autores de revisões sistemáticas, assim como editores e revisores de periódicos em como conduzir, descrever, fazer análise crítica e interpretar esse tipo de delineamento de pesquisa.

Potentialities of newly isolated Bacillus subtilis and Lactobacillus sp for curd preparation and a comparative study of its physico-chemical parameters with other marketed curds.

Two Bacillus sp. were isolated from the local fermented milk and identified on the basis 16S rRNA sequence profile as Bacillus subtilis AKL1 and by biochemical process as Lactobacillus acidophilus AKL2. These isolates were used as fresh inoculums for curd preparation individually and in combinations. Different physico-chemical and therapeutic properties of the newly prepared curd were examined and compared with marketed local (sweet and sour) and branded (Mother Dairy and Thackar) curds. The total hydrolyzed peptides, free amino acids, lactic acid were significantly higher, whereas, total solid, ash content, syneresis and free reducing sugar were lower in the curd prepared by a mixture of AKL1 and AKL2 (0.5:0.5, v/v). The antioxidant activity against ABTS+, DPPH•, OH• and Fe3+ were also higher in the newly formulated curd. Polyphenols (85.5µg/g), flavonoids (12.5µg/g) and free aromatic amino acids contents were also higher in AKL1+AKL2. All these components prevent excess protein oxidation that was revealed by SDS-PAGE. The curd also exhibited potent antimicrobial activity against some entero-pathogens like Clostridium perfringens, Escherichia coli, Shigella dysentery, Vibrio cholerae and Staphylococcus aureus. It can be concluded that the combination of these Lactobacillus sp. will be a fruitful inoculum for the preparation of curd having better health promoting effects.

Expression of virulence genes by Listeria monocytogenes J0161 in natural environment

Majority of studies concerning the gene expression of Listeria monocytogenes have been done on pure culture states. Our objective was to study L.monocytogenes in a co-cultured state and to understand if microbes in their natural state of existence are different in their expression than that of the purely cultured lab grown forms. For a long period discussions have been on the expression of prfA, (which is a virulence gene regulator) in a mammalian host and its role in causing the switch from a saprophytic to pathogenic form of L.monocytogenes. We, in this paper for the first time report the expression of prfA and other virulence genes by L.monocytogenes under different extracellular conditions, and also as a pure culture biofilms, that is different from the previous reports. We also report that the expression of prfA seems to vary considerably when co-cultured with Bacillus subtilis.

Molecular cloning, purification and characterization of thermostable -1,3-1,4 glucanase from Bacillus subtilis A8-8.

A gene encoding a -1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant -1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60oC, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60oC and retained 30% of its original activity at 70oC for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.

Molecular detection of lipase A gene in putative bacillus subtilis strains isolated from soil

The present study was undertaken to screen the soil samples collected in Iran for the presence of the Bacillus subtilis lipase A gene. The bacterial colonies obtained from the collected soil samples were examined by physical appearance, biochemical tests and the polymerase chain reaction [PCR]. Only four colonies were identified as putative B. subtilis strains and all contained the lipase A gene. However, the intensities of the DNA bands were different and correlated with the differences obtained from the biochemical tests. Polymorphism of the lipase gene was also determined in samples using SSCP assay. In conclusion, this study demonstrates an easy and reliable method for detection of the lipase gene in B. subtilis strains. Further screening of the soil by this method will enable the detection and identification of industrially more favorable lipases

The mutagenic and antimutagenic effects of the traditional phytoestrogen-rich herbs, Pueraria mirifica and Pueraria lobata

Pueraria mirifica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism. We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indicator strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and antimutagenic tests. Both extracts at a final concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence of anti-mutagenic activities of the two plant extracts were confirmed in rec-assays and further supported by a micronucleus test where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder) failed to exhibit significant micronucleus formation in rats. The tests confirmed the non-mutagenic but reasonably antimutagenic activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.

Cloning of protective antigen gene from Bacillus anthracis into Bacillus subtilis

Protective antigen [PA] of Bacillus anthracis is used as anthrax vaccine. Cloning and expression of the PA gene in various strain such as E.coli and Bacillus subtilis was reported that most expression was in B. subtilis up to 160 microg/ml. The objectives of this study were: to clone the gene of PA in an expression vector [pWB980] and then transformation into the B. subtilis WB600 strain. The pXOl plasmid was separated from the strain stern of B. anthracis with alcalin method and the PA gene with 2.4kb sequence amplified by PCR. Then the amplified fragment was directly cloned into pTZ57R plasmid as T-vector and transferred into E.coli DH5 alpha using CaC12 method. After that the gene was separated from the T-vector by enzymatic digestion [Sa1I and KpnI]. Ligation between the purified gene fragment of the PA and the vector was carried out. Then it was transferred into B. subtilis WB600 by electroporation method in 1000 V. In this study we isolated PA gene from B. anthracis strain stern with PCR and was cloned into pTZ57R plasmid. The Presence of the gene was confirmed by restriction analysis, PCR and sequencing. Then the PA gene was cloned into pWB980 and B. subtilis and the presence of the gene in two kanamycin resistant colonies [AMN1 and AMN3] was confirmed by restriction analysis and PCR. We may conclude that by making modification in the methods used and using pWB980 expression vector, we were able to clone the PA gene into B. subtilis. This is the first research project in Iran that the PA gene is isolated and cloned and B. subtilis is used as host

Isolation and characterization of stable mutants of Streptomyces peucetius defective in daunorubicin biosynthesis.

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, epsilon-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed by dnrE (mutants SPAK and SPMAG), dnrS (SPFS and SPRHO) and doxA (SPDHC) gene products.

Microbial production of L-tyrosine: a review.

Microbial production of L-tyrosine by direct fermentation and by enzymatic methods has been reviewed. Achievements in this regard made through recombinant DNA techniques have also been included. The review also includes biosynthesis and regulation of tyrosine.

Viabilidad de Bacillus subtilis auxotrofos en ausencia de sus metabolitos esenciales / Viability of Bacillus subtilis auxotrophs in the absence of their essential metabolites

Se estudió el efecto de la privación de triptofano y uracilo sobre la viabilidad del tansformante Bacillus subtilis BSA trp ura y de las cepas parentales Bacillus subtilis PB 168 trp-C y Bacillus subtilis PB 3308 ura. Los ensayos se llevaron a cabo en las condiciones descriptas para el desarrollo de competencia para transformación, durante 16 h. Los resultados obtenidos demostraron que Bacillus subtilis BSA 170 trp ura es resistente a la muerte por carencia de triptofano durante el tiempo del ensayo y a la muerte por carencia de uracilo durante 3h. Luego ocurre una disminución de la recuperación de UFC, acompañada por una reduccción de las absorbancias de los cultivos. La muerte por carencia de uracilo es prevenida en ausencia de triptofano, sugiriendo que reuiere síntesis de proteínas. Las cepas parentales mostraron un comportamiento semejante. Bacillus subtilis PB 168 trp-C mostró ser resistente a la muerte por carencia de triptofano y Bacillus subtilis PB 3308 uta sufrió una reducción de la capacidad para formar colonias, debida a la privación de uracilo, comparable a la de la cepa transformante