Optimization of fermentation parameters to improve the biosynthesis of selenium nanoparticles by Bacillus licheniformis F1 and its comprehensive application.

BACKGROUND: Selenium nanoparticles (SeNPs) are increasingly gaining attention due to its characteristics of low toxicity, high activity, and stability. Additionally, Bacillus licheniformis, as a probiotic, has achieved remarkable research outcomes in diverse fields such as medicine, feed processing, and pesticides, attracting widespread attention. Consequently, evaluating the activity of probiotics and SeNPs is paramount. The utilization of probiotics to synthesize SeNPs, achieving large-scale industrialization, is a current hotspot in the field of SeNPs synthesis and is currently the most promising synthetic method. To minimize production costs and maximize yield of SeNPs, this study selected agricultural by-products that are nutrient-rich, cost-effective, and readily available as culture medium components. This approach not only fulfills industrial production requirements but also mitigates the impact on downstream processes. RESULTS: The experimental findings revealed that SeNPs synthesized by B. licheniformis F1 exhibited a spherical morphology with diameters ranging from 110 to 170 nm and demonstrating high stability. Both the secondary metabolites of B. licheniformis F1 and the synthesized SeNPs possessed significant free radical scavenging ability. To provide a more robust foundation for acquiring large quantities of SeNPs via fermentation with B. licheniformis F1, key factors were identified through single-factor experiments and response surface methodology (RSM) include a 2% seed liquid inoculum, a temperature of 37 ℃, and agitation at 180 rpm. Additionally, critical factors during the optimization process were corn powder (11.18 g/L), soybean meal (10.34 g/L), and NaCl (10.68 g/L). Upon validating the optimized conditions and culture medium, B. licheniformis F1 can synthesize nearly 100.00% SeNPs from 5 mmol/L sodium selenite. Subsequently, pilot-scale verification in a 5 L fermentor using the optimized medium resulted in a shortened fermentation time, significantly reducing production costs. CONCLUSION: In this study, the efficient production of SeNPs by the probiotic B. licheniformis F1 was successfully achieved, leading to a significant reduction in fermentation costs. The exploration of the practical applications of this strain holds significant potential and provides valuable guidance for facilitating the industrial-scale implementation of microbial synthesis of SeNPs.

Effect of Different Selenium Species on Indole-3-Acetic Acid Activity of Selenium Nanoparticles Producing Strain Bacillus altitudinis LH18.

Acting as a growth regulator, Indole-3-acetic acid (IAA) is an important phytohormone that can be produced by several Bacillus species. However, few studies have been published on the comprehensive evaluation of the strains for practical applications and the effects of selenium species on their IAA-producing ability. The present study showed the selenite reduction strain Bacillus altitudinis LH18, which is capable of producing selenium nanoparticles (SeNPs) at a high yield in a cost-effective manner. Bio-SeNPs were systematically characterized by using DLS, zeta potential, SEM, and FTIR. The results showed that these bio-SeNPs were small in particle size, homogeneously dispersed, and highly stable. Significantly, the IAA-producing ability of strain was differently affected under different selenium species. The addition of SeNPs and sodium selenite resulted in IAA contents of 221.7 µg/mL and 91.01 µg/mL, respectively, which were 3.23 and 1.33 times higher than that of the control. This study is the first to examine the influence of various selenium species on the IAA-producing capacity of Bacillus spp., providing a theoretical foundation for the enhancement of the IAA-production potential of microorganisms.

Exploration of Streptomyces fradiae J1-021 as a Potential Host for the Heterologous Production of Spinosad.

Spinosad is a potent insecticide produced by Saccharopolyspora spinosa. However, it harbors certain limitations of a low growing rate and unfeasible genetic manipulation that can be overcome by adopting a superior platform, such as Streptomyces. Herein, we exploited the industrial tylosin-producing Streptomyces fradiae J1-021 for the heterologous production of spinosad. An engineered strain (HW01) with deletion of the tylosin biosynthetic gene cluster (BGC) was constructed and then transformed with the natural spinosad BGC. The distribution and expression levels of the tylosin BGC operons were assessed to construct a natural promoter library. The rate-limiting steps of spinosad biosynthesis were identified by analyzing the transcriptional expression of the spinosad biosynthetic genes. The stepwise engineering work involved the overexpression of the biosynthetic genes participating in rate-limiting pathways using strong promoters, affording an increase in spinosad production to 112.4 µg/L. These results demonstrate that strain HW01 has the potential to be used as a chassis for the heterologous production of polyketides.

Insect multimeric G-quadruplexes fold into antiparallel structures of different compactness and stability in K+ and Na+ solutions.

Human telomere sequences (TTAGGG)n fold into G-quadruplexes with different conformations in K+ and Na+ solutions, which are highlighted for their potential as antitumor drug targets. Moreover, human multimeric G-quadruplexes have been broadly studied potentially for screening ligands with higher selectivity than monomeric G-quadruplexes. Most insects have telomeres consisting of pentanucleotide (TTAGG) repeats, which fold into an antiparallel structured G-quadruplex with a two-layer G-planar in a K+ solution. However, the structure of insect telomeric G-quadruplexes in Na+ solutions and their higher-order structures have not been explored. The quinoline derivative BMPQ-1 has been reported to bind human multimeric G-quadruplex. This study compared the stability and compactness of insect monomeric and multimeric G-quadruplex structures in K+ and Na+ solutions and further validated the interaction between BMPQ-1 and insect multimeric G-quadruplexes. Circular dichroism (CD) spectral scanning analysis revealed that although the insect telomeric G-quadruplex folds into an antiparallel structure in both K+ and Na+ solutions, all the insect telomeric G-quadruplexes are more stable in Na+ solutions. Fluorescence resonance energy transfer (FRET) analysis indicated insect telomeric G-quadruplexes have a more compact structure in Na+ solutions. BMPQ-1 exhibited higher selectivity for insect multimeric G-quadruplex Bom37 than monomeric G-quadruplex Bom17, and had a different binding pattern to Bom37 G-quadruplex in K+ and Na+ solutions. Finally, BMPQ-1 was found to have a significant inhibitory effect on the proliferation of pest cells. This study contributes to our comprehensive understanding of insect telomeric G-quadruplexes.

Effects of Molecular Crowding on the Structure, Stability, and Interaction with Ligands of G-quadruplexes.

G-quadruplexes (G4s) are widely found in cells and have significant biological functions, which makes them a target for screening antitumor and antiviral drugs. Most of the previous research on G4s has been conducted mainly in diluted solutions. However, cells are filled with organelles and many biomolecules, resulting in a constant state of a crowded molecular environment. The conformation and stability of some G4s were found to change significantly in the molecularly crowded environment, and interactions with ligands were disturbed to some extent. The structure of the G4s and their biological functions are correlated, and the effect of the molecularly crowded environment on G4 conformational transitions and interactions with ligands should be considered in drug design targeting G4s. This review discusses the changes in the conformation and stability of G4s in a physiological environment. Moreover, the mechanism of action of the molecularly crowded environment affecting the G4 has been further reviewed based on previous studies. Furthermore, current challenges and future research directions are put forward. This review has implications for the design of drugs targeting G4s.

Molecular crowding promotes the aggregation of parallel structured G-quadruplexes.

G-quadruplexes are widely distributed in cells and are usually essential in mediating biological processes. The intracellular environment is often in a state of molecular crowding, and the current research considerably focuses on the effect of molecular crowding on the conformation of telomeric G-quadruplexes. However, G-quadruplex-forming oligonucleotides are primarily located in the promoter region of the proto-oncogene and on mRNA inside the cell and are reported to fold into parallel structures. Thus, studying the interaction mechanism between ligands and parallel structured G-quadruplexes under crowding conditions is crucial for the design of drugs targeting G-quadruplexes. In our study, molecular crowding was simulated through polyethylene glycol with an average molecular weight of 200 (PEG200) to investigate the parallel structure of the canonical G-quadruplexes c-KIT1, c-MYC, and 32KRAS and their interactions with ligands. Circular dichroism (CD) spectral scanning, fluorescence resonance energy transfer (FRET), and native polyacrylamide gel electrophoresis (PAGE) analysis revealed that molecular crowding failed to induce oligonucleotides to form parallel G-quadruplex structures in the explored model sequences while induced telomeric G-rich sequences to form antiparallel G-quadruplexes in solution without K+. Molecular crowding did not induce changes in their parallel structures but promoted the formation of G-quadruplex aggregates. Moreover, to some extent, molecular crowding also induced a looser structure of the monomer G-quadruplexes. Further studies showed that molecular crowding did not alter the binding stoichiometry of the ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino [4,3,2-kl] acridinium methosulfate (RHPS4) to c-KIT1, while it inhibited its interaction with parallel structured G-quadruplexes. This work provides new insights into developing anticancer drugs targeting parallel structured G-quadruplexes.

Highly Efficient Biotransformation and Production of Selenium Nanoparticles and Polysaccharides Using Potential Probiotic Bacillus subtilis T5

Selenium is an essential microelement required for human health. The biotransformation of selenium nanoparticles has attracted increasing attention in recent years. However, little of the literature has investigated the comprehensive evaluation of the strains for practical application and the effect on the functional properties in the existence of Se. The present study showed the selenite reduction strain Bacillus subtilis T5 (up to 200 mM), which could produce high yields of selenium polysaccharides and selenium nanoparticles in an economical and feasible manner. Biosynthesized selenium nanoparticles by B. subtilis T5 were characterized systematically using UV-vis spectroscopy, FTIR, Zeta Potential, DLS, and SEM techniques. The biosynthesized SeNPs exhibited high stability with small particle sizes. B. subtilis T5 also possessed a tolerance to acidic pH and bile salts, high aggregation, negative hemolytic, and superior antioxidant activity, which showed excellent probiotic potential and can be recommended as a potential candidate for the selenium biopharmaceuticals industry. Remarkably, B. subtilis T5 showed that the activity of α-amylase was enhanced with selenite treatment to 8.12 U/mL, 2.72-fold more than the control. The genus Bacillus was first reported to produce both selenium polysaccharides with extremely high Se-content (2.302 g/kg) and significantly enhance the activity to promote α-amylase with selenium treatment. Overall, B. subtilis T5 showed potential as a bio-factory for the biosynthesized SeNPs and organ selenium (selenium polysaccharide), providing an appealing perspective for the biopharmaceutical industry.

RHPS4 shifted the conformation ensemble equilibrium of Tel24 by preferentially stabilizing the (3 + 1) hybrid-2 conformation.

Telomeric G-quadruplexes have been a promising target for developing antitumor drugs with fewer side effects. The intracellular environment is usually in a state of molecular crowding. Studying the interaction mechanism among ligands and telomeric G-quadruplexes under crowded conditions is important for designing drugs that target telomeric G-quadruplexes. In the present study, the telomeric G-quadruplex Tel24 (TTAGGG)4 was found to fold into a conformational ensemble of parallel and (3 + 1) hybrid-2 conformations in solution with molecular crowding conditions created by PEG200. G-quadruplex-ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl] acridinium methosulfate (RHPS4) preferentially stabilized the (3 + 1) hybrid-2 conformation and shifted the conformational ensemble equilibrium of Tel24 towards the hybrid conformation. We also found that the (3 + 1) hybrid-2 conformation of Tel24 was more likely to form as compared to the parallel conformation in the conformational ensemble of Tel24. Overall, this study provides new insights into the conformation of telomere G-quadruplexes and their interactions with ligands in a physiological environment.

Enhancing the Activity of Carboxymethyl Cellulase Enzyme Using Highly Stable Selenium Nanoparticles Biosynthesized by Bacillus paralicheniformis Y4.

The inorganic selenium is absorbed and utilized inefficiently, and the range between toxicity and demand is narrow, so the application is strictly limited. Selenium nanoparticles have higher bioactivity and biosafety properties, including increased antioxidant and anticancer properties. Thus, producing and applying eco-friendly, non-toxic selenium nanoparticles in feed additives is crucial. Bacillus paralicheniformis Y4 was investigated for its potential ability to produce selenium nanoparticles and the activity of carboxymethyl cellulases. The selenium nanoparticles were characterized using zeta potential analyses, Fourier transform infrared (FTIR) spectroscopy, and scanning electron microscopy (SEM). Additionally, evaluations of the anti-α-glucosidase activity and the antioxidant activity of the selenium nanoparticles and the ethyl acetate extracts of Y4 were conducted. B. paralicheniformis Y4 exhibited high selenite tolerance of 400 mM and the selenium nanoparticles had an average particle size of 80 nm with a zeta potential value of -35.8 mV at a pH of 7.0, suggesting that the particles are relatively stable against aggregation. After 72 h of incubation with 5 mM selenite, B. paralicheniformis Y4 was able to reduce it by 76.4%, yielding red spherical bio-derived selenium nanoparticles and increasing the carboxymethyl cellulase activity by 1.49 times to 8.96 U/mL. For the first time, this study reports that the carboxymethyl cellulase activity of Bacillus paralicheniforis was greatly enhanced by selenite. The results also indicated that B. paralicheniformis Y4 could be capable of ecologically removing selenite from contaminated sites and has great potential for producing selenium nanoparticles as feed additives to enhance the added value of agricultural products.

Comparative low lethal effects of three insecticides on demographical traits and enzyme activity of the Spodoptera exigua (Hübner).

Many species of devastating insect pests have acquired a high degree of resistance to insecticides in the field during the last few decades. Spodoptera exigua, for example, is the most damaging pests of economic crops with a worldwide spread. In a present study, the comparative growth, reproduction, and detoxification enzyme activity were evaluated along with exposure to three insecticides at low lethal doses of lufenuron, indoxacarb, and spinosad as compared to the control. Results indicate that the larval developmental time was significantly extended on lufenuron (21.5 ± 29 days) followed by indoxacarb (20.28 ± 0.24 days) and spinosad (19.74 ± 0.23 days) as compared to that on the control (18.13 ± 0.13 days). Similarly, the lowest number of eggs of S. exigua females were recorded on lufenuron (328.75 ± 50.81 eggs) followed by spinosad (367 ± 36.4 eggs) and indoxacarb (411.58 ± 42.38 eggs) as compared to that on the control (560.2 ± 13.47). Interestingly, the lowest intrinsic rate of increase (r) (0.121 ± 0.009) and highest mean generation time (T) (36.2 ± 0.35 days) were observed when larvae were treated to a low lethal concentration (LC20) of lufenuron as compared to that of indoxacarb, spinosad, and control. In addition, considerably lower activity of all detoxification enzymes in larvae was recorded on lufenuron after control as compared to that on indoxacarb and spinosad. Our study serves as a reference and basis for the toxicity and low lethal evaluation of lufenuron, indoxacarb, and spinosad on life table parameters and enzymatic properties in S. exigua, which may contribute to identifying targets for effective control of S. exigua.

Minimization and optimization of α-amylase terminator for heterologous protein production in Bacillus licheniformis.

Terminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.

Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.

Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.

Toxicity and possible mechanisms of action of honokiol from Magnolia denudata seeds against four mosquito species.

This study was performed to determine the toxicity and possible mechanism of the larvicidal action of honokiol, extracted from Magnolia denudata seeds, and its 10 related compounds against third-instar larvae of insecticide-susceptible Culex pipiens pallens, Aedes aegypti, and Aedes albopictus and Anopheles sinensis resistant to deltamethrin and temephos. Honokiol (LC50, 6.13-7.37 mg/L) was highly effective against larvae of all of the four mosquito species, although the toxicity of the compound was lower than that of the synthetic larvicide temephos. Structure-activity relationship analyses indicated that electron donor and/or bulky groups at the ortho or para positions of the phenol were required for toxicity. Honokiol moderately inhibited acetylcholinesterase and caused a considerable increase in cyclic AMP levels, indicating that it might act on both acetylcholinesterase and octopaminergic receptors. Microscopy analysis clearly indicated that honokiol was mainly targeted to the midgut epithelium and anal gills, resulting in variably dramatic degenerative responses of the midgut through sequential epithelial disorganization. Honokiol did not affect the AeCS1 mRNA expression level in Ae. aegypti larvae, but did enhance expression of the genes encoding vacuolar-type H+-ATPase and aquaporin 4, indicating that it may disturb the Na+, Cl- and K+ co-transport systems. These results demonstrate that honokiol merits further study as a potential larvicide, with a specific target site, and as a lead molecule for the control of mosquito populations.

A Clade II-D Fungal Chimeric Diterpene Synthase from Colletotrichum gloeosporioides Produces Dolasta-1(15),8-diene.

Based on a terpenoid overproduction platform in yeast for genome mining, a chimeric diterpene synthase from the endophytic fungus Colletotrichum gloeosporioides ES026 was characterized as the (5R,12R,14S)-dolasta-1(15),8-diene synthase. The absolute configuration was independently verified through the use of enantioselectively deuterated terpene precursors, which unequivocally established the predicted C1-III-IV cyclization mode for this first characterized clade II-D enzyme. Extensive isotopic labeling experiments and isolation of the intermediate (1R)-δ-araneosene supported the proposed cyclization mechanism.

Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase.

Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which codes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC Finally, the engineered strain DWc9nΔ7 (Δepr ΔwprA Δmpr ΔaprE Δvpr ΔbprA ΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis This tool could be applied to strain improvement for future research.IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

Expression and functional analysis of the lysine decarboxylase and copper amine oxidase genes from the endophytic fungus Colletotrichum gloeosporioides ES026.

Huperzine A (HupA) isolated from Huperzia serrata is an important compound used to treat Alzheimer's disease (AD). Recently, HupA was reported in various endophytic fungi, with Colletotrichum gloeosporioides ES026 previously isolated from H. serrata shown to produce HupA. In this study, we performed next-generation sequencing and de novo RNA sequencing of C. gloeosporioides ES026 to elucidate the molecular functions, biological processes, and biochemical pathways of these unique sequences. Gene ontology and Kyoto Encyclopedia of Genes and Genomes assignments allowed annotation of lysine decarboxylase (LDC) and copper amine oxidase (CAO) for their conversion of L-lysine to 5-aminopentanal during HupA biosynthesis. Additionally, we constructed a stable, high-yielding HupA-expression system resulting from the overexpression of CgLDC and CgCAO from the HupA-producing endophytic fungus C. gloeosporioides ES026 in Escherichia coli. Quantitative reverse transcription polymerase chain reaction analysis confirmed CgLDC and CgCAO expression, and quantitative determination of HupA levels was assessed by liquid chromatography high-resolution mass spectrometry, which revealed that elevated expression of CgLDC and CgCAO produced higher yields of HupA than those derived from C. gloeosporioides ES026. These results revealed CgLDC and CgCAO involvement in HupA biosynthesis and their key role in regulating HupA content in C. gloeosporioides ES026.

Larvicidal activity of Magnolia denudata seed hydrodistillate constituents and related compounds and liquid formulations towards two susceptible and two wild mosquito species.

BACKGROUND: Anopheles sinensis, Aedes aegypti and Aedes albopictus and Culex pipiens pallens mosquitoes transmit malaria, dengue fever and West Nile virus diseases respectively. This study was conducted to determine the toxicity of 17 constituents from Magnolia denudata seed hydrodistillate (MD-SHD) and four experimental MD-SHD liquid formulations (10-50 mg L(-1) liquid) to third-instar larvae from insecticide-susceptible Cx. p. pallens and Ae. aegypti as well as wild Ae. albopictus and An. sinensis. RESULTS: 2,4-Di-tert-butylphenol was the most toxic constituent (LC50 1.98-3.90 mg L(-1)), followed by linoleic acid (7.19-10.49 mg L(-1)), towards larvae of the four mosquito species. High toxicity was also produced by nerolidol, (±)-limonene, α-terpinene and γ-terpinene (LC50 9.84-36.42 mg L(-1)). The toxicity of these compounds was virtually identical towards larvae of the four mosquito species, even though An. sinensis larvae were resistant to deltamethrin and temephos. The MS-SHD 50 mg L(-1) liquid resulted in 92-100% control towards larvae of the four mosquito species, while commercial temephos 200 g L(-1) emulsifiable concentrate was almost ineffective towards An. sinensis larvae (30% mortality). CONCLUSION: Reasonable mosquito control in the aquatic environment can be achieved by MD-SHD 50 mg L(-1) liquid as a potential larvicide.

Ethanol and methanol can improve huperzine A production from endophytic Colletotrichum gloeosporioides ES026.

Huperzine A (HupA) is a plant alkaloid that is of great interest as a therapeutic candidate for the treatment of Alzheimer's disease. However, the current production of HupA from plants in large quantity is unsustainable because the plant resource is scarce and the content of HupA in plants is extremely low. Surprisingly, this compound was recently found to be produced by various endophytic fungi, which are much more controllable than the plants due to simpler genetics and ease of manipulation. However, it might be due to the innate properties of endophytic symbiosis, that production of this chemical in large quantity from endophytes has not yet been put into practice. Endophytic Colletotrichum gloeosporioides ES026 was previously isolated from a HupA producing plant and the fungi also proved to produce HupA. In this study, various fermentation conditions were tried to optimize the production of HupA from C. gloeosporioides ES026. Optimization of these parameters resulted in a 25.58% increase in HupA yield. Potato extracts supplemented with glucose or sucrose but not maltose facilitated HupA producing from the fungi. A final concentration of 0.5-2% ethanol stimulated the growth of fungi while methanol with the same treatment slightly inhibited the growth. However, both methanol and ethanol greatly increased the HupA production with the highest yield of HupA (51.89% increment) coming from ethanol treatment. Further analysis showed that both ethanol and methanol were strong inducers of HupA production, while ethanol was partially used as a carbon source during fermentation. It was noticed that the color of that ethanol treated mycelia gradually became dark while methanol treated ones stayed grey during fermentation. The present study sheds light on the importance of optimizing the fermentation process, which, combined with effective inducers, maximizes production of chemicals of important economic interest from endophytic fungi.

Larvicidal activity of Cnidium monnieri fruit coumarins and structurally related compounds against insecticide-susceptible and insecticide-resistant Culex pipiens pallens and Aedes aegypti.

BACKGROUND: An assessment was made of the toxicity of imperatorin and osthole identified in Cnidium monnieri fruit, 11 related compounds and five insecticides to larvae from insecticide-susceptible Culex pipiens pallens (KS-CP strain) and Aedes aegypti and wild C.p. pallens (YS-CP colony) using a direct-contact mortality bioassay. Results were compared with those of the conventional larvicide temephos. RESULTS: Imperatorin (LC(50) = 3.14 and 2.88 mg L(-1) ) was 1.9-, 3.7- and 4.2-fold and 2.4-, 4.5- and 4.6-fold more toxic than isopimpinellin, isoimperatorin and osthole against susceptible C. p. pallens and A. aegypti larvae respectively. Overall, all of the compounds were less toxic than temephos (0.011 and 0.019 mg L(-1) ). The toxicity of these compounds was virtually identical against larvae from the two Culex strains, even though YS-CP larvae were resistant to fenthion (resistance ratio RR = 390), deltamethrin (RR = 164), cyfluthrin (RR = 14) and temephos (RR = 14). This finding indicates that the coumarins and the insecticides do not share a common mode of action. The structure-activity relationship indicates that the chemical structure and alkoxy substitution and length of the alkoxyl side chain at the C8 position are essential for imparting toxicity. CONCLUSION: The C. monnieri fruit-derived coumarins and the related coumarins described merit further study as potential insecticides or lead molecules for the control of insecticide-resistant mosquito populations.

Contact and fumigant toxicity of cinnamaldehyde and cinnamic acid and related compounds to Dermatophagoides farinae and Dermatophagoides pteronyssinus (Acari: Pyroglyphidae).

Toxicities of (E)-cinnamaldehyde and (E)-cinnamic acid and their 41 structurally related compounds to adult Dermatophagoides farinae Hughes and Dermatophagoides pteronyssinus Trouessart (Acari: Pyroglyphidae) were examined using fabric-circle contact plus fumigant and vapor-phase mortality bioassays. Results were compared with those of two acaricides, benzylbenzoate and dibutyl phthalate. In contact plus fumigant mortality bioassays, the most toxic compounds were (E)-cinnamaldehyde, methyl (E)-cinnamate, cinnamyl acetate, and hydrocinnamaldehyde against adult D.farinae (17.5-23.3 mg/m2) and D. pteronyssinus (19.0-24.0 mg/m2), based on 24-h 50% lethal concentration (LC50) values. These compounds were significantly more toxic than either benzyl benzoate (LC50, 64.9 and 60.5 mg/m2) or dibutyl phthalate (218.9 and 232.3 mg/m2). The toxicity of allyl cinnamate versus benzyl benzoate was not significantly different. Structure-activity relationship indicates that structural characteristics, such as types of functional groups, carbon skeleton, and saturation, appear to play a role in determining the compound toxicities. In vapor-phase mortality bioassays, these compounds were effective against adult D. farinae in closed, but not in open containers, indicating that their mode of delivery was largely a result of vapor action. The active compounds described merit further study as potential house dust mite control fumigants with contact action in light of global efforts to reduce the level of highly toxic synthetic acaricides in indoor environments.