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Phomopsis sp. XP-8 (an endophytic fungus) was previously found to produce pinoresinol diglucoside (PDG), a major antihypertensive compound of Tu-Chung (the bark of Eucommia ulmoides Oliv.), which is widely used in Chinese traditional medicines. In the present study, two bioconversion systems were developed for the production of PDG in Tris-HCl buffer containing glucose and Phomopsis sp. XP-8 cells (both resting and freeze-dried). When other factors remained unchanged, the bioconversion time, glucose concentration, cell ages, cell dosage, pH, temperature, and stirring speed influenced PDG production in a similar and decreasing manner after an initial increase with increasing levels for each factor. Considering the simultaneous change of various factors, the optimal conditions for PDG production were established as 70 g/l cells (8-day-old), 14 g/l glucose, 28°C, pH 7.5, and 180 rpm for systems employing resting cells, and 3.87 g/l cells, 14.67 g/l glucose, 28°C, pH 7.5, and 180 rpm for systems employing freeze-dried cells. The systems employing freeze-dried cells showed lower peak PDG production (110.28 μg/l), but at a much shorter time (12.65 h) compared with resting cells (23.62 mg/l, 91.5 h). The specific PDG production levels were 1.92 and 24 μg per gram cells per gram glucose for freeze-dried cells and resting cells, respectively. Both systems indicated a new and potentially efficient way to produce PDG independent of microbial cell growth.

A novel approach for the synthesis of trisubstituted isoxazoles from nitroenamines and aromatic aldehydes is developed. L-Proline/potassium carbonate system was employed to promote this process. The reaction underwent nucleophilic attack of nitroenamines to aromatic aldehydes, intramolecular denitration, tautomerization and elimination of H2O to furnish the target compounds.

Developing efficient controlled release system of insecticide can facilitate the better use of insecticide. We described here a first example of photo-controlled release of an insecticide by linking fipronil with photoresponsive coumarin covalently. The generated coumarin-fipronil (CF) precursor could undergo cleavage to release free fipronil in the presence of blue light (420nm) or sunlight. Photophysical studies of CF showed that it exhibited strong fluorescence properties. The CF had no obvious activity against mosquito larvae under dark, but it can be activated by light inside the mosquito larvae. The released Fip from CF by blue light irradiation in vitro retained its activity to armyworm (Mythimna separate) with LC50 value of 24.64μmolL-1. This photocaged molecule provided an alternative delivery method for fipronil.

Spirotetramat metabolizes to its active enol form in the plant. We described here a photocaged pesticide delivery system that can release insecticidal spirotetramat enol form upon light irradiation. Covalently linking spirotetramat-enol with photoresponsive coumarin generated the caged insecticide. The photophysical and photochemical properties, deprotection photolysis and insecticidal activities of the caged spirotetramat enol were studied. This light-triggered system can undergo cleavage to release free spirotetramat enol form at the presence of blue light (420 nm) or sunlight. Bioassays indicated that the triggered molecule has no obvious insecticidal activity against Aphis craccivora Koch at dark and could be activated by light to release the insecticidal ingredients, which provides precise control over insecticide delivery.

Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (Eucommia ulmoides Oliv) showed capability to biosynthesize pinoresinol (Pin) and pinoresinol diglucoside (PDG) from glucose (glu) and phenylalanine (Phe). To verify the mass flow in the biosynthesis pathway, [13C6]-labeled glu and [13C6]-labeled Phe were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry. As results, [13C6]-labeled Phe was incorporated into [13C6]-cinnamylic acid (Ca) and p-coumaric acid (p-Co), and [13C12]-labeled Pin, which revealed that the Pin benzene ring came from Phe via the phenylpropane pathway. [13C6]-Labeled Ca and p-Co, [13C12]-labeled Pin, [13C18]-labeled pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glu was used, demonstrating that the benzene ring and glucoside of PDG originated from glu. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study identified the occurrence of phenylalanine- lignan biosynthesis pathway in fungi at the level of mass flow.

Foodborne pathogens are serious challenges to food safety and public health worldwide. Fermentation is one of many methods that may be used to inactivate and control foodborne pathogens. Many studies have reported that lactic acid bacteria (LAB) can have significant antimicrobial effects. The current review mainly focuses on the antimicrobial activity of LAB, the mechanisms of this activity, competitive growth models, and application of LAB for inhibition of foodborne pathogens.

An enzymatic reaction system was developed and optimized for bioconversion of resveratrol from glucose. Liquid enzyme extracts were prepared from Alternaria sp. MG1, an endophytic fungus from grape, and used directly or after immobilization with sodium alginate. When the enzyme solution was used, efficient production of resveratrol was found within 120 min in a manner that was pH-, reaction time-, enzyme amount-, substrate type-, and substrate concentration-dependent. After the optimization experiments using the response surface methodology, the highest value of resveratrol production (224.40 µg/l) was found under the conditions of pH 6.84, 0.35 g/l glucose, 0.02 mg/l coenzyme A, and 0.02 mg/l ATP. Immobilized enzyme extracts could keep high production of resveratrol during recycling use for two to five times. The developed system indicated a potential approach to resveratrol biosynthesis independent of plants and fungal cell growth, and provided a possible way to produce resveratrol within 2 h, the shortest period needed for biosynthesis of resveratrol so far.

Background Phomopsis sp. XP-8 is an endophytic fungus with the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application in biosynthesis of PDG independent of plants. In order to enhance the production of PDG, 18 different natural materials were tested in solid-state cultivation of Phomopsis sp. XP-8. Results Most of the tested natural materials promoted the production of PDG. A supplement derived from mung beans produced the highest PDG yield and better fungal growth than the other materials. Also, pinoresinol monoglucoside, pinoresinol and other substrates (phenylalanine, p-coumaric acid, cinnamic acid, caffeic acid, and ferulic acid) were obtained after fermentation on mung beans. Furthermore, PDG production was much higher when mung beans were incorporated into solid state agar versus a liquid medium. The highest pinoresinol diglucoside production (72.1 mg kg(-1) in fresh culture) was obtained in 9 days using a solid state culture of Phomopsis sp. XP-8 on a mung bean grain medium containing 100 g kg(-1) glucose. Mung bean water-soluble polysaccharide was identified as a major promoter of PDG production by Phomopsis sp. XP-8. Conclusion Mung bean, especially its water-soluble polysaccharide fraction, was an efficient natural material to promote PDG production by Phomopsis sp. XP-8. © 2015 Society of Chemical Industry.

Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (EucommiaulmoidesOliv), revealed the pinoresinol diglucoside (PDG) biosynthetic pathway after precursor feeding measurements and genomic annotation. To verify the pathway more accurately, [13C6]-labeled glucose and [13C6]-labeled phenylalanine were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high performance liquid chromatography-quantitative time of flight mass spectrometry. As results, [13C6]-labeled phenylalanine was found as [13C6]-cinnamylic acid and p-coumaric acid, and [13C12]-labeled pinoresinol revealed that the pinoresinol benzene ring came from phenylalanine via the phenylpropane pathway. [13C6]-Labeled cinnamylic acid and p-coumaric acid, [13C12]-labeled pinoresinol, [13C18]-labeled pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glucose was used, demonstrating that the benzene ring and glucoside of PDG originated from glucose. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study verified the occurrence of the plant-like phenylalanine and lignan biosynthetic pathway in fungi.ImportanceVerify the phenylpropanoid-pinoresinol biosynthetic pathway and its glycosides in an endophytic fungi.

Phomopsis sp. XP-8 is an endophytic fungus that has the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application for the biosynthesis of PDG independent of plants. When cultivated in mung bean medium, PDG production was significantly improved and pinoresinol monoglucoside (PMG) and pinoresinol (Pin) were also found in the culture medium. In this experiment, starch, protein, and polysaccharides were isolated from mung beans and separately used as the sole substrate in order to explore the mechanism of fermentation and identify the major substrates that attributed to the biotransformation of PDG, PMG, and Pin. The production of PDG, PMG, and Pin was monitored using high-performance liquid chromatography (HPLC) and confirmed using HPLC-MS. Activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) were analyzed and tracked during the cultivation. The reaction system contained the compounds isolated from mung bean in the designed amount. Accumulation of phenylalanine, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin and the activities of PAL, C4H, and 4CL were measured during the bioconversion. PMG was found only when mung bean polysaccharide was analyzed, while production of PDG and Pin were found when both polysaccharide and starch were analyzed. After examining the monosaccharide composition of the mung bean polysaccharide and the effect of the different monosaccharides had on the production of PMG, PDG, and Pin, galactose in mung bean polysaccharide proved to be the major factor that stimulates the production of PMG.

The quality of kiwifruit became worse as a result of the abuse of plant growth regulators (PGRs). The safety of the fruits treated with PGRs also worried consumers. Therefore, the present study analyzed the structure of thidiazuron [TDZ, (1-phenyl-3-(1,2,3-thidiazol-5-yl)-urea)] (1) and its metabolites of biotransformation in kiwifruits using liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC–IT–TOF–MS). Standard compounds were also synthesized and used for structural identification of those metabolites. In addition, cytotoxicity of TDZ and its metabolites was tested through sulforhodamine B assays against normal Chinese hamster ovary (CHO) cells. Four metabolites were identified. They were 4-hydroxy-thidiazuron (2), 3-hydroxy-thidiazuron (3), thidiazuron-4-O-β-d-glucoside (4), and thidiazuron-3-O-β-d-glucoside (5). Values of IC50 of compounds 1, 2, and 3 to CHO cells were 18.3 ± 1.8, 37.56 ± 1.5, and 23.36 ± 1.59 μM, respectively. Compounds 4 and 5 had no effect on CHO cells.

Forchlorfenuron (1-(2-chloro-4-pyridyl)-3-phenylurea, FCF) is a plant growth regulator, being extensively used for increasing kiwifruit size. The toxicological properties of its may persist in their transformation products (TPs) or even higher toxicity than FCF. TPs elucidation of FCF in postharvest kiwifruit (Actinidia chinensis, Chinese gooseberry) by the liquid chromatography ionization hybrid ion trap and time-of-flight mass spectrometry (LC-ESI-IT-TOF/MS) in positive mode was the objective of the present study. Fifteen days after full bloom, kiwifruits were dipped for 5s with high dosage FCF solution (60 mg/L), so that sufficient peaks could be detected. The chemical structure of unknown TPs was analyzed in combination of functions of LCMS-IT-TOF, such as high-accurate MSn, formula predictor, metabolite structural analysis software MetID Solution, profiling solution metabolomics software, and neutral loss, characteristic isotopic patterns of chlorine, the fragmentation pattern and retention time of standard substances, nitrogen rule, chemical components of kiwifruit. Total 17 TPs were detected via comparisons of their accurate MSn data of commercial analytical standards and synthesized standards with high purity, such as 4-amino-2-chloropyridine, phenylurea, 2-hydroxy-FCF, 1-(2-chloro-6-((3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) pyridin-4-yl)-3-phenylurea, 1, 3-bis (2-chloropyridin-4-yl) urea, 1,3-diphenylurea, 1-(2-chloropyridin-4-yl)urea, FCF-2-O-β-D-glucoside, and so on. The major transformation pathways of FCF in kiwifruit were biochemical and photochemical cleavage pathway. The experimental results indicate that LCMS-IT-TOF is powerful and effective tool for identification of FCF TPs.

Alternaria sp. MG1, an endophytic fungus previously isolated from Merlot grape, produces resveratrol from glucose, showing similar metabolic flux to the phenylpropanoid biosynthesis pathway, currently found solely in plants. In order to identify the resveratrol biosynthesis pathway in this strain at the gene level, de novo transcriptome sequencing was conducted using Illumina paired-end sequencing. A total of 22,954,434 high-quality reads were assembled into contigs and 18,570 unigenes were identified. Among these unigenes, 14,153 were annotated in the NCBI non-redundant protein database and 5,341 were annotated in the Swiss-Prot database. After KEGG mapping, 2,701 unigenes were mapped onto 115 pathways. Eighty-four unigenes were annotated in major pathways from glucose to resveratrol, coding 20 enzymes for glycolysis, 10 for phenylalanine biosynthesis, 4 for phenylpropanoid biosynthesis, and 4 for stilbenoid biosynthesis. Chalcone synthase was identified for resveratrol biosynthesis in this strain, due to the absence of stilbene synthase. All the identified enzymes indicated a reasonable biosynthesis pathway from glucose to resveratrol via glycolysis, phenylalanine biosynthesis, phenylpropanoid biosynthesis, and stilbenoid pathways. These results provide essential evidence for the occurrence of resveratrol biosynthesis in Alternaria sp. MG1 at the gene level, facilitating further elucidation of the molecular mechanisms involved in this strain’s secondary metabolism.

Identification and evaluation of safety of forchlorfenuron ((1-(2-chloro-4-pyridyl)-3-phenylurea)), 1, metabolites after biotransformation in kiwifruit is the objective of this study. To elucidate properties of these metabolites, liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) was applied, with MetID Solution and Formula Predictor Software in positive mode. Cytotoxicity of forchlorfenuron and its metabolites were tested through sulforhodamine B assays against normal Chinese hamster ovary cells (CHO). As deduced from characteristic fragment ions of forchlorfenuron, then confirmed by comparison with synthetic standards, as well as characterized by NMR and mass spectrometry techniques, results indicate the presence of 4-hydroxyphenyl-forchlorfenuron, 2, 3-hydroxyphenyl-forchlorfenuron, 3, and forchlorfenuron-4-O-β-D-glucoside, 5. Forchlorfenuron (IC50 = 12.12 ± 2.14 μM) and 4-hydroxyphenyl-forchlorfenuron (IC50 = 36.15 ± 1.59 μM), exhibits significant cytotoxicity against CHO, while 3-hydroxyphenyl-forchlorfenuron and forchlorfenuron-4-O-β-D-glucoside show no cytotoxicity.

Pinoresinol diglucoside (PDG) and pinoresinol (Pin) are normally produced by plant cells via the phenylpropanoid pathway. This study reveals the existence of a related pathway in Phomopsis sp. XP-8, a PDG-producing fungal strain isolated from the bark of the Tu-chung tree (Eucommiaulmoides Oliv.). After addition of 0.15 g/L glucose to Phomopsis sp. XP-8, PDG and Pin formed when phenylalanine, tyrosine, leucine, cinnamic acid, and p-coumaric acid were used as the substrates respectively. No PDG formed in the absence of glucose, but Pin was detected after addition of all these substrates except leucine. In all systems in the presence of glucose, production of PDG and/or Pin and the accumulation of phenylalanine, cinnamic acid, or p-coumaric acid correlated directly with added substrate in a time- and substrate concentration- dependent manner. After analysis of products produced after addition of each substrate, the mass flow sequence for PDG and Pin biosynthesis was defined as: glucose to phenylalanine, phenylalanine to cinnamic acid, then to p-coumaric acid, and finally to Pin or PDG. During the bioconversion, the activities of four key enzymes in the phenylpropanoid pathway were also determined and correlated with accumulation of their corresponding products. PDG production by Phomopsis sp. exhibits greater efficiency and cost effectiveness than the currently-used plant-based system and will pave the way for large scale production of PDG and/or Pin for medical applications.