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Rash is one of the primary dose-limiting toxicities of Akt (protein kinase B) inhibitors in clinical trials. Here, we demonstrate the inhibition of Akt2 isozyme may be a driver for keratinocyte apoptosis, which promotes us to search for new selective Akt inhibitors with an improved cutaneous safety property. According to our previous research, compound 2 is selected for further optimization for overcoming the disadvantages of compound 1, including high Akt2 inhibition and high toxicity against HaCaT keratinocytes. The dihedral angle-based design and molecular dynamics simulation lead to the identification of Hu7691 (B5) that achieves a 24-fold selectivity between Akt1 and Akt2. Hu7691 exhibits low activity in inducing HaCaT apoptosis, promising kinase selectivity, and excellent anticancer cell proliferation potencies. Based on the superior results of safety property, pharmacokinetic profile, and in vivo efficacy, the National Medical Products Administration (NMPA) approved the investigational new drug (IND) application of Hu7691.

The gain of cell motility is an essential prerequisite for cancer metastasis. The ubiquitin ligase subunit WD repeat and SOCS box-containing 1 (WSB1) has been demonstrated to regulate hypoxia-driven tumor cell migration. However, there is still a lack of methods for discovering inhibitors targeting the WSB1 axis. Here, we employed phenotypic screening models and identified compound 4 that displayed migration inhibitory activity against WSB1-overexpressing cells. Further studies indicated that it may function as a WSB1 degrader, thus leading to the accumulation of the Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2) protein, reversing the expression of downstream F-actin and formation of membrane ruffles, and disturbing the migration capacity of cancer cells. Moreover, compound 4 exhibited a promising in vivo anticancer metastatic effects. Our findings show the discovery of a new WSB1 degrader, providing a unique solution for the treatment of cancer metastasis.

Phenylpropanoid (PPPN) compounds are widely used in agriculture, medical, food, and cosmetic industries because of their multiple bioactivities. Alternaria sp. MG1, an endophytic fungus isolated from grape, is a new natural source of PPPNs. However, the PPPN biosynthesis pathway in MG1 tends to be suppressed under normal growth conditions. Starvation has been reported to stimulate the PPPN pathway in plants, but this phenomenon has not been well studied in endophytic fungi. Here, metabolomics analysis was used to examine the profile of PPPN compounds, and quantitative reverse transcription-polymerase chain reaction was used to detect the expression of key genes in the PPPN biosynthesis pathway under starvation conditions. Starvation treatment significantly increased the accumulation of shikimate and PPPN compounds and upregulated the expression of key genes in their biosynthesis pathways. In addition to previously reported PPPNs, sinapate, 4-hydroxystyrene, piceatannol, and taxifolin were also detected under starvation treatment. These findings suggest that starvation treatment provides an effective way to optimize the production of PPPN compounds and may permit the investigation of compounds that are undetectable under normal conditions. Moreover, the diversity of its PPPNs makes strain MG1 a rich repository of valuable compounds and an extensive genetic resource for future studies.

A series of pyrazole-thiophene derivatives exhibiting good Akt inhibitory activities were obtained on the basis of conformational restriction strategy, leading to the discovery of compound 1d and 1o which showed excellent in vitro antitumor effect against a variety of hematologic cancer cells and their potential of inducing apoptosis, blocking the cell cycles at S phase and significantly inhibiting the phosphorylation of downstream biomarkers of Akt kinase of cancer cells. Amongst, compound 1o also exhibited good PK profiles and inhibited about 40% tumor growth in MM1S xenograft model. Compound 1o might be a potential candidate for further development.

A series of 3,4-disubstituted piperidine derivatives were obtained based on a conformational restriction strategy and a lead compound, A12, that exhibited potent in vitro and in vivo antitumor efficacies; however, obvious safety issues limited its further development. Thus, systematic exploration of the structure-activity relationship of compound A12, involving the phenyl group, hinge-linkage, and piperidine moiety, led to the discovery of the superior 3,4,6-trisubstituted piperidine derivative E22. E22 showed increased potency in Akt1 and cancer cell inhibition, remarkably reduced human ether-a-go-go-related gene blockage, and significantly improved safety profiles. Compound E22 also exhibited good kinase selectivity, had a good pharmacokinetic profile, and displayed very potent in vivo antitumor efficacy, with over 90% tumor growth inhibition in the SKOV3 xenograft model. Further mechanistic studies were conducted to demonstrate that compound E22 could significantly inhibit the phosphorylation of proteins downstream of Akt kinase in cells and tumor tissue from the xenograft model.

Tumor imaging tools with high specificity and sensitivity are needed to aid the boundary recognition in solid tumor diagnosis and surgical resection. In this study, we developed a near infra-red (NIR) probe (P6) for in vitro/in vivo tumor imaging on the basis of the dual strategy of cancer cell targeting and stimulus-dependent activation. The selective imaging capacity towards cancer cells of P6 was thoroughly investigated, and the potential mechanisms of endocytosis were preliminary explored. Methods: GSH-activated biotin labelled NIR probe (P6) was designed, synthesized and characterized. The GSH responsive properties were systematically illustrated through UV-vis, fluorescent tests and LC-MS analysis. In vitro fluorescent imaging of probe P6 was collected in various living cancer cell lines (i.e. SW480, HGC-27, H460, BxPC-3, KHOS) and normal cell lines (i.e. BEAS-2B, HLF-1, THP1) under confocal laser scanning microscopy. Probe P6 was further applied to image primary human cancer cells which were freshly isolated from the peritoneal carcinoma and rectal cancer patients. Serial sections of human tumor tissues were collected and sent for H&E (hematoxylin-eosin) staining and P6 imaging. Live fluorescent and photoacoustic imaging were used to investigate the in vivo imaging of P6 in both tumor and normal tissues in HGC-27 and KHOS xenograft model. Results: Probe P6 could be recognized and transported into cancer cells by tumor specific biotin receptors and efficiently be triggered by GSH to release fluorophore 4. In fact, the cellular uptake of P6 could be partially blocked by the addition of free biotin. Furthermore, probe P6 could image various cancer cell lines, as well as primary cancer cells, exhibiting a ten-fold increase in fluorescence intensity over normal cells. In freshly dissected cancer tissues, P6 fluorescent imaging distinguished the cancerous area under confocal laser scanning microscopy, which was exact the same area as indicated by H&E staining. We also found that P6 exhibited superior selectivity against cancer tissues by local injection. Conclusion: In this study, we developed a dual-modal NIR probe P6 with enhanced cellular uptake into cancer cells and environmental stimulus triggered fluorescence. Our strategy provided a novel insight into the development of imaging tools that could be potentially used for fluorescent image-guided cancer boundary recognition and possibly cancer diagnosis.

The potential of specific proteasome inhibitors to act as anti-cancer agents has attracted intensive investigations. The proteasome can be covalently inhibited by epoxyketone derivatives via a two-step reaction. Several computational approaches have been developed to mimic the covalent binding event. Compound 1 composed of a six-membered heterocyclic ring was designed by using covalent docking. With a possible different binding mode from the clinical compound Carfilzomib, it occupied the S5 pocket of 20S proteasome and showed favorable inhibitory activity. Subsequently optimization and evaluation were taken place. Among these compounds, 11h demonstrated extraordinary in vitro inhibitory activity and selectivity, and good in vivo proteasome inhibitory activity, a favorable pharmacokinetic profile and xenograft tumor inhibition. The possible binding pattern of compound 11h against proteasome was further fully explored via calculations, providing a theoretical basis for finding potent proteasome inhibitors.

A series of furan/thiophene-derived Cr-MIL-101s were obtained via visible-light-mediated C-C bond-forming catalysis within photosensitizing porous materials. The guest trapping process was achieved under very mild and metal-free conditions, affording newly functionalized MOFs with more π-π stacking, hydrogen bonding properties and excellent adsorption capacity in removing herbicides from aquatic environments.

The emergence of imazalil (IMZ) resistance in Penicillium digitatum has become a great threat for controlling citrus green mold. In this paper, we investigated the antifungal efficiency and mechanism of an alkaloid antofine against an IMZ-resistant P. digitatum strain Pdw03. Results showed that antofine exhibited a strong antifungal activity against the mycelial growth of strain Pdw03, with a minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of 1.56 × 10-3 and 1.25 × 10-2 g/L, respectively. In vivo application of antofine effectively delayed the disease progress and reduced the incidence of green mold in citrus fruit. The disease incidence of 10 × MFC antofine-treated fruit after 6 days of storage was only 11% ± 4%, which was significantly lower than that of the control (100% ± 0%). Antofine treatment altered mycelial morphology of strain Pdw03 without affecting the cell wall integrity. Although the ergosterol contents remained stable, a decrease in the total lipid content induced by lipid peroxidation was observed at 30 min of exposure, indicating disruption of cell membrane permeability of strain Pdw03. In addition, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) contents were also decreased at 60 min of exposure. These results indicated that antofine inhibited the growth of strain Pdw03 by disrupting cell membrane permeability and impairing energy metabolism induced by oxidative burst. PRACTICAL APPLICATIONS: One of the most economically important postharvest diseases of citrus fruit is green mold caused by Penicillium digitatum. The pathogen is mainly controlled by using imazalil, but the prolonged and extensive application of this chemical fungicide has led to emergence of numerous IMZ-resistant strains among P. digitatum isolates. Consequently, new and safe strategies for controlling citrus green mold caused by IMZ-resistant P. digitatum strains are urgently needed. In this study, an alkaloid antofine effectively inhibited the growth of IMZ-resistant P. digitatum strain Pdw03 and significantly decreased green mold incidence in the affected citrus fruits. Antofine induced membrane lipid peroxidation of Pdw03 mycelia, resulting in damage to the cell membrane and impairment of energy metabolism. Antofine is therefore a potential antifungal agent for the control of green mold, which provide theoretical guidance for the food industry.

Metal–organic framework (MOF)-based asymmetric heterogeneous catalysts have attracted increasing attention; however, some challenges need to be addressed, such as the rigidity of chiral auxiliary groups within MOFs and the lack of a versatile methodology for the facile construction of chiral MOFs. To address this issue, in this study, a mechanistically distinct approach was developed for heterogenizing linear chiral catalysts in the MOF cavities, rather than the synthetic modification of MOFs. This strategy involves the facile in situ polymerization of pre-impregnated chiral monomers within MOFs, affording the hybrid composites featuring a locally homogeneous and globally heterogeneous structure. The introduced chiral catalytic sites would be flexible in the pores, offering an opportunity to exploit its cooperative effect with the nearby catalytic metal nodes. The advantages of the chiral polymer/MOF composites were clarified by their excellent diastereo- and enantioselectivities and recycling capacity for catalyzing the asymmetric Aldol reaction, which were superior to those of individually heterogeneous and homogeneous catalysts.

Programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) is one of the most promising targets in the field of immune checkpoint blockade therapy. Beginning with our exploration of linkers and structure-activity relationship research, we found that the aromatic ring could replace the linker and aryl group to maintain the satisfactory activity of classic triaryl scaffold inhibitor. Based on previous studies, we designed and synthesized a series of C2-symmetric phenyl-linked compounds, and further tail optimization afforded the inhibitors, which displayed promising inhibitory activity against the PD-1/PD-L1 interaction with IC50 value at the single nanomolar range (C13-C15). Further cell-based PD-1/PD-L1 blockade bioassays indicated that these C2-symmetric molecules could significantly inhibit the PD-1/PD-L1 interaction at the cellular level and restore T cells' immune function at the safety concentrations. The discovery of these phenyl-linked symmetric small molecules showed the potential of simplified-linker and C2-symmetric strategy and provided a basis for developing symmetric small molecule inhibitors of PD-1/PD-L1 interaction. Moreover, C13 and C15 performed stable binding modes to PD-L1 dimeric after computational docking and dynamic simulation, which may serve as a good starting point for further development.

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.

AIMS This study investigated the inhibitory effect of glutaraldehyde (GA) on sour rot in citrus fruit and the underlying antifungal mechanism on mycelial growth of the causative pathogen Geotrichum citri-aurantii. METHODS AND RESULTS Glutaraldehyde exhibited a strong inhibitory effect on G. citri-aurantii, with a minimum inhibitory and fungicidal concentration (MFC) of 1·00 µl ml-1 . In addition, in vivo application of GA (1 × MFC and 5 × MFC) reduced the disease incidence of sour rot in citrus fruit by 60 and 80% respectively. Scanning electron microscopy results revealed that the morphology of G. citri-aurantii mycelia was greatly altered by GA treatment. Propidium iodide and Calcofluor White Staining revealed that the membrane permeability, rather than the cell wall integrity, of G. citri-aurantii mycelia was severely disrupted after the addition of GA. Massive accumulation of malonaldehyde and reactive oxygen species as well as an increase in lipoxygenase activity were observed. CONCLUSION These results indicate that GA may inhibit the mycelia growth of G. citri-aurantii through a membrane damage mechanism induced by membrane peroxidation. SIGNIFICANCE AND IMPACT OF THE STUDY Glutaraldehyde is expected to be a novel fungicide for controlling sour rot in citrus fruit.

Background Alternaria sp. MG1, an endophytic fungus isolated from grape, is a native producer of resveratrol, which has important application potential. However, the metabolic characteristics and physiological behavior of MG1 still remains mostly unraveled. In addition, the resveratrol production of the strain is low. Thus, the whole-genome sequencing is highly required for elucidating the resveratrol biosynthesis pathway. Furthermore, the metabolic network model of MG1 was constructed to provide a computational guided approach for improving the yield of resveratrol. Results Firstly, a draft genomic sequence of MG1 was generated with a size of 34.7 Mbp and a GC content of 50.96%. Genome annotation indicated that MG1 possessed complete biosynthesis pathways for stilbenoids, flavonoids, and lignins. Eight secondary metabolites involved in these pathways were detected by GC–MS analysis, confirming the metabolic diversity of MG1. Furthermore, the first genome-scale metabolic network of Alternaria sp. MG1 (named iYL1539) was reconstructed, accounting for 1539 genes, 2231 metabolites, and 2255 reactions. The model was validated qualitatively and quantitatively by comparing the in silico simulation with experimental data, and the results showed a high consistency. In iYL1539, 56 genes were identified as growth essential in rich medium. According to constraint-based analysis, the importance of cofactors for the resveratrol biosynthesis was successfully demonstrated. Ethanol addition was predicted in silico to be an effective method to improve resveratrol production by strengthening acetyl-CoA synthesis and pentose phosphate pathway, and was verified experimentally with a 26.31% increase of resveratrol. Finally, 6 genes were identified as potential targets for resveratrol over-production by the recently developed methodology. The target-genes were validated using salicylic acid as elicitor, leading to an increase of resveratrol yield by 33.32% and the expression of gene 4CL and CHS by 1.8- and 1.6-fold, respectively. Conclusions This study details the diverse capability and key genes of Alternaria sp. MG1 to produce multiple secondary metabolites. The first model of the species Alternaria was constructed, providing an overall understanding of the physiological behavior and metabolic characteristics of MG1. The model is a highly useful tool for enhancing productivity by rational design of the metabolic pathway for resveratrol and other secondary metabolites. Electronic supplementary material The online version of this article (10.1186/s12934-019-1063-7) contains supplementary material, which is available to authorized users.

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.

Tremendous economic losses in the citrus industry are caused by infestations of Penicillium digitatum. Previous research has shown that the terpene myrcene in citrus essential oils could stimulate the spore germination and mycelial growth of P. digitatum. However, the underlying mechanism is poorly known. Here, how myrcene induces the spore germination of P. digitatum was elucidated. The addition of myrcene significantly promoted the spore germination of P. digitatum and green mold in citrus fruit. As the incubation time increased, the intracellular and extracellular myrcene contents both significantly decreased in myrcene-treated samples, and no downstream products were detected through a Gas Chromatography-Mass Spectrometer. Metabolomics analysis revealed that the contents of metabolites involved in central carbon metabolism, such as glycolysis, pentose phosphate pathway, and citrate cycle, were significantly affected after the addition of myrcene. The adenosine triphosphate content and the intracellular energy charge were also drastically increased. Overall, our results suggest that the ability of myrcene to stimulate the spore germination of P. digitatum might be mediated by the upregulation of central carbon and energy metabolism.

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 present study describes the isolation of the alkaloid “antofine” from Cynanchum atratum BUNGE as well as its anti-fungal potential against the citrus postharvest pathogen Penicillium digitatum. Antofine, which was extracted using an ultrasonic-assisted method and identified by 1H, 13C-NMR, and LC/MS analysis, showed strong antifungal activity against P. digitatum, with an observed minimum inhibitory concentration (MIC) and a minimum fungicidal concentration (MFC) of 1.56 × 10−3 and 1.25 × 10-2 g L-1, respectively. In vivo assays showed that antofine could significantly reduce the incidence of green mold. Furthermore, the total lipid and ergosterol contents of P. digitatum decreased after antofine treatment, indicating the disruption of membrane integrity. In addition, antofine caused a significant reduction of the intracellular adenosine triphosphate (ATP) content during the entire exposure period. These results suggest that the antifungal activity of antofine against P. digitatum can be attributed to the disruption of the cell membrane integrity and energy metabolism.

Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT-PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT-PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species. Electronic supplementary material The online version of this article (doi:10.1186/s13568-016-0283-z) contains supplementary material, which is available to authorized users.

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.

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.