Yanhua Lu

Enhanced production of fumigaclavine C by ultrasound stimulation in a two-stage culture of Aspergillus fumigatus CY018

Stimulation by physical means including ultrasound is important to cell morphology and the product yield. In this work, the effect of ultrasound on the production of fumigaclavine C (FC), a conidiation-associated alkaloid with strong anti-inflammatory activity, was investigated in a newly developed two-stage culture of Aspergillus fumigatus CY018. The optimum ultrasonication conditions consisted of exposing cultures (at 12h of growth phase) to 10-min repeated irradiation (4 times) with a 24-h interval at the fixed power (500 W). Under this condition, FC production reached 118.09 mg/L, which was 89% higher than the control and much higher than previous reported values. Morphological analysis demonstrated that mycelia morphology from ultrasonication was in the form smaller and looser pellets as compared to that of the control. In addition, conidia that is closely related to FC biosynthesis were significantly increased after ultrasound stimulation, with 3 folds of that from the control.

Enhanced production of Fumigaclavine C in liquid culture of Aspergillus fumigatus under a two-stage process

Fumigaclavine C (FC) produced by Aspergillus fumigatus is a conidiation associated ergot alkaloid with strong anti-inflammatory activity. However, its wide application has been severely limited by low FC production from submerged culture. In this work, a novel two-stage culture process by combining shake culture with static culture was proposed to enhance the production of FC. After the process optimization, the FC production reached 62.7 mg/L, which was significantly higher than ever report. For scaling up this new culture process, the gas-liquid interfacial area per unit volume (Agas-liq) was identified as the key factor. The results showed that in a combined stirred-static bioreactor system, a maximum FC production (58.97 mg/L) was obtained at an Agas-liq value of 1.30 cm(2)/mL. These results demonstrated that two-stage culture is an efficient strategy to enhance FC production and the information obtained will be useful to production of this powerful bioactive compound on a large scale.

Adsorption characteristics and preparative separation of chaetominine from Aspergillus fumigatus mycelia by macroporous resin

Chaetominine (CHA) is a quinazolinone alkaloid with strong anti-cancer activity produced by Aspergillus fumigatus CY018. For recovering CHA from A. fumigates efficiently, adsorption and desorption capacities of eight macroporous resins were tested in this work. Based on batch experiments, XAD-16 resin was revealed the best adsorption and desorption performance among all the tested resins. Then, adsorption kinetics and adsorption isotherms were constructed on XAD-16 resin, and the experimental data were fitted well to the pseudo first-order kinetics and Freundlick isotherm model. In the dynamic adsorption and desorption, the purity of CHA increased from 0.0314% (w/w) in the crude extract to 57.86% in the final product with recovery yield of 70.56% by a one-step treatment. Moreover, the experiments were also performed in a lab scale-up scale, in which the purity and recovery of CHA were 56.12% (w/w) and 68.02%, respectively. In addition, XAD-16 resin could be recycled 3 times for CHA separation after regeneration without adverse effects on adsorption/desorption performance. These results suggested that XAD-16 resin adsorption could act as a useful and economic method for recovering CHA from A. fumigatus.

Assessment of the Cytotoxic and Apoptotic Effects of Chaetominine in a Human Leukemia Cell Line

Chaetominine is a quinazoline alkaloid originating from the endophytic fungus Aspergillus fumigatus CY018. In this study, we showed evidence that chaetominine has cytotoxic and apoptotic effects on human leukemia K562 cells and investigated the pathway involved in chaetominine-induced apoptosis in detail. Chaetominine inhibited K562 cell growth, with an IC50 value of 35 nM, but showed little inhibitory effect on the growth of human peripheral blood mononuclear cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Under chaetominine treatment, the Bax/Bcl-2 ratio was upregulated (from 0.3 to 8), which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway. Chaetominine inhibited K562 cell growth and induced apoptotic cell death through the intrinsic pathway, which suggests that chaetominine might be a promising therapeutic for leukemia.

Preparative separation and purification of fumigaclavine C from fermented mycelia of Aspergillus fumigatus CY018 by macroporous adsorption resin

In this work, the separation and purification of fumigaclavine C (FC), an ergot alkaloid with strong anti-inflammatory activity from fermented mycelia of Aspergillus fumigatus was systematically evaluated. Among the eight tested resins, the non-polar resin D101 displayed the best adsorption and desorption based on of static adsorption and desorption tests. Adsorption isotherms were constructed on D101 resin and fitted well to the Freundlich model. Dynamic adsorption and desorption tests on a column packed with D101 resin have been investigated for optimization of chromatographic parameters. Under optimized conditions, the contents of FC increased from 7.32% (w/w) in the crude extract to 67.54% in the final product with a recovery yield of 90.35% (w/w) via one run. Furthermore, a lab scale-up separation was carried out, in which the FC content and recovery yield were 65.83% and 90.13%, respectively. These results demonstrated that this adsorption-desorption strategy by using D101 resin was simple and efficient, thus showing potential for large scale purification and preparation of FC in the future.

Essential oil composition of Allium tuberosum seed from China

Since ancient times, many Allium species, such as onion, garlic, leek, and chives, have been used as food, spice, and herbal remedies in widespread areas of the world, especially in the northern hemisphere. The scientific name of Chinese chive is Allium tuberosum Rottler ex Spreng. (Chinese chive, Liliaceae). Chinese chive is one of the daily edible green vegetables for Chinese. Allium tuberosum is distributed all over mainland China and used not only as food but also as medicine. China is the largest Chinese chive–producing country. It is widely cultivated in China, whose seeds have been reputedly used as a traditional Chinese medicine for treating both impotence and nocturnal emissions [1, 2]. According to the dictionary of Chinese drugs [3], the leaves have been used for treatment of abdominal pain, diarrhea, hematemesis, snakebite, and asthma, while the seeds of this plant are used in Chinese folk medicine as a tonic and aphrodisiac. With regard to the constituents of Allium tuberosum, many new and known steroidal saponins, alkaloids, and amides have been reported on the Allium genus seed [4–7]. In order to clarify its bioactive compounds, we studied the chemical constituents of the seeds of Allium tuberosum systematically. We reported fatty acid compositions of seed oil of Allium tuberosum [8]. However, no chemical analysis has been carried out on the seed essential oil of this plant. In this paper we describe the results of GC/MS analyses of the volatile oil from the seeds of Allium tuberosum for the first time. The seed of Allium tuberosum was collected from the Chinese Chive Seed Company, which is subordinate to the Agriculture Science Research Institute, Henan Province, China. The plant seed sample was identified by Prof. Wu Shifu, College of Life and Environment Science, Shanghai Normal University, Shanghai. A voucher specimen is deposited in the Institute of New World Biotechnology, East China University of Science & Technology, Shanghai, P. R. China. Chinese chive seed was milled into a fine powder before water distillation. The essential oil was obtained by hydrodistillation using a Clevenger-type apparatus for 3 h, yielding 0.8% (v/w) oil for the seed on a moisture-free basis. The essential oil was dried over anhydrous sodium sulfate and studied by GC-MS. Analysis of the oil was performed using a gas chromatography (Agilent 6890) mass spectroscopy (Micromass GCT) (GC-MS) technique under the following operating conditions: chromatogram column (DB-5) length 60 m; column diameter 0.25 mm; film thickness 0.25 m; carrier gas helium; initial oven temperature 35 C for 3 min, rising to 240 C at 8 C/min and held for 20 min; injector temperature 240 C; electron ionization mass spectra were acquired over the mass rage of 30–800 amu; ionization voltage 70 eV; ionization electric current 30 A. Kovats retention indices were calculated using co-chromatographed standard hydrocarbons. The individual compounds were identified by MS, and their identity was confirmed by comparing their retention indices relative to n-alkanes and by comparing their mass spectra and retention times with those of authentic samples or with data already available in the library search, which was carried out using the Wiley GC-MS Library. The essential oil of Allium tuberosum leaves was reported to contain disulphide compounds (64.9%) and trisulphide compounds (18.9%) as main constituents [9]. The flower was reported to contain trisulphide compounds (34.0%) and disulphide compounds (20.2%) as main constituents (Wei and Wan, 2003). Trisulphide compounds (47.3%) were the main constituents for the essential oil of the Allium tuberosum rhizome. The volatile constituents of the seed of Allium tuberosum are shown in Table 1.

Enhancement of dalesconols A and B production via upregulation of laccase activity by medium optimization and inducer supplementation in submerged fermentation of Daldinia eschscholzii

Dalesconols (dalesconols A and B) are novel polyketides with strong immunosuppressive activity produced by Daldinia eschscholzii. In this work, the effects of different media (M1, M2, and M3) on fungus growth and dalesconols biosynthesis were firstly tested and compared. Intermediates and enzyme analysis indicated that laccase had the major contribution to dalesconols biosynthesis. The key role of laccase on dalesconols biosynthesis was further experimentally confirmed, which suggested that the modified M2 was more favored for laccase and dalesconols production. Thereafter, the medium composition was optimized by RSM with a fermentation titer of 36.66 mg/L obtained. Furthermore, Ca(2+) induction was employed to up-regulate of laccase activity and further enhanced dalesconols production (76.90 mg/L), which was 308% higher than that in M2. In addition, dalesconols production reached 63.42 mg/L in scale-up experiments. This work indicated great potential of laccase as a key enzyme on regulation of dalesconols production.

A Steroidal Saponin from the Seeds of Allium tuberosum

In order to investigate its bioactive compounds, we studied the chemical constituents of the seeds of Allium tuberosum systematically. Our detailed investigation of its seeds has led to the discovery of a new saponin. The new steroidal saponin, named tuberoside B, was isolated from the seeds of Allium tuberosum Rottl. ex Spreng. On the basis of acid hydrolysis, comprehensive spectroscopic analyses and comparison with spectral data of the new compound, its structure was established as (24S,25S)-5β-spirostan-2α,3β,5,24-tetraol 3-O-α-L-rhamnopyranoyl-(1 → 2)-O-[α-L-rhamnopyranosyl-(1 → 4)]-β-D-glucopyranoside. The new compound was identified by direct comparison with authentic samples (co-TLC, IR, MS, NMR) and with reported spectral and physical data. This paper deals with the isolation and structural elucidation of the new saponin.

Resveratrol improves fungal ribosylation capacity through a unique mechanism

Ribosylation is a significant modification conferring new complex and broadened cellular roles of compounds, but small organic molecules are rarely ribosylated. Here, we report the 3-O- and 4′-O-α-ribosylation of resveratrol, a phytoalexin that is exogenous to the fungus Daldinia eschscholzii IFB-TL01. The ribosylation is mechanistically due to the resveratrol-activated expression of the silent or less active fungal genes governing the ribosylation of non-fungal phytophenols. The resveratrol-induced ribosylation is also characterized by an increased expression of ribosyltransferases in concert with a rare ribosylating reaction using nicotinamide mononucleotide (NMN) as a ribose donor. The ribosylation-reduced toxicity of resveratrol to D. eschscholzii, along with the involvement of at least two p-glycoproteins in the glycosylation, suggests that such a glycosylation process may be a general strategy for the fungal detoxification of phenolic chemicals. The findings present an updated view of the ribosylation of small molecules, and provide direct chemogenetic evidence that will aid understanding of how phytophenols such as resveratrol function differently in the plant, microbial and animal kingdoms.

Improvement of fumigaclavine C production in a two-stage culture of Aspergillus fumigatus with molasses as a cost-effective ingredient

Fumigaclavine C (FC), which is produced by Aspergillus fumigatus, is a conidiation-associated ergot alkaloid with significant medical benefits. However, its application is restricted by low yields from submerged cultures. In this study, the technical feasibility of using molasses as a cost-effective ingredient for FC production in a two-stage culture of A. fumigatus was evaluated. The results indicated that molasses supplementation significantly enhanced FC accumulation by promoting conidiation and up-regulating hydroxymethylglutaryl-CoA reductase activity. Via the optimization of the two-stage process in the presence of molasses, FC production in shake flasks reached 226.9 mg/L, which was approximately three times that in the original medium (75.9 mg/L). The use of molasses as a cost-effective ingredient for FC fermentation was also successfully reproduced in a lab-scale bioreactor system in which the maximum FC production reached 215.0 mg/L. The FC production obtained in this study is the highest ever reported. This increased efficiency will enable large-scale production of FC and extend the application of molasses as a low-cost substrate for producing other conidiation-related secondary metabolites.