Efficient strategy for maintaining and enhancing the huperzine A production of Shiraia sp. Slf14 through inducer elicitation.

Huperzine A (HupA), a naturally occurring lycopodium alkaloid, is a potent, highly specific and reversible inhibitor of acetylcholinesterase and is a potential treatment for Alzheimer's disease. However, isolating HupA from Huperziaceae plants is inefficient; thus, extracting this compound from endophytic fungi may be more controllable and sustainable. However, the large-scale production of this chemical from endophytes is limited by the innate instability of endophytic fungi. In this study, we maintained the stability and viability of the HupA-producing endophytic fungus Shiraia sp. Slf14 and enhanced the HupA titers during fermentation by adding Huperzia serrata extracts (HSE), L-lysine, and acetic acid into the culture as inducers. Adding trace amounts of HupA clearly improved the HupA production of Shiraia sp. Slf14, reaching a maximum content of approximately 40 µg g(-1). Moreover, the addition of HSE and L-lysine promoted HupA production in the flask fermentation. The aforementioned bioprocessing strategy may be potentially applied to other endophytic fungal culture systems for the efficient production of plant secondary metabolites.

[Effects of electron acceptor and light on the abundance of microbial function gene related to soil CH4 emission]. / ç”µå­å—ä½“å’Œå ‰ç §å¯¹åœŸå£¤CH4æŽ’æ”¾ç›¸å ³å¾®ç”Ÿç‰©åŠŸèƒ½åŸºå› ä¸°åº¦çš„å½±å“.

To explore the effects of different electron acceptors on soil methane emission and responses of soil microorganisms to different light conditions, a strict anaerobic 20-day incubation experiment was conducted with eight treatments: darkness + Fe3+ (DF); darkness + NO3- (DN); darkness +SO42- (DS); darkness + distilled water (DCK); light + Fe3+ (LF); light + NO3- (LN); light +SO42- (LS); light + distilled water (LCK). The changes of methane concentration in the anaerobic incubation flask and the variation of the abundance of bacteria, archaea, fungi and six soil functional genes were analyzed. Results showed that soil methane emission under NO3-, SO42- addition and control (CK) was significantly lower under light conditions than dark, except the Fe3+ treatment. DN, DCK and LF treatments had the highest abundance of bacteria, fungi and archaea genes, respectively. The gene abundance of methanogenic mcrA, sulfate-reducing bacteria Dsr, and carbon-fixing CbbL were significantly up-regulated in the LF, while that of methanotrophs pmoA, iron-reducing bacteria Geo, and denitrifying bacteria nosZ were significantly up-regulated in the LN, DCK and LCK, respectively. Results of Pearson correlation and RDA analysis showed that CH4 emission was significantly positively correlated with CO2 concentration, pH, ammonium-nitrogen, and total N contents, and negatively correlated with N2O concentration, Eh, nitrate, and total C contents. Under dark condition, methane emission was positively correlated with archaea and pmoA genes abundance, and negatively correlated with other genes abundance. Under light condition, methane emission was negatively correlated with the abundance of soil microbe and functional genes. In general, methane emission under light condition was significantly lower than that under dark condition (except for the Fe3+ treatment). These results showed that it was helpful to reduce methane emission under light condition, but the increase or decrease of methane emission was closely related to the type of electron acceptors and the functional responses of soil micro-organisms.

[Isolation of endophytic fungi from Huperzia serrata and their acetylcholinesterase inhibitory activity].

A total of 127 strains of endophytic fungi were isolated from roots, branches and leaves of Huperzia serrata. These strains were identified into 19 genera based on morphological characters and ribosomal DNA (rDNA) sequence analysis, there into Penicillium, Aspergillus and Podospora were dominant populations in H. serrata. From analysis results we found some endophytic fungi showed a certain degree of tissue preference. The isolation rate and colonization rate of stems were both larger than those of leaf and roots. After testing the acetylcholinesterase (AChE) inhibitory activity of these endophytic fungi, a total of 39 endophytic fungi belonging to 15 genera showed AChE inhibition. Eleven endophytic fungi showed potent AChE inhibition, 7 of which were isolated from leaf. The research not only provided theoretical basis for developing and utilizing the resources of endophytic fungi in H. serrata but also showed a new path for searching medicines resource which has AChE inhibitory activity.

[Accumulation of flavonoids and chlorogenic acid in callus and suspension cell of Eucommia ulmoides].

OBJECTIVE: To analyze and compare the cell growth and accumulation of flavonoids and chlorogenic acid in the callus and suspension cell of Eucommia ulmoides. METHOD: The callus induced from the leaf of E. ulmoides seedlings were suspended in liquid medium. The time courses of cell growth and yields of flavonoids and chlorogenic acid were studied. RESULT: The highest contents of flavonoids and chlorogenic acid in the callus were 13.46, 1.712 mg x g(-1), respectively, while the contents of these two secondary metabolites were 16.63, 3.93 mg x g(-1) in suspension cell culture correspondingly. CONCLUSION: Comparing with callus, the suspension cell showed a short growth period and high growth rate with a remarkable high content of flavonoids and chlorogenic acid.

[Detection of cytokeratin20 mRNA in peripheral blood from colonic cancer patients by fluorescence quantitative polymerase chain reaction].

OBJECTIVE: To investigate the expression level of cytokeratin20 mRNA (CK20 mRNA) of cancer cells in peripheral blood from colorectal cancer patients, detected by fluorescence quantitative polymerase chain reaction (FQ-PCR), and to evaluate its clinical value. METHODS: To systemically study the reproducibility, quantitative range and amplification efficiency of CK20 mRNA detection by FQ-PCR, analyze and compare the result consistency with conventional RT-PCR and immunohistochemistry, separately. The expression level of CK20 mRNA of cancer cells in peripheral blood was examined in 136 colorectal cancer cases with or without hepatic metastasis. RESULTS: The within-run and between-run CV of FQ-PCR to assay CK20 mRNA were 3.6% and 5.3%, respectively, quantitative range was from 10(3) copies/ml to 10(8) copies/ml and amplification efficiency was 87.4%. Comparing with traditional RT-PCR and immunohistochemistry, the Kappa value was 0.87 and 0.83, respectively. The expression level of CK20 mRNA of cancer cells in peripheral blood from colorectal cancer patients was (3.52 +/- 1.47) x 10(4) copies/ml, and the expression positive rate was 48.5%. None was found among the 75 cases in the control group. The positive rate of CK20 mRNA of cancer cells in peripheral blood was 9.5%, 25.0%, 48.8% and 87.5% in the patients at Dukes stage A, B, C and D, respectively (P < 0.05). The positive rate of CK20 mRNA was 87.5% in patients with hepatic metastasis and 32.3% in patients without hepatic metastasis (P < 0.05). CK20 mRNA showed a tendency to decline in 35 cases of colorectal cancer within the 1st, 3rd and 5th week after operation. There was no difference among the data of pre-operation cases and on the 1st, 3rd week (P > 0.05), but a significant difference between pre-operation and the 5th week (P < 0.05). CONCLUSION: FQ-PCR is a rapid and sensitive method for quantitating CK20 mRNA. The expression of CK20 mRNA of cancer cells in peripheral blood from colorectal cancer patients has a correlation with recurrence and metastasis of the tumors. Detection of CK20 mRNA is helpful to monitor hematogenous dissemination of colorectal cancers.

[Improved production of microbial lipids in the two-liquid phase fermentation system].

In the present study, we developed a two-liquid phase fermentation system by adding 1% n-dodecane as oxygen-vector to enhance the microbial lipids productivity of Trichosporon fermentans using cassava starch hydrolysate. Results suggest that the oxygen-vector could alleviate the oxygen shortage in flask fermentation. The cell mass and lipids concentration were 101.2 g/L and 50.28 respectively in 2 L fermenter with the presence of 1% n-dodecane. Additionally, gas chromatography analysis also reveals that the microbial lipids produced by T. fermentans contained a higher percentage of saturated fatty acid in the oxygen-vector case.

[Microbial oil production by Trichosporon cutaneum B3 using cassava starch].

Microbial oil, as raw material for biodiesel, can be produced by Trichosporon cutaneum B3 using cassava starch hydrolysate. Batch cultures demonstrated that there was little inhibitory effect with the concentration of cassava starch hydrolysate up to 90 g/L. The favorable initial pH, C/N molar ratio, nitrogen source and its concentration were 6.0, 116, yeast extract and 3.0 g/L, respectively. Under the optimized conditions, dry biomass reached 15.2 g/L and lipid content reached 40.9% after culture for 144 h in flask. Batch cultures in a 2 L stirred-tank fermenter were run for 44 h and resulted in dry biomass, lipid content and lipid yield of 28.7 g/L, 42.8% and 12.27 g/L, respectively. The chemical compositions of biodiesel prepared from lipids of T cutaneum B3 mainly included palmitic acid methyl ester, stearic acid methyl ester, oleic acid methyl ester and linoleic acid methyl ester etc., and its main physicochemical properties were in compliance with relevant national diesel standards. Therefore, the biodiesel prepared from lipids of T cutaneum B3 can serve as a potential fossil fuel alternatives.

[Carbon metabolism and energetic utilization of Synechococcus sp. PCC7942 under mixotrophic condition].

To investigate the energy utilization efficiency of Synechococcus sp. PCC7942 under mixotrophic conditions, we studied its growth characteristics in mixotrophic cultures with glucose and acetic acid respectively and discussed the carbon metabolism and energy utilization based on metabolic flux analysis. Results showed that both glucose and acetate could better enhance the growth of Synechococcus sp. PCC7942, and the latter was more effective. The metabolic flux through glycolytic pathway in mixotrophic cultures was stimulated by glucose whereas depressed by acetate, while the flux through the tricarboxylic acid cycle increased in both cases. Under mixotrophic conditions, glucose makes more significant impact on the diminishment of photochemical efficiency of Synechococcus sp. PCC7942. Although the contribution of light energy was smaller, the cell yields based on total energy in mixotrophic cultures were higher comparing with photoautotrophic culture. The energy conversion efficiencies based on ATP synthesis in photoautotrophic culture, mixotrophic cultures with glucose and with acetate were evaluated to be 6.81%, 7.43% and 8.77%, respectively.