| Brefeldin A （BFA） is a natural antibiotic that has a 13-membered macrocyclic lactone and was first isolated from the cuture broth of Pencillium decumben in 1958. Brefeldin A possesses an array of biological activities including antiviral, antifugal, antimitotic and anticancer activities, finding its way into many useful clinical, agricultural and fundemental research applications. It has been reported that brefeldin A can drive Golgi complex disassembly and redistribution to the endoplasmic reticulum and can inhibit protein transport to post-Golgi compartments in the cell. Recently, in addition to being developed as an immunosuppressive agent, brefeldin A has received increasing attention due to its potential application as an anticancer agent, based on an NCI finding that BFA is a potential hemotherapeutic agent. Due to these diverse functions, large scale production of brefeldin A of critical importance to provide abundant reagent for both fundamental research and antitumor drug development.We isolated an endophytic fungus A1163 from microbial samples collected across the nation, whose broth displayed strong antifugal activity. The active component was purified from the fermentation broth of strain A1163, and further identified as brefeldin A using IR, HPLC-MS, NMR, X-ray crystallography techniques. Besides a convenient TLC method, a fast, precise HPLC procedure for quantitatively detecting brefeldin A content in culture broth was established.Strain Al 163 was identifed belonging to Eupenicillium brefeldianum by genetic identification along with morphology observation. As the producibility of wild E. brefeldianum A1163 was not high enough for large scale production, it was subjected to UV mutagenesis, nitrogen ion implantation. After several rounds of mutation, E. brefeldianum ZJB082702 was found to be most active, and preserved at CCTCC with a number of M 208113. The maximal concentration of E. brefeldianum CCTCC M 208113 reached approximately 78.4 mg/l prior to cultivation optimization.Single factor experiments and responses surface methodology were adopted to optimize conditions for E. brefeldianum CCTCC M 208113 biosynthesizing brefeldin A. The optimized culture medium was composed of 13.33 g/l starch, 26.66 g/l glucose, 9.7×10-1 g/l yeast extract, 1 g/l corn steep liquor, 4.6×10-1 g/l soybean cake meal, 2.5 g/l malt extract, 7.4×10-1 g /lNaNO3, 1.0×10-2 g/l CuSO4, 6.0 g /l CaCO3, 3.0 g/l MgSO4·7H2O, 4.0 g/l KH2PO4. The optimal conditions for brefeldin A biosynthesis were as follows: shaking flask with acumination baffles, culture temperature 28℃, initial medium pH 6.0, shaker speed 180rpm, inoculum size of 3 % （v/v）, loading volume at 140ml per 500ml, and fermentation time 144 h. Under these optimal conditions, the maximal brefeldin A content achieved 1301.45mg/l.Results from fermentation study showed that after 72 h preculture, addition of 0.7 % maltose raised brefeldin A yield by 10.9 %. Additionally, n-propanol and isopropanol promoted brefeldin A synthesis. Compared with the control, isopropanol improved brefeldin A production by 21.7 %. Also, Tween 80 had a positvie effect on brefeldin A concentration, approching up tol586.0 mg/l in the presence of Tween 80.Inspired by the results from fermentation in shaking flaskes, we further conducted brefeldin A fermentation with E. brefeldianum CCTCC M 208113 in 51 and 151 stirred fermentor, and the productivities for each were up to 375.2 mg/l and 648.17 mg/l respectively, which were lower than those from fermentation in shaking flaskes.Based on Logistic, Luedeking-Piret equation and the data from batch fermentation in 500ml shaking-flaskes, a mathematical model was built up describing kinetics of E. brefeldianum CCTCC M 208113 growth and brefeldin A formation. Besides, the parameters of the models were obtained through fitting the established model into experimental data. The regressed models were as follows.E. brefeldianum CCTCC M 208113 growth model: dX/dt=1.7×10-1X（1-X/（17.0））Brefeldin A formation model: dP/dt = 8.5×10-1XSubstrate depletion model: -dS/dt = 9.8×10-1dX/dt+3.4×10-3dP/dt... |
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