| Stromata of Shiraia bambusicola were used as folk drug in China,and the main active chemicals of them were hypocrellins with antibacterial,anti-HIV pharmacological activity and so on.There became a currently research hotspot which using the methods of microbial fermentation improving production of hypocrellin,due to the productivity of wild Shiraia bambusicola resources is subject to various factors,which are not enable to meet the demand for large-scale clinical trials and industrial applications.In this paper,Shiraia sp.Slf14,an endophytic fungus was isolated from Huperzia serrata,was used to further study the yield of perylene quinone compounds by the addition of exogenous metal ions.We also study the molecular mechanism of calcium signal regulation of Shiraia sp.Slf14 synthesis of Perylenequinones(PQ).This study provides a reference for research on the mechanism of PQ biosynthesis and lays a foundation for future research on combinatorial biosynthesis of PQ.The study main conclusions are as follows:(1)The effects of metal ions on the synthesis of PQ in Shiraia sp.Slf14 were studied by adding different kinds of metal ions into culture during the process of liquid fermentation and the results show that the addition of K+,Na+ and Ca2+ were beneficial to the synthesis of PQ,but Cu2+,Zn2+,Fe3+ were significantly inhibited the synthesis of PQ.Adding 6 g/L CaCl2 into the culture on the fourth day of fermentation,the total amount of PQ in the end was to 1894.66 ± 21.93 mg/L,compared with the control group increased 4.83 folds and the contents of five individual PQ(EA,EB,EC,HA and HB)were increased 5.39,3.77,7.44,4.76,2.09 folds as control respectively.(2)Study the regulation of the calcium signal on the synthesis of PQ by adding calcineurin inhibitor chlorpromazine and calcineurin inhibitor tacrolimus during the fermentation process.The results show that 1 mM chlorpromazine and 10 ?M tacrolimus significantly inhibited the synthesis of PQ while under the condition of calcium supplementation,the degree of inhibition can be relieved.These results suggest that calcium can mediate calcium signaling to regulate the synthesis of PQ.In order to further understand intracellular calcium,calcium signals and PQ relationship,we investigated the dynamic changes of intracellular Ca2 + in the fermentation process.When adding the calcium ions,the calcium content in the cells was maintained at a high level.Under the condition of adding the inhibitor,the content of intracellular Ca2+ was significantly decreased.When the inhibitor and Ca2+ were added in the culture medium simultaneously,the intracellular calcium content recuperated.The accumulation of PQ in the cells under different culture conditions proved that calcium can mediate calcium signaling to regulate the synthesis of perylene quinone.(3)Detection the transcription level changes of genes about calcium-induction and perylenequinone synthesis for the further study PQ synthesis by calcium signaling.The results showed that addition Ca2+ induced three key calcium-induction genes(cam,cna and crz1)and PQ synthesis genes(PKS?,omef and hyd)up-regulated.However,under the condition of adding inhibitors,the transcription levels of these genes were significantly down-regulated.When both the inhibitors and the Ca2+ were added to the culture medium,the transcription level of these genes restored to varying degrees relative to the case where only the inhibitor is added.The changes of the total amount of PQ and each component in the cells in different conditions indicated that the Ca2+ inhance the transcription level of the genes involved in calcium signal transduction and PQ synthesis genes that lead to improve the total amount and each component of PQ.On basis of elucidating preliminarily the regulative mechanism of PQ production,this study provides a simple and effective strategy to improve the PQ production and lays a foundation for its industrial production in future. |
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