摘要
【目的】千层塔中分离得到的内生真菌胶孢炭疽Cg01可合成石杉碱甲(huperzine A,HupA),但产量较低,且随着继代的增加,产量下降,菌株退化严重。研究表明,表观遗传修饰与次生代谢产物的合成密切相关。本研究旨在提高HupA的产量,改善退化菌株的品质,并从表观遗传修饰的角度探讨次生代谢产物合成的机理。【方法】通过改变培养基碳源、添加生物诱导子,根据胶孢炭疽Cg01的菌落形态、菌丝生长速度、生物量及HupA产量等筛选复壮培养基;添加不同浓度的组蛋白甲基化转移酶抑制剂,检测HupA的产量,筛选提高HupA产量的小分子抑制剂;检测相关表观遗传修饰基因的表达。【结果】添加同源刺激物千层塔茎叶汁,对胶孢炭疽Cg01的菌落形态、生长速度、形态特征及生物量无显著影响,但可提高HupA的产量,传代至第5代时为对照组的1.67倍(125.7μg/L)。添加千层塔茎叶汁能显著降低组蛋白甲基化转移酶Cg12377、组蛋白去乙酰化酶Cg15620、DNA甲基化转移酶Cg02440基因的表达,提高组蛋白去乙酰化酶Cg02312基因的表达。UNC0224对内生真菌胶孢炭疽菌的HupA产量无显著影响;2‒15μmol/L BRD4770能显著提高HupA的产量(169.57‒152.10μg/L)。BRD4770组处理后,相关表观遗传基因Cg12377、Cg02440、Cg02312和Cg15620的表达量都显著下降。【结论】添加千层塔茎叶汁培养胶孢炭疽Cg01可维持其合成次生代谢产物的能力;添加组蛋白甲基化转移酶抑制剂BRD4770可提高HupA的产量。本研究为解决内生真菌大规模生产过程中的菌株退化问题提供了参考,并为组蛋白甲基化影响次生代谢产物的合成提供了依据。
[Objective]The endophytic fungus Colletotrichum gloeosporioides Cg01,isolated from Huperzia serrata,can produce huperzine A(HupA)with a low yield.As the strain was subcultured for generations,the yield decreased and the strain degraded.Studies have shown that epigenetic modification is associated with the synthesis of secondary metabolites.This study aims to increase the yield of HupA,improve the quality of degraded strains,and decipher the mechanism of secondary metabolite synthesis from the perspective of epigenetic modification.[Methods]The carbon sources and biotic elicitors for the rejuvenation media were screened according to the colony morphology,mycelial growth rate,biomass,and HupA yield.The histone methyltransferase inhibitors UNC0224 and BRD4770 were screened to improve the HupA yield.The expression of genes involved in epigenetic modification was determined.[Results]The addition of the water extract of H.serrata did not significantly affect the growth rate,morphological characteristics,or biomass of C.gloeosporioides Cg01,while it increased the yield of HupA,which was 1.67 times(125.7μg/L)that of the control group in the fifth generation.Furthermore,the addition significantly down-regulated the expression of the histone methyltransferase gene Cg12377,the histone deacetylase gene Cg15620,and the DNA methyltransferase gene Cg02440,while it up-regulated the expression of the histone deacetylase gene Cg02312.UNC0224 had no significant effect on the HupA production,while 2–15μmol/L BRD4770 increased the HupA yield(152.10–169.57μg/L)and down-regulated the expression of Cg12377,Cg02440,Cg02312,and Cg15620.[Conclusion]The secondary metabolite production of C.gloeosporioides Cg01 could be maintained by adding the water extract of H.serrata.The addition of BRD4770,a histone methyltransferase inhibitor,could increase the yield of HupA.This study provides a reference for addressing the strain degradation during the large-scale production of endophytic fungi and studying the influence of histone methylation
作者
肖郑鹏
朱亚婷
张雨轩
罗杰
刘东波
康信聪
XIAO Zhengpeng;ZHU Yating;ZHANG Yuxuan;LUO Jie;LIU Dongbo;KANG Xincong(College of Horticulture,Hunan Agricultural University,Changsha 410128,Hunan,China;State Key Laboratory of Subhealth Intervention Technology,National Administration of Traditional Chinese Medicine,Changsha 410128,Hunan,China;Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization,Changsha 410128,Hunan,China;Hunan Co-innovation Center for Utilization of Botanical Functional Ingredients,Changsha 410128,Hunan,China)
出处
《微生物学报》
CAS
CSCD
北大核心
2023年第11期4372-4382,共11页
Acta Microbiologica Sinica
基金
湖南省自然科学基金(2020JJ5266)。
关键词
胶孢炭疽
复壮
组蛋白甲基化抑制剂
甘露醇
石杉碱甲
Colletotrichum gloeosporioides
rejuvenation
histone methylation inhibitors
mannitol
huperzine A
