College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China; National Engineering Research Center of JUNCAO Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China.
Food Res Int. 2018 Apr;106:626-635. doi: 10.1016/j.foodres.2018.01.037. Epub 2018 Feb 8.
Monascus spp. have been used for thousands of years as a traditional food additive in China. This mold can produce many different types of commercially valuable secondary metabolites of biological activity. Soluble starch and glycerol are the two principal carbon sources universally utilized by Monascus for the production of beneficial metabolites. In this study, the effects and regulation mechanisms of soluble starch and glycerol for M. purpureus FAFU618 on Monascus azaphilone pigments (MonAzPs) were investigated through ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS), comparative proteomics and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). The production of intracellular and extracellular pigments was significantly different between the soluble starch group (SSG) and glycerol group (GCG). Additionally, the components of intracellular pigments revealed by UPLC-QTOF-MS/MS showed that Monascin and Ankaflavin increased significantly in the GCG, while Rubropunctatin and Monascorubrin increased in the SSG. Differentially expressed proteins of mycelia between SSG and GCG were analyzed by two-dimensional gel electrophoresis (2-DE) and MALDI-TOF/TOF MS. We identified 27 proteins with statistically altered expression, of which 18 proteins associated with the EMP (glycolytic pathway), translation, energy generation, proteolysis, etc. were up-regulated, and 9 proteins, including ribosomal proteins, heat shock proteins (HSPs) and others, were down-regulated in GCG. Meanwhile, the expression levels of MonAzP biosynthetic genes were also analyzed by RT-qPCR, and the results showed that mppA, mppC, mppR1 and mppR2 were down-regulated, whereas genes MpPKS5, MpFasA2, MpFasB2, mppB, mppD and mppE were up-regulated. Collectively, these findings illustrate that the regulation of MonAzPs is not only closely related to the expression levels of certain proteins in the polyketide synthesis pathway but also closely related to the concentration of primary metabolism-generated molecules that are used as substrates for polyketide synthesis. The present study provides insights into the regulation of different carbon sources on the metabolism of MonAzPs in M. purpureus FAFU618. These results may promote further development of functional foods or medicines from Monascus spp. fermented products.
红曲菌已在中国被使用了数千年，作为一种传统的食品添加剂。这种霉菌可以产生许多不同类型的具有生物活性的商业价值次生代谢物。在生产有益代谢物方面，可溶性淀粉和甘油是红曲菌普遍使用的两种主要碳源。在这项研究中，通过超高效液相色谱-四极杆飞行时间质谱联用仪（UPLC-QTOF-MS/MS）、比较蛋白质组学和定量实时逆转录聚合酶链反应（RT-qPCR），研究了可溶性淀粉和甘油对红曲菌 FAFU618 产生的 Monascus azaphilone 色素（MonAzPs）的影响及其调控机制。在可溶性淀粉组（SSG）和甘油组（GCG）之间，细胞内和细胞外色素的产生有明显的差异。此外，UPLC-QTOF-MS/MS 揭示的细胞内色素成分表明，GCG 中 Monascin 和 Ankaflavin 显著增加，而 SSG 中 Rubropunctatin 和 Monascorubrin 增加。通过二维凝胶电泳（2-DE）和 MALDI-TOF/TOF MS 分析 SSG 和 GCG 之间菌丝体的差异表达蛋白。我们鉴定了 27 个具有统计学差异表达的蛋白质，其中 18 个与 EMP（糖酵解途径）、翻译、能量产生、蛋白水解等相关的蛋白质上调，9 个包括核糖体蛋白、热休克蛋白（HSPs）和其他蛋白在内的蛋白质下调。同时，通过 RT-qPCR 分析 MonAzP 生物合成基因的表达水平，结果表明 mppA、mppC、mppR1 和 mppR2 下调，而 MpPKS5、MpFasA2、MpFasB2、mppB、mppD 和 mppE 上调。综上所述，这些发现表明，MonAzPs 的调控不仅与聚酮合成途径中某些蛋白质的表达水平密切相关，还与作为聚酮合成底物的初级代谢产物分子的浓度密切相关。本研究为深入了解不同碳源对红曲菌 FAFU618 中 MonAzPs 代谢的调控提供了依据。这些结果可能会促进进一步开发来自红曲菌发酵产品的功能性食品或药物。
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