RESUMO
Monascus-type azaphilone pigments (MonAzPs) are produced in multi-thousand ton quantities each year and used as food colorants and nutraceuticals in East Asia. Several groups, including ours, described MonAzPs biosynthesis as a highly complex pathway with many branch points, affording more than 110 MonAzP congeners in a small group of fungi in the Eurotiales order. MonAzPs biosynthetic gene clusters (BGCs) are also very complex and mosaic-like, with some genes involved in more than one pathway, while other genes playing no apparent role in MonAzPs production. Due to this complexity, MonAzPs BGCs have been delimited differently in various fungi. Since most of these predictions rely primarily on bioinformatic analyses, it is possible that genes immediately outside the currently predicted BGC borders are also involved, especially those whose function cannot be predicted from sequence similarities alone. Conversely, some peripheral genes presumed to be part of the BGC may in fact lay outside the boundaries. This study uses a combination of computational and transcriptional analyses to predict the extent of the MonAzPs BGC in Monascus ruber M7. Gene knockouts and analysis of MonAzPs production of the mutants are then used to validate the prediction, revealing that the BGC consists of 16 genes, extending from mrpigA to mrpigP. We further predict that two strains of Talaromyces marneffei, ATCC 18224 and PM1, encode an orthologous but non-syntenic MonAzPs BGC with 14 genes. This work highlights the need to use comprehensive, integrated approaches for the more precise determination of secondary metabolite BGC boundaries.
RESUMO
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.