No abstract

The Monascus azaphilone (MAz) pigment is a wellknown food colorant that has yellow, orange and red components. The structures of the yellow and orange MAz differ by two hydride reductions, with yellow MAz being the reduced form. Orange MAz can be non-enzymatically converted to red MAz in the presence of amine derivatives. It was previously demonstrated that mppE and mppG are involved in the biosynthesis of yellow and orange MAz, respectively. However, ΔmppE and ΔmppG knockout mutants maintained residual production of yellow and orange MAz, respectively. In this study, we deleted the region encompassing mppD, mppE and mppG in M. purpureus and compared the phenotype of the resulting mutant (ΔmppDEG) with that of an mppD knockout mutant (ΔmppD). It was previously reported that the ΔmppD strain retained the ability to produce MAz but at approximately 10% of the level observed in the wildtype strain. A chemical analysis demonstrated that the ΔmppDEG strain was still capable of producing both yellow and orange MAz, suggesting the presence of minor MAz route(s) not involving mppE or mppG. Unexpectedly, the ΔmppDEG strain was observed to accumulate fast-eluting pigments in a reverse phase high-performance liquid chromatography analysis. A LC-MS analysis identified these pigments as ethanolamine derivatives of red MAz, which had been previously identified in an mppE knockout mutant that produces high amounts of orange MAz.Although the underlying mechanism is largely unknown, this study has yielded an M. purpureus strain that selectively accumulates red MAz.

Di-and tri-methylation of lysine 4 on histone H3 (H3K4me2 and H3K4me3, respectively) are epigenetic markers of active genes. Complex associated with Set1 (COMPASS) mediates these H3K4 methylations. The involvement of COMPASS activity in secondary metabolite (SM) biosynthesis was first demonstrated with an Aspergillus nidulans cclA knockout mutant. The cclA knockout induced the transcription of two cryptic SM biosynthetic gene clusters, leading to the production of the cognate SM. Monascus spp. are filamentous fungi that have been used for food fermentation in eastern Asia, and the pigment Monascus azaphione (MAz) is their main SM. Monascus highly produces MAz, implying that the cognate biosynthetic genes are highly active in transcription. In the present study, we examined how COMPASS activity modulates MAz biosynthesis by inactivating Monascus purpureus cclA (Mp-cclA) and swd1 (Mp-swd1). For both ΔMp-cclA and ΔMp-swd1, a reduction in MAz production, accompanied by an abated cell growth, was observed. Suppression of MAz production was more effective in an agar culture than in the submerged liquid culture. The fidelity of the ΔMp-swd1 phenotypes was verified by restoring the WT-like phenotypes in a reversion recombinant mutant, namely, trpCp: Mp-swd1, that was generated from the ΔMp-swd1 mutant. Realtime quantitative Polymerase chain reaction analysis indicated that the transcription of MAz biosynthetic genes was repressed in the ΔMp-swd1 mutant. This study demonstrated that MAz biosynthesis is under the control of COMPASS activity and that the extent of this regulation is dependent on growth conditions.

Monascus is a source of food colorant with high productivity of the pigment azaphilone. Monascus azaphilone (MAz) is biosynthesized through a single non-reducing polyketide pathway, the major components of which are ankaflavin (1), monascin (2), rubropunctatin (3) and monascorubrin (4); valuable biological activities have been reported for these compounds. Thus, various culture conditions were explored to reduce the cost of culture ingredients, enhance productivity and modulate compound composition. In the present study, we examined an extractive fermentation (EF) method with Diaion HP-20 resin (HP20) in direct comparison to a previously explored method involving Triton X-100 (TX100) to explore the modulated production of the major MAzs. We employed wild-type Monascus purpureus as well as two derivative recombinant strains (ΔmppG and ΔmppE) that are known to have differential MAz profiles as that of the wild-type strain. The HP20 resin was capable of modulating the MAz profile in favor of orange MAzs 3 and 4, oxidized congeners in this class, as was TX100-a phenomenon not previously observed for TX100 EF with Monascus anka. These finding substantiate that HP20 can be employed for the selective production of oxidized MAz and for diversifying the culture conditions used for Az production.

Advances in bacterial and fungal genome mining uncover a plethora of cryptic secondary metabolite biosynthetic gene clusters. Guided by the genome information, targeted transcriptional derepression could be employed to determine the product of a cryptic gene cluster and to explore its biological role. Monascus spp. are food grade filamentous fungi popular in eastern Asia and several genome data belong to them are now available. We achieved transcription activation of a cryptic fungal polyketide synthase-nonribosomal peptide synthase gene Mpfus1 in Monascus purpureus ΔMpPKS5 by inserting Aspergillus gpdA promoter at the upstream of Mpfus1 through double crossover gene replacement. The gene cluster with Mpfus1 show a high similarity to those for the biosynthesis of conjugated polyene derivatives with 2-pyrrolidone ring and the mycotoxin fusarin is the representative member of this group. The ΔMpPKS5 is incapable of producing azaphilone pigment, providing an excellent background to identify chromogenic and UV-absorbing compounds. Activation of Mpfus1 resulted in a yellow hue on mycelia and its methanol extract exhibit a maximum absorption at 365 nm. HPLC analysis of the organic extracts indicated the presence of a variety of yellow compounds in the extract. This implies that the product of MpFus1 is metabolically or chemically unstable. LC-MS analysis guided us to predict the MpFus1 product and to propose that the Mpfus1-containing gene cluster encode the biosynthesis of a desmethyl analogue of fusarin. This study showcases the genome mining in Monascus and the possibility to unveil new biological activities embedded in it. Keywords Activation of cryptic gene • Aspergillus gpdA promoter • Double crossover gene replacement • Fusarin analogue • Monascus purpureus • Polyketide synthase-nonribosomal peptide synthase hybrid 서 론