Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster

Abstract

Monascus spp. produce several well-known polyketides such as monacolin K, citrinin, and azaphilone pigments. In this study, the azaphilone pigment biosynthetic gene cluster was identified through T-DNA random mutagenesis in Monascus purpureus. The albino mutant W13 bears a T-DNA insertion upstream of a transcriptional regulator gene (mppR1). The transcription of mppR1 and the nearby polyketide synthase gene (MpPKS5) was significantly repressed in the W13 mutant. Targeted inactivation of MpPKS5 also gave rise to an albino mutant, confirming that mppR1 and MpPKS5 belong to an azaphilone pigment biosynthetic gene cluster. This M. purpureus sequence was used to identify the whole biosynthetic gene cluster in the Monascus pilosus genome. MpPKS5 contains SAT/KS/AT/PT/ACP/MT/R domains, and this domain organization is preserved in other azaphilone polyketide synthases. This biosynthetic gene cluster also encodes fatty acid synthase (FAS), which is predicted to assist the synthesis of 3-oxooactanoyl-CoA and 3-oxodecanoyl-CoA. These 3-oxoacyl compounds are proposed to be incorporated into the azaphilone backbone to complete the pigment biosynthesis. A monooxygenase gene (an azaH and tropB homolog) that is located far downstream of the FAS gene is proposed to be involved in pyrone ring formation. A homology search on other fungal genome sequences suggests that this azaphilone pigment gene cluster also exists in the Penicillium marneffei and Talaromyces stipitatus genomes.

Selective production of red azaphilone pigments in a Monascus purpureus mppDEG deletion mutant

“…When O-4 was administrated to an albino mutant of M. purpureus (ΔMpPKS5) (Balakrishnan et al 2013), the mycelial pellet readily exhibited a red color, but the red colorant was not extractible with methanol (data not shown). Trials to extract this red colorant that included additional steps, such as acidifying and boiling, did not make a difference.…”

“…Yellow MAz contains a reduced pyranoquinone core, and has a visible absorption peak at 390 nm. Orange MAz, bearing a typical Az pyranoquinone core, has a maximum absorption at 470 nm and is converted into red MAz in the presence of amine (Balakrishnan et al 2013). A characteristic absorption peak of red MAz can be found at approximately 410 and 500 nm (Feng et al 2012;Shi et al 2016).…”

“…We previously identified MAz biosynthetic gene clusters in Monascus pilosus (Balakrishnan et al 2013) and M. purpureus (Balakrishnan et al 2014a) (Fig. 2).…”

“…2). Subsequent gene knockout studies in M. purpureus led us to the isolation of MAz biosynthetic intermediates, helping us to elucidate the biosynthetic pathway (Balakrishnan et al 2013;2014a;2014b;Bijinu et al 2014;Balakrishnan et al 2017a;2017b) (Fig. 1).…”

“…Az is synthesized by a non-reducing fungal polyketide synthase with a reductive release domain (NR-fPKS-R) and its collaborating enzymes (Zabala et al 2012). The following chemical modifications generate structural diversity in Az compounds (Winter et al 2012;Balakrishnan et al 2013).…”