Tetrahedron Letters 55 (2014) 1640–1643

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Tetrahedron Letters

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Mpp7 controls regioselective Knoevenagel condensation during thebiosynthesis of Monascus azaphilone pigments

http://dx.doi.org/10.1016/j.tetlet.2014.01.0900040-4039/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +82 31 330 6470; fax: +82 31 336 0870.E-mail address: hjink@mju.ac.kr (H.-J. Kwon).

Bijinu Balakrishnan a, Chien-Chi Chen b, Tzu-Ming Pan c, Hyung-Jin Kwon a,⇑a Department of Biological Science, Myongji University, Yongin-si, Gyunggi-do 449-728, Republic of Koreab Bioinformatics Group, Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwanc Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Roosevelt Road, Taipei, Taiwan

a r t i c l e i n f o

Article history:Received 6 December 2013Revised 14 January 2014Accepted 22 January 2014Available online 31 January 2014

Keywords:Monascus azaphilone pigment biosynthesisMonascus purpureusKnoevenagel aldol condensationTargeted inactivation of mpp7

a b s t r a c t

Targeted inactivation of the mpp7 gene in the Monascus azaphilone pigment (MAzP) biosynthetic genecluster resulted in the accumulation of monasfluol A (7) and B (8), of which the latter was a novel com-pound, and the abolition of the main MAzPs. It is thus proposed that 7 and 8 are the products of non-enzymatic Knoevenagel condensation followed by a reduction and that Mpp7 assists in regioselectiveKnoevenagel aldol condensation during MAzP biosynthesis.

� 2014 Elsevier Ltd. All rights reserved.

Monascus extract has served as a traditional food colorant ineastern Asia, and the main colored substances of this extract areazaphilone compounds. These Monascus azaphilone pigments(MAzPs), produced from some Monascus species, such as Monascusruber, Monascus pilosus, and Monascus purpureus, comprise ankafla-vin (1), monascin (2), rubropunctatin (3), monascorubrin (4),rubropuctamine (5), and monascorubramine (6), along with vari-ous minor compounds (Scheme 1).1 MAzPs can be divided intothree classes depending on their colors: yellow, orange, and red.MAzPs 1 and 2 are yellow-colored, and the orange pigments 3and 4 turn into the red pigments 5 and 6, respectively, when re-acted with ammonia. The yellow and orange MAzPs display diversebiological properties including anti-diabetic, anti-inflammatory,anti-atherosclerosis, and anti-cancer activities.2

T-DNA random mutagenesis studies have previously localizedthe MAzP biosynthesis loci in M. ruber and M. purpureus,3 the latterof which was used to identify the MAzP biosynthetic gene clusterfrom the M. pilosus genome sequence (GenBank accession no.KC148521).3b The genome sequences of M. ruber and M. purpureusrecently became available in the genome portal of the Departmentof Energy-Joint Genome Institute (DOE-JGI). These M. ruber and M.purpureus genome sequences were used to compare the geneticorganizations of the MAzP biosynthesis clusters from these threeMonascus species (Fig. 1). The clusters encode the blueprints for

four oxidoreductases, two transcription factors, an acyltransferase,and an esterase between MpPKS5 and MpfasB2 (Fig. 1).4 MpPKS5encodes a non-reducing fungal type I polyketide synthase (NR-fPKS) with a reductive releasing domain and its inactivationresulted in the loss of MAzP biosynthesis.3b MpfasA2/MpfasB2 ispredicted to encode a canonical fungal fatty acid synthase and isproposed to be involved in the formation of the short chain fattyacyl thioester for MAzP biosynthesis.3b Among the genes locatedoutside this central region, mppF is suggested to mediate thehydroxylation step for the pyran-ring closure (Scheme 1 andFig. 1). This type of hydroxylation is a prerequisite for the forma-tion of the azaphilone structure.5 Between MpfasB2 and mppF, se-ven ORFs (mpp1–7) are found in M. pilosus and M. ruber, but theirroles in MAzP biosynthesis are elusive. The genetic organizationof the M. purpureus cluster bears a notable difference to that ofM. pilosus and M. ruber in this region, as the M. purpureus clustershares mpp7 with the two other clusters, but not mpp1 through6. A putative MAzP biosynthetic gene cluster was also previouslyidentified in the genome sequence of Talaromyces stipitatus,3b andthis putative cluster harbors an mpp7 homologue at the right bor-der of the cluster (Fig. 1). A conserved domain search in the Na-tional Center for Biotechnology Information (NCBI) interface6

predicts that Mpp7 belongs to an uncharacterized protein familywith a non-ribosomal peptide synthase (NRPS) condensation do-main (COG4908).

In the present study, we generated the Dmpp7::hyg mutant toassess the role of mpp7 in MAzP biosynthesis (see Supplementary

Scheme 1. Proposed biosynthetic pathway of azaphilone pigment in Monascus spp. depicting the role of Mpp7 in Knoevenagel aldol condensation.

Figure 1. Organization of the azaphilone pigment biosynthetic gene clusters from M. pilosus, M. ruber, and M. purpureus, with the putative cluster gene from T. stipitatus. TheM. purpureus MAzP biosynthetic gene cluster is included in the genome draft scaffold 1 at the nucleotide position from 368,313 to 301,716 (http://genome.jgi.doe.gov/Monpu1/Monpu1.home.html).

B. Balakrishnan et al. / Tetrahedron Letters 55 (2014) 1640–1643 1641

data for experimental procedures). HPLC analysis of the mpp7 mu-tant showed that the main MAzPs 1–4 were not detectable andthat new compounds 7 and 8 accumulated (Fig. 2). It appeared that7 and 8 were minor components in the wile-type culture andhighly accumulated upon the loss of 1–4. Notably, 8 appearedabundant inside the cell, and 7 is readily secreted into the medium,whereas 1 to 4 generally accumulated in the cell. Citrinin was con-tained in the organic extracts of the supernatants, eluting at thesame retention time as 8 under the given HPLC conditions, but sil-ica gel chromatography clearly separated 8 from citrinin. Com-pound 7 and 8 were isolated through silica gel chromatographyof the supernatant extracts; approximately 120 mg and 30 mg of

7 and 8, respectively, were obtained from a 1.5 L culture of thempp7 mutant. In 1H NMR analysis (Table 1), 7 and 8 appear closelyrelated, showing three olefinic singlet hydrogen signals at 7.38,6.10/6.09 (7/8), and 5.38 ppm. These downfield signals, togetherwith a singlet methyl hydrogen signal at 1.58 ppm, are character-istic to the azaphilone structure (Scheme 1).5a Several 1H NMR up-field signals indicate saturated acyl chains in 7 and 8. The 13C NMRspectra are also comparable between 7 and 8, except for two addi-tional methylene carbons (29.0 and 28.8 ppm) in 8, as determinedby HSQC and 13C DEPT experiments. In the HMBC spectrum of 8,the singlet methyl hydrogen signal of C10 (1.58 ppm) correlatesto a carbonyl carbon at 191.5 ppm (C6), a quaternary carbon at

Figure 2. HPLC chromatograms for the mpp7 mutant (Dmpp7::hyg) (A) and wild-type M. purpureus (B). Organic extracts of the culture supernatants (�1) and themycelial pellet (�2) were obtained from potato dextrose broth cultures. Y-Axesrepresent absorbance and have the same scale for all four spectra.

1642 B. Balakrishnan et al. / Tetrahedron Letters 55 (2014) 1640–1643

82.8 ppm (C6a), and a tertiary carbon at 42.6 ppm (C9a). Themethine hydrogen at C9a correlates to another methine hydrogenat C9 (JHH, 12.2 Hz) in COSY experiment. The 13C signal at168.7 ppm is assigned as an ester carbon (C8) and this carbon cor-relates to these two methine hydrogens in the HMBC spectrum.This observation suggests the presence of a c-butyrolactone moi-ety composed of C6a, C8, C9, and C9a. The two methine hydrogensof C9 and C9a also correlate to a carbonyl carbon found at202.2 ppm (C14), which correlates to the methylene hydrogenson a-carbon (C15) of the fatty acyl chain. In addition, the olefinicC1 carbon correlates to the C9a hydrogen. The NMR spectra of 7are found comparable to those of 8. These NMR spectroscopy anal-yses determined the structures of 7 and 8 (Scheme 1 and Table 1).Mass analysis using fast atom bombardment ionization confirmedthat the molecular formulas were C21H26O6 (7) and C23H30O6 (8) bythe detection of 375.1802 and 403.2126 m/z signals for [M+H]+,where the calculated exact masses of C21H27O6 and C23H31O6 are375.1808 and 403.2121 for 7 and 8, respectively. Both 7 and 8 havea UV–vis absorption peak at 349 nm in methanol. Compound 7 was

Table 11H (600 MHz) and 13C (125 MHz) spectral data (CDCl3) for compounds 7 and 8

C no. d 1H (ppm) (multi., JHH in Hz)

7 8

1 7.38 (s) 7.38 (s)34 6.10 (s) 6.09 (s)4a5 5.38 (s) 5.38 (s)66a89 3.75 (d, 12.2) 3.73 (d, 12.2)9a 3.86 (d, 12.2) 3.86 (d, 12.2)9b10 1.58 (s) 1.58 (s)11 2.55 (dd, 14.6 & 4.1), 2.51 (n.d.) 2.55 (dd, 14.6 & 4.2), 212 4.17 (m) 4.17 (m)13 1.31 (d, 6.2) 1.34 (d, 6.4)1415 3.15 (ddd, 6.7 & 8.5 & 15.2), 2.48 (n.d.) 3.15 (ddd, 6.4 & 8.5 &16 1.60 (n.d.) 1.60 (n.d.)17 1.27 (n.d.) 1.27 (n.d.)18 1.27 (n.d.) 1.27 (n.d.)19 0.88 (t, 7.0) 1.27 (n.d.)20 1.27 (n.d.)21 0.87 (t, 6.7)

n.d., not determined.* The proton signals from C9 and C9a overlap in this HMBC experiment (Fig. S4).

previously reported in a mutant strain of M. purpureus,7a and 8 is anovel MAzP. Monasfluore A (9) and B (10), which were previouslyreported in Monascus AS3.4444, are closely related to 7 and 8(Scheme 1).7b Although 7 was previously reported, there is no triv-ial name assigned. In this Letter, we coin the names monasfluol Aand B for 7 and 8, respectively.

The proposed MAzP biosynthetic pathway involves an intramo-lecular Knoevenagel aldol condensation of the presumed interme-diate 11 (Scheme 1). The disappearance of 1–4 and accumulationof 7 and 8 in the mpp7 mutant suggest the involvement of Mpp7in a regioselective Knoevenagel aldol condensation for the installa-tion of 2-furanone moiety. The c-butyrolactone structure in 7 and8 (lactone ring installed on C9a) can be found in some azaphilonecompounds, as exemplified by chaetoviridins and chaetomug-lins.1,8 In vitro enzyme experiments demonstrated that chaetovir-idin C was synthesized by CazE (acyltransferase) and CazF(polyketide synthase) when they were incubated with an azaphi-lone intermediate called cazisochromene and the precursors forthe CazF-mediated 3-oxo-acyl-ACP synthesis.8 This observationindicates that cazisochromene with a 3-oxo-acyl moiety installedon the C6a hydroxyl group undergoes a spontaneous Knoevenagelcondensation at the C9a position. It is thus proposed that Mpp7mediates regioselective aldol condensation on the C6 ketone groupand that the subsequent dehydration step is spontaneous, whereasthe absence of Mpp7 leads to lactone ring formation on C9a. Thesubsequent dehydration and double bond reduction of theC9AC9a double bond are proposed to yield 7 and 8 (Scheme 1).Chaetoviridin biosynthesis is predicted to involve an enoyl reduc-tion step for c-butyrolactone moiety, as proposed for the genera-tion of 7 and 8.8 The MAzP biosynthetic gene cluster encodesfour oxidoreductase candidates, such as MppA, MppC, MppE, andMppG, but none of them show a significant homology to the geneproducts of the chaetoviridin biosynthesis cluster. Thus, the iden-tity of the enoyl reductase in the biosynthesis of 7 and 8 is veiled.Although MppE belongs to an enoyl reductase family, MppE seemsunlikely to be involved in the C9AC9a double bond reduction be-cause of its significant homology (47% identity/62% similarity) toAzaJ of azanigerone biosynthesis and the absence of such an enoyl

d 13C (ppm) HMBC

7 8 7 8

147.7 147.7 9* 9a160.6 160.3 1, 4, 11 1, 4, 11108.3 108.6 5 5, 11145.2 144.8 1, 9* 1, 9a104.7 105.6 4 4191.1 191.5 9*, 10 9a, 1082.5 82.8 5, 9*, 10 9a, 10168.7 168.7 9* 9, 9a55.9 56.4 9* 9a42.2 42.6 9*, 10 10114.1 114.6 1, 5 1, 5, 9a23.0 23.2

.51 (n.d.) 42.4 42.8 13 1364.8 65.4 11, 13 11, 1322.8 23.6201.8 202.2 15 9, 15

15.2), 2.48 (n.d.) 42.3 42.822.1 22.9 15 1530.5 28.8 1921.9 29.0 1913.4 31.5 21

22.5 2114.0

B. Balakrishnan et al. / Tetrahedron Letters 55 (2014) 1640–1643 1643

reductive modification in azanigerone biosynthesis.5a MppG is pre-dicted to encode a flavin-containing amine oxidase and is unlikelyto be the catalyst for an enoyl reduction. For now, it is thus pro-posed that MppA or MppE mediates the enoyl reduction step ifthe catalyst is encoded within the biosynthetic gene cluster. Con-sideration of the biosynthetic origin leads us to assume that 7and 8 retain the same S stereochemistry at C6a as compounds 1–4. Circular dichroism spectra of 7 and 8 appeared indistinguishableand displayed a maximum negative ellipticity at 326 nm (see Sup-plementary data), which supports that 7 and 8 retain an S config-uration at the C6a position.9

The mpp7 gene has homologs in some fungal genomes inaddition to the aforementioned genomes of T. stipitatus and itsclose relative Penicillium marneffei. These mpp7 homologs areEPUS_08303 (% identities/% positives to Mpp7 in the deducedamino acid sequence) (52/68) from Endocarpon pusillum Z07020,TRIATDRAFT_176483 (42/58) from Trichoderma atroviride IMI206040, and CHGG_10018 (37/50) from Chaetomium globosumCBS 148.51. Notably, in all three cases, azaphilone-type (reductivereleasing domain) NR-fPKS genes are located near the mpp7 homo-logues; the NR-fPKS genes EPUS_08300 and CHGG_10027 arelocated near EPUS_08303 and CHGG_10018, respectively, whereasthere is 6,895 nt gap between the NR-fPKS and TRIAT-DRAFT_176483 (TRIATDRAFT locus tags are not annotated alongthe genome locations). This observation suggests that Mpp7-typecatalysts are also involved in the biosynthesis of other azaphilonepolyketide, and the present information will guide our understand-ing of the biosynthetic potentials of these cryptic polyketide bio-synthetic gene clusters.

As mentioned above, several MAzPs were successfully purifiedand evaluated for their biological activities.2 However, the prepara-tion of minor components often demands tedious experimentalprocedures. This study demonstrates the usefulness of the geneticengineering of MAzP biosynthetic blueprints for the developmentof strains that accumulate select MAzP derivatives. A similarachievement in MAzP production was recently reported in M.ruber.10 The targeted inactivation of oxidoreductase gene (MpigEor mppC) switched the MAzP profile and induced the yellow pig-ments, but the structural identities of these yellow pigments werenot resolved in that report. The present study provides a strain thatselectively accumulates compound 7 and 8, features a novel cata-lyst, Mpp7, which controls the regioselectivity of 2-furanone ringformation during azaphiolone biosynthesis.

Acknowledgments

This research was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF) fundedby the Ministry of Education (2011-0021169; 2013R1A1A2059458).

Supplementary data

Supplementary data (detailed experimental procedures, NMRspectra, UV–vis scanning, and circular dichroism) associated withthis article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.01.090.

References and notes

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4. It was previously suggested that mppD encoded amine oxidase and esterasedomain by a gene prediction analysis, although these two domains were foundin distinctive proteins in BLAST search analysis. PCR experiments with M.purpureus cDNA substantiated the esterase domain region of mppD constitutesan individual transcript (data not shown), leading us to annotate the amineoxidase domain gene as mppG.

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