Exploring the Subcellular Localization of Monascus Pigments Biosynthases: Preliminary Unraveling of the Compartmentalization Mechanism

Abstract

Monascus pigments (MPs), a class of secondary metabolites produced by Monascus spp., can be classified into yellow, orange, and red MPs according to their differences in the wavelength of the maximum absorption. However, the biosynthetic sequence and cellular biosynthesis mechanism of different MPs components are still not yet completely clear in Monascus spp. In this study, the subcellular localization of five MPs synthases was investigated using fluorescent protein fusion expression. The results revealed that the proteins encoded by the MPs biosynthetic gene cluster were compartmentalized in various subcellular locations, including the mitochondrial polyketide synthase MrPigA, cytosolic enzymes consisting of the ketoreductase MrPigC, the oxidoreductase MrPigE, and the monooxygenase MrPigN, and the cell-wall-bound oxidoreductase MrPigF. Moreover, the correct localization of MrPigF to the cell wall was crucial for the synthesis of orange MPs. Lastly, we discussed the compartmentalized biosynthetic pathway of MPs. This study will not only be helpful in clarifying the biosynthetic sequence and biosynthesis mechanism of different MPs but also provides new insights into the cellular biosynthesis of secondary metabolites in filamentous fungi.

Keywords: Monascus pigments; Monascus pigments biosynthetic proteins; Monascus spp.; compartmentalization; subcellular localization.

Figure 1

Analysis of spontaneous fluorescence of M. ruber M7 and characteristics of ∆mrpigA+lig4+G::G mutants. (A) Analysis of spontaneous fluorescence of M. ruber M7. Full-wavelength fluorescence scanning of M. ruber M7 mycelia was carried out using a confocal laser scanning microscope. Different levels of intrinsic fluorescence were observed in the M. ruber M7 mycelia when excited with wavelengths of 405 nm, 488 nm, and 561 nm. (B) Colony morphology and spontaneous fluorescence observation of M7, ∆mrpigA+lig4+G::G, and ∆mrpigN mutants under different channels. TD: transmitted light. (C) Verification of the gene transcription in MPs biosynthetic gene cluster of ∆mrpigA+lig4+G::G mutants. The cDNA from the ∆mrpigA+lig4+G::G mutants was used as a template to confirm the transcription of multiple genes within the MPs gene cluster utilizing the primers illustrated in Table S1. m: Trans 2K marker; A: mrpigA; B: mrpigB; C: mrpigC; D: mrpigD; E: mrpigE; F: mrpigF; G: mrpigG; H: mrpigH; I: mrpigI; J: mrpigJ; K: mrpigK; L: mrpigL; M: mrpigM; N: mrpigN; O: mrpigO; P: mrpigP. (D) M7PKS-1 feeding experiment. ∆mrpigA+lig4+G::G mutants were cultivated on PDA medium, PDA + ACN medium, and PDA + M7PKS-1 medium. ACN: acetonitrile. The content of MPs was analyzed using HPLC, with the chromatogram at a wavelength of 380 nm presented on the right.

Figure 2

Subcellular locations of five key enzymes involved in the biosynthesis of MPs. (A) High-resolution confocal images for subcellular localizations of MrPigA, MrPigC, MrPigE, MrPigF, and MrPigN. The mutants were cultivated on PDA media at 28 °C for 7 d. TD: transmitted light. (B) Observations of TD, EGFP, and Mito-Tracker Red and merged image of the three channels in MrPigA-EGFP mutants are shown. TD: transmitted light. (C) Observations of merged EGFP and Mito-Tracker Red in MrPigA-EGFP mutants are shown; the white arrow indicates the position and direction of the fluorescence intensity analysis. Bars equal 10 μm.

Figure 3

Bioinformatics analysis of MrPigF. (A) The gene structure diagram of mrpigF. The full-length cDNA sequence of mrpigF was determined through RACE, and it is presented in Figure S2. (B) Prediction of signal peptide sequence in MrPigF using SignalP 6.0. (C) The protein structure diagram of MrPigF. (D) Multiple sequence alignment of MrPigF protein of nineteen Monascus strains.

Figure 4

Subcellular localization analysis of MrPigF. (A) Subcellular localization analysis of MrPigF. (B) Subcellular localization analysis of MrPigF^∆SP. TD: transmitted light. Bars equal 10 μm.

Figure 5

Effects of subcellular localization of MrPigF on MPs production. (A) Colony morphology on PDA media. (B) The expression level of mrpigF in the indicated strains was determined by qRT-PCR. Error bars represent the standard error of three biological replicates; different letters in the graph indicate statistical significance between strains (one-way ANOVA) (p < 0.05). (C) MPs species analysis of different strains cultivated on PDA media for 7 days. (D) MPs from different strains were analyzed and quantified using HPLC. Error bars represent the standard error of three biological replicates. Different letters in the graph indicate statistical significance of each MPs among different mutants. In alphabetical order, a preceding position signifies a greater magnitude of statistical variance (one-way ANOVA) (p < 0.05).

Figure 6

Schematic compartmentalized MPs biosynthesis.