| Filamentous fungus Monascus spp. is a traditional fermentation microorganism, usually used to manufacture Hongqu in China and Southeast Asian countries. It is able to produce several beneficial secondary metabolites, such as cholesterol-lowering monacolins, antihypertensive γ-amino butyric acid, and food-grade colorants-Monascus pigments (MPs), and can also secrete a mycotoxin, citrinin that is nehprotoxic to humans, and pollutes Hongqu.MPs, one kind of the main secondary metabolites produced by M. spp, are a mixture of azaphilone compounds with three colors (Red, orange, and yellow colors). Up to now, more than 50 MPs have been obtained and investigated, providing various functions, such as antioxidant, anti-inflammatory, and anti-cancer activities. Although MPs were thought to be synthesized following a polyketide biosynthetic pathway, unfortunately, any intermediate or gene responsible for their biosynthesis have not been reported to date.Polyketide synthase (PKS), mainly found in plants, bacteria, and fungi, is a key enzyme of polyketide biosynthetic pathway. According to the structure and catalytic mechanism, PKS can be divided into four types, that is modular PKS (type Ⅰ), iterative PKS (type Ⅱ), chalcone synthase-like PKS (type Ⅲ), and iterative type Ⅰ PKS. Most of fungal PKS belong to the iterative type I PKS. The iterative type Ⅰ PKS is multifunctional enzyme that harbors a set of iteratively acting domains responsible for the catalysis of every cycle of polyketide chain elongation. Based on the difference of domains, the iterative type I PKS can be further divided into eight types including four non-reduced PKS (Non-reduced type-Ⅰ, type-Ⅱ, type-Ⅰ& Ⅱ, and type-Ⅲ) and four reduced PKS (Reduced type-Ⅰ, type-Ⅱ, type-Ⅲ, and type Ⅳ). Despite differences in number and type of domains, all of the iterative type Ⅰ PKS have three basic conserved domains, that is β-ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), based on which neo-PKS genes have been cloned.Present studies revealed that genes related to the biosynthesis of any polyketides usually linked together as gene clusters in fungi and at least one PKS gene and one pathway-specific regulatory gene occurs in such a cluster, which gives a clue to clone PKS gene clusters for some secondary metabolites when the PKS gene was gained. The biosynthetic pathways of some polyketides have been explored based on the analysis of their gene clusters.On the basis of these theories, we cloned a PKS gene for MPs biosynthesis from M. ruber M-7 using degenerate primers that were designed according to the conserved domain of KS. Later, MPs biosynthetic gene cluster was obtained through analyzing the genomic sequence of M-7. In the paper, the functions of genes within MPs gene cluster were predicted using bioinformatics’technology and some of them were given a further identification. Besides, we constructed a mutant showing higher MPs production and lower citrinin here. These results will be elaborated as follows.1 Clone of the PKS gene responsible MPs biosynthesisA genealogy of PKS was constructed based on the analysis of 253 KS domains, which came from Aspergillus, Penicillium, and Monascus, and were retrieved from NCBI. It was used for designing degenerate primers to clone PKS genes from the genome of M-7.4 PKS genes were finally cloned and one of them (pksPT) was proved to be vital for MPs biosynthesis.2 Analysis of MPs biosynthetic gene clusterThe sequencing of M-7 genomic DNA provided us the probable MPs biosynthetic gene cluster including 17 genes. The pksPT, regulatory gene (pigR), fatty acid synthase a and ∞ subunit genes (fas∝ and fas∞) were analyzed, showing that protein encoded by pksPT is a non-reduced type III PKS and has some active domains with the arrangement of KS-AT-ACP-ACP-ME (Methyltransferase); protein encoded by pigR is a Zn(II)2Cys6-type regulator and contains a DNA binding motif (CdnCrkkkvkCdakkpaCs, [C represents cysteinyl residue; d, n, r, k, a, p, v, and s are abbreviations of other amino acids]); protein encoded by fasa includes several active domains like ACP, KS, KR (β-ketoreductase), and PPT (Phosphopantetheinyl transferase); protein encoded by fasβ consists of AT、ER (Enoylreductase)、DH (Dehydratase), and MT (Malonyl transferase).3 Construction and identification of gene-deleted mutantsHomologous recombination method was used to delete pksPT, pigR, fasa, and fasβ in M-7, respectively, generating ApksPT, ApigR, Afasa, and △fasβ. Colonial morphology, conidial germination, pigment and citrinin production, and growth rate of these mutants were analyzed and the results were list as follows. On PDA plate, all the mutants could generate cleistothecum and conidum normally; Compared to the colony of M-7, ApksPT and ApigR have nonpigmented colonies while Afasa and △fasβ have yellow colonies. Certain yellow compound was accumulated not in M-7 but in Afasa and △fasβ, indicating that the yellow compound might be related to some intermediate of MPs biosynthesis. In rice medium, ApksPT, ApigR, Afasa, and △fasβ were unable to produce MPs. In YES medium, biomasses of the four mutants were obviously higher than that of M-7 and the growth rate ApksPT was fastest; Citrinin production in YES fermented broth was also detected by HPLC, showing that ApksPT, ApigR, Afasa, and △fasβ were about 3.8,2.3, 2.2, and 2.4-fold higher than that of M-7. These results indicate that pksPT, pigR, fasa, and fasβ were of extremely importance to the biosynthetic pathways of MPs.4 Construction of pigR-overexpressing strainsA pigR-overexpressing vector containing an entire pigR driven by constitutive trpC promoter was introduced into M-7 and ApigR, forming ptrpC∷pigR and pigR-complemented ApigR, respectively. Colonial morphology, pigment and citrinin production, and pigR expression of pigR-overexpressing strains were determined and the results were shown as follows. Colonial diameters of the two mutants were obviously smaller than that of M-7. After 7 days’cultivation in PDB medium, ptrpC∷pigR had a 9-times increase in red pigment production and 1-fold increase in yellow pigment production, while pigR-complemented ApigR produced 10-times higher red pigment production than M-7 and 0.5-fold higher yellow pigment production than M-7. At that time, M-7 had not begin to synthesize orange pigments whereas both of ptrpC∷pigR and pigR-complemented ApigR had produce 7 orange pigments with the production of 2.5 and 4.2 U/g-dried mycelia, respectively. pigR expression in ptrpC∷pigR and pigR-complemented ApigR was also measured by Real-time qPCR, showing that it were increased by 2 × 104 and 3.5 × 104, respectively, compared to that in M-7. Citrinin production in YES fermented broth was also analyzed by HPLC, indicating that the ability of ptrpCv∷pigR and pigR-complemented ApigR to produce citrinin were extremely lower than that of M-7, both decreased by about 30%. These results elaborate that it is an effective method to increase pigment production and decrease citrinin production via overexpression of pigR gene.5 Analysis of an intermediate of MPs biosynthesis and the exploration of MPs biosynthetic pathwayA mutant that could secrete an intermediate of MPs biosynthesis was screened during the experiment of constructing the Afasa and named as ISM (Intermediate secreting mutant). The intermediate was confirmed by co-cultivation experiment and intermediate-feeding experiment. Co-cultivation experiment showed that ApksPT could reproduce some pigments when it was cultivated with ISM, implying that ISM could produce certain intermediate of MPs biosynthesis. HPLC analysis of the compounds accumulated around the colonies of mutants showed that a compound was produced by ISM not by ApksPT, indicating it might be the intermediate of MPs biosynthesis. It was directly confirmed that the compound was the intermediate of MPs biosynthesis by the result that ApksPT reproduced some MPs after the supplement of the purified putative intermediate to the fermented broth of ApksPT.ISM was also co-cultivated with Afasfi and the intermediate contents in △fasβ and M-7 were compared using HPLC method, showing that the intermediate was unable to induce Afasfi reproduce MPs and the content of the intermediate in Afasfi was similar to that of M-7. These results above showed that the intermediate could force ApksPT not △fasβ to regain the ability to produce MPs, proving that the enzyme catalyzing the biosynthesis of intermediate located at the upstream of FAS and downstream of PKS. |
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