Monacolin K, also known as lovastatin, is able to act on cholesterol biosynthesis, which can reduce the function of HMG-CoA reductase as a competitive inhibitor. The monacolin K biosynthetic gene clusters in Monascus have received much attention because of the various biological activities of this compound. The monacolin K biosynthetic gene cluster has been identified according to the similarities with lovastatin synthetic genes (LNKS) in Aspergillus. Nine genes (mokA–mokI) have proposed the functions of these genes which were associated with the monacolin K synthesis. The mokA–deficient mutant in M. pilosus BCRC38072 cannot produce monacolin K, indicating that mokA encodes the polyketide synthase responsible for monacolin K biosynthesis in M.pilosus BCRC38072. Additionally, the mokB-deficient mutant of M. pilosus NBRC4480 cannot produce monacolin K, but exhibits accumulation of monacolin J, indicating that mokB is responsible for the synthesis of the diketide side chain of monacolin K (Sakai et al. 2009; Chen et al. 2015). Overexpression of the mokH gene in M. pilosus results in significantly higher monacolin K production than that in wild-type strains, indicating that mokH positively regulates monacolin K production (Chen et al. 2010). Based on previous reports, many factors, particularly nitrogen sources, such as amino acids, can influence the production of secondary metabolites in Monascus. For example, monacolin K production is increased in glutamic acid or leucine culture conditions; an ideal nitrogen source can be selected to control the low final pH and then produce citrinin-free Monascus pigments (Kang et al. 2013b). This is the first report on the inhibition of citrinin biosynthesis by controlling an extremely low pH. Previous also showed that lowering the pH value to 2.5 would result in high monacolin K and citrinin concentrations as well as high biomass in fixed dioscorea amount, implying that pH value may stimulate the formation of monacolin K and citrinin through increasing Monascus cell amount (Lee et al. 2007).

Previous studies have shown that the most suitable pH value for Monascus growth is about 4 (Lee et al. 2007). Interestingly, in this study, we found that the pH varied during different stages of growth; during the adjustment and logarithmic phases of Monascus growth, the pH was lower in glutamic acid-containig medium (4.68 and 4.88, respectively) than that of the original medium (5.15 and 6.02, respectively). Thus the amount of Monascus mycelia was greater in glutamic acid-containing medium than that in original medium. We hypothesized that glutamate may increase monacolin K production by increasing the density of Monascus.

In this study, we demonstrated, for the first time, the correlation between the expression levels of monacolin K biosynthetic genes and monacolin K production in Monascus. At any stage of cell growth, we found that glutamic acid enhanced monacolin K production in Monascus M1, compared with cultivation in original medium. When Monascus M1 was grown in glutamic acid-containing medium rather than original medium, monacolin K production increased from 48.4 to 215.4 mg l^?1. Thus, these data showed that glutamic acid promoted the production of moncolinK.

Monascus expresses nine genes related to monacolin K synthesis, and monacolin K accumulation was found to be positively correlated with the expression of the monacolin K biosynthetic gene cluster. Indeed, RT-qPCR analysis showed that the maximal monacolin K biosynthesis quality was reached on day 8, at which point mostgenes related to monacolinK synthesis showed higher transcription in glutamic acid-containing medium, with the exception of mokA. These data indicated that mokB–mokI were eight key genes mediating monacolin K production in the presence of glutamic acid. According to the speculation on the function of monacolin K synthesis genes, we speculated that, mokH acts as a transcription factor, it will promoted the expression of key genes in monacolin K biosynthesis. MokI acts as an efflux pump, it will promoted the process of transferring monacolin K out of the Monascus cells, reduced the content of monacolin K in cells and promoted the final mknacolin K content. MokB–mokG participated in the monacolin K biosynthesis process and promoted the production of monacolin K in glutamic acid medium directly. Thus, based on the expression of monacolin K synthesis-related transcripts, these data supported that glutamic acid promoted the production of moncolin K has an internal power and the promoting effect is stable.

In summary glutamic acid increased the content of Monascus mycelia, altered the pH value in fermentation broth, changed the permeability of Monascus mycelia, enhanced the secretion of monacolin K to the outside of the cell, and reduced monacolin K content in the Monascus mycelia, thereby enhancing monacolin K production. In addition, glutamic acid may not only be used to provide energy for Monascus growth and metabolize, but also generate the production of acetyl coenzyme A, which is a substrate for monacolin K, and ultimately increase the content of metabolites. Our findings also demonstrated that glutamic acid could enhance monacolin K production by upregulating the expression of mokB, mokC, mokD, mokE, mokF, mokG, mokH,and mokI. So further studies are needed to elucidate the molecular pathways through which glutamic acid regulates monacolin K production.

Monacolin K, also known as lovastatin, is able to act on cholesterol biosynthesis, which can reduce the function of HMG-CoA reductase as a competitive inhibitor. The monacolin K biosynthetic gene clusters in Monascus have received much attention because of the various biological activities of this compound. The monacolin K biosynthetic gene cluster has been identified according to the similarities with lovastatin synthetic genes (LNKS) in Aspergillus. Nine genes (mokA–mokI) have proposed the functions of these genes which were associated with the monacolin K synthesis. The mokA–deficient mutant in M. pilosus BCRC38072 cannot produce monacolin K, indicating that mokA encodes the polyketide synthase responsible for monacolin K biosynthesis in M.pilosus BCRC38072. Additionally, the mokB-deficient mutant of M. pilosus NBRC4480 cannot produce monacolin K, but exhibits accumulation of monacolin J, indicating that mokB is responsible for the synthesis of the diketide side chain of monacolin K (Sakai et al. 2009; Chen et al. 2015). Overexpression of the mokH gene in M. pilosus results in significantly higher monacolin K production than that in wild-type strains, indicating that mokH positively regulates monacolin K production (Chen et al. 2010). Based on previous reports, many factors, particularly nitrogen sources, such as amino acids, can influence the production of secondary metabolites in Monascus. For example, monacolin K production is increased in glutamic acid or leucine culture conditions; an ideal nitrogen source can be selected to control the low final pH and then produce citrinin-free Monascus pigments (Kang et al. 2013b). This is the first report on the inhibition of citrinin biosynthesis by controlling an extremely low pH. Previous also showed that lowering the pH value to 2.5 would result in high monacolin K and citrinin concentrations as well as high biomass in fixed dioscorea amount, implying that pH value may stimulate the formation of monacolin K and citrinin through increasing Monascus cell amount (Lee et al. 2007).

Previous studies have shown that the most suitable pH value for Monascus growth is about 4 (Lee et al. 2007). Interestingly, in this study, we found that the pH varied during different stages of growth; during the adjustment and logarithmic phases of Monascus growth, the pH was lower in glutamic acid-containig medium (4.68 and 4.88, respectively) than that of the original medium (5.15 and 6.02, respectively). Thus the amount of Monascus mycelia was greater in glutamic acid-containing medium than that in original medium. We hypothesized that glutamate may increase monacolin K production by increasing the density of Monascus.

In this study, we demonstrated, for the first time, the correlation between the expression levels of monacolin K biosynthetic genes and monacolin K production in Monascus. At any stage of cell growth, we found that glutamic acid enhanced monacolin K production in Monascus M1, compared with cultivation in original medium. When Monascus M1 was grown in glutamic acid-containing medium rather than original medium, monacolin K production increased from 48.4 to 215.4 mg l^?1. Thus, these data showed that glutamic acid promoted the production of moncolinK.

Monascus expresses nine genes related to monacolin K synthesis, and monacolin K accumulation was found to be positively correlated with the expression of the monacolin K biosynthetic gene cluster. Indeed, RT-qPCR analysis showed that the maximal monacolin K biosynthesis quality was reached on day 8, at which point mostgenes related to monacolinK synthesis showed higher transcription in glutamic acid-containing medium, with the exception of mokA. These data indicated that mokB–mokI were eight key genes mediating monacolin K production in the presence of glutamic acid. According to the speculation on the function of monacolin K synthesis genes, we speculated that, mokH acts as a transcription factor, it will promoted the expression of key genes in monacolin K biosynthesis. MokI acts as an efflux pump, it will promoted the process of transferring monacolin K out of the Monascus cells, reduced the content of monacolin K in cells and promoted the final mknacolin K content. MokB–mokG participated in the monacolin K biosynthesis process and promoted the production of monacolin K in glutamic acid medium directly. Thus, based on the expression of monacolin K synthesis-related transcripts, these data supported that glutamic acid promoted the production of moncolin K has an internal power and the promoting effect is stable.

In summary glutamic acid increased the content of Monascus mycelia, altered the pH value in fermentation broth, changed the permeability of Monascus mycelia, enhanced the secretion of monacolin K to the outside of the cell, and reduced monacolin K content in the Monascus mycelia, thereby enhancing monacolin K production. In addition, glutamic acid may not only be used to provide energy for Monascus growth and metabolize, but also generate the production of acetyl coenzyme A, which is a substrate for monacolin K, and ultimately increase the content of metabolites. Our findings also demonstrated that glutamic acid could enhance monacolin K production by upregulating the expression of mokB, mokC, mokD, mokE, mokF, mokG, mokH,and mokI. So further studies are needed to elucidate the molecular pathways through which glutamic acid regulates monacolin K production.