Metabolism and secretion of yellow pigment under high glucose stress with Monascus ruber
© The Author(s) 2017
Received: 29 March 2017
Accepted: 3 April 2017
Published: 11 April 2017
The biosynthesis of microbial secondary metabolites is induced by a wide range of environmental stresses. In this study, submerged fermentation of Monascus yellow pigments by Monascus ruber CGMCC 10910 under high glucose stress was investigated. The increase of lipid content was the major contributor to the increase of dry cell weight (DCW), and the lipid-free DCW was only slightly changed under high glucose stress, which benefited the accumulation of intracellular hydrophobic pigments. The fatty acid composition analysis in Monascus cell membranes showed that high glucose stress significantly increased the ratio of unsaturated/saturated fatty acid and the index of unsaturated fatty acid (IUFA) value, which would improve the fluidity and permeability of the cell membrane. As a consequence, high glucose stress increased extracellular yellow pigments production by enhancing secretion and trans-membrane conversion of intracellular pigments to the broth. The total yield of extracellular and intracellular yellow pigments per unit of lipid-free DCW increased by 94.86 and 26.31% under high glucose stress compared to conventional fermentation, respectively. A real-time quantitative PCR analysis revealed that the expression of the pigment biosynthetic gene cluster was up-regulated under high glucose stress. The gene mppE, which is associated with yellow pigment biosynthesis, was significantly up-regulated. These results indicated that high glucose stress can shift the Monascus pigment biosynthesis pathway to accumulate yellow pigments and lead to a high yield of both extracellular and intracellular yellow pigments. These findings have potential application in commercial Monascus yellow pigment production.
KeywordsMonascus ruber High glucose stress Pigments secretion Gene expression Yellow pigments Lipids
Monascus pigments are secondary metabolites with polyketide structures that are produced by Monascus spp. (Feng et al. 2012), and are usually classified by color (yellow, orange or red) (Patakova 2013). Monascus yellow pigments have been widely researched due to their hypolipidemic (Lee et al. 2010), anti-obesity (Lee et al. 2013), anti-inflammation (Hsu et al. 2012), anti-tumor (Su et al. 2005; Lee et al. 2013), anti-diabetic and anti-oxidative stress (Shi et al. 2012), which are related to the molecular structures of yellow pigments (Su et al. 2005).
It has long been known that the biosynthesis of microbial secondary metabolites is induced by stress (Ranby 1978). Under stress inducing conditions, microorganisms shift from producing primary metabolites to secondary ones in order to preserve energy sources and essential metabolites for more favorable growth conditions. For example, high temperature (>45 °C) can increase the production of Monascus yellow pigments, and a high concentration of sodium chloride inhibited mycelia growth but caused an increase in the production of Monascus red pigments (Babitha et al. 2007). Klebsiella oxytoca fermented with a high concentration of molasses exhibited increased production of 2, 3-butanediol (Afschar et al. 1991). Increased production of monacolin K was observed when a high concentration of glycerol was used as the sole carbon source for Monascus purpureus fermentation with the agricultural residue bagasse used as an inert carrier (Lu et al. 2013). In past studies of Monascus pigment fermentation, research has mainly focused on improving cell densities and pigment production in fed-batch cultures with long incubation times (Krairak et al. 2000; Lee et al. 2013; Chen et al. 2015). In fed-batch fermentation of Monascus, compared with low glucose concentration, high glucose concentration had different impact on the production of Monascus pigments (Chen and Johns 1994), and the characteristics of pigments were shifted in Monascus anka fed-batch culture with high cell densities (Chen et al. 2015). Cell membrane is the first barrier of microorganism coping with environmental stress, not only for the nutrients absorption but also for the extracellular products excretion, the absorption and excretion ability of microorganism cell response to the fluidity and permeability of the cell membrane (Zhang and Cheung 2011). Glutamic acid could promote the monacolin K production by regulating the permeability of Monascus mycelium and then the secretion of monacolin K was promoted without feedback inhibition from intracellular product (Zhang et al. 2017). The permeability and fluidity of cell membrane depended on the saturability of the containing fatty acid (Wang et al. 2013). As high carbon source but low oxidoreduction potential (ORP) could benefit the production of extracellular water-soluble yellow pigments with Monascus ruber CGMCC 10910 (Wang et al. 2017), multifaceted mechanisms of high glucose stress that had impacted the metabolism and secretion of Monascus yellow pigments should be further investigated.
Recently, the biosynthetic gene cluster of azaphilone pigments in the Monascus pilosus genome and the functions of some critical genes involved in the pigment biosynthetic pathway were reported (Balakrishnan et al. 2013). In the present study, the effect of high glucose stress on the fermentation characteristics of M. ruber CGMCC 10910 was investigated. Cell growth and lipid production were analyzed to investigate the relationship between pigment production and lipid metabolism. The fatty acid composition of Monascus cell membrane under high glucose stress was analyzed using GC–MS to study the influence of high glucose stress on the fluidity and permeability of the cell membrane. The expression levels of pigment biosynthetic genes under high glucose stress were measured by real-time quantitative PCR with a simultaneous analysis of extracellular and intracellular pigment compositions. By undertaking these investigations, we hoped that the regulatory mechanisms of pigment metabolism during high glucose stress would be revealed.
Materials and methods
Microorganism and culture conditions
All experiments in this study were performed with M. ruber CGMCC 10910 (China General Microbiological Culture Collection Center, CGMCC 10910), which was cultivated on PDA medium at 30 °C for 7 days and then stored at 4 °C. The seed medium contained (g/L): glucose, 20; yeast extract, 3; peptone, 10; KH2PO4, 4; KCl, 0.5; and FeSO4·7H2O, 0.01. The inoculum was incubated in a 250-mL Erlenmeyer flask containing 50 mL of seed medium at 30 °C and was shaken at 180 rpm for 25 h. The conventional fermentation medium contained (g/L): glucose, 50; (NH4)2SO4, 5; KH2PO4, 5; MgSO4·7H2O, 0.5; KCl, 0.5; MnSO4·H2O, 0.03; ZnSO4·7H2O, 0.01; and FeSO4·7H2O, 0.01. Fermentation medium containing a higher initial glucose concentration (up to 200 g/L) was used for glucose concentration stress experiments. The fermentation experiment was conducted at 30 °C with shaking at 180 rpm for 8 days in a 250-mL Erlenmeyer flask containing 25 mL of fermentation media and using 2 mL of inoculum. All experiments were performed in triplicate.
Measurements of pigment and residual glucose concentration, DCW, lipid weight and lipid-free DCW
After fermentation, the spent medium was vacuum filtered through a 0.8 mm mixed cellulose esters membrane, after which the filtrate was diluted. Extracellular pigment production was assessed using a UV–Visible spectrophotometer (Unico, USA) scanning from 300 to 550 nm at 1-nm intervals (Shi et al. 2015). The absorbance units (AU) at the peak wavelength (350 nm) multiplied by the dilution ratio was used as an index of the extracellular yellow pigments concentration (Wang et al. 2017). The residual glucose was determined by the standard 3,5-dinitrosalicylic acid (DNS) method. The mycelia was washed for three times and then dried to a constant weight at 60 °C to determine biomass (dry cell weight, DCW). Some of those dry mycelia were submitted for estimation of lipid content. Lipid content in DCW was determined following the standard method by Bligh and Dyer (1959) with some modifications: 0.2 g of dry mycelia was re-suspended in 6 mL hydrochloric acid solution (4 mol/L), and then the mixture was heated to 100 °C and incubated for 3 min. After this, the mixture was immediately cooled down to have the intact cell structure broken down. A 12 mL of fresh extraction solution (methanol/chloroform, 1:1 v/v) was added into the cooled mixture and mixed for 30 s. After centrifugation at 5000 rpm for 15 min, the lower (chloroform) phase was collected to a new test tube containing 5 mL of 0.1% NaCl solution. After a centrifugation at 3500 rpm for 5 min, the lower (chloroform) phase was collected and evaporated with flushing nitrogen to get the lipid residual. Then the lipid residual was oven dried at 60 °C to a constant weight to determine the lipid weight. The lipid content was the extracted lipid weight (g) from per 100 g DCW. The lipid-free DCW was calculated by deducing the lipid weight from the total DCW (Wang et al. 2015a).
The intracellular pigment concentration was determined following those procedure as follows: mycelia were washed and re-suspended in 25 mL of acidic aqueous ethanol (70% v/v pH 2 with hydrochloric acid); the mixture was then incubated for 1 h and then passed through filter paper; finally, the filtrate (intracellular extract) was diluted for determining the intracellular pigment concentration. A UV–Visible absorbance spectrum of intracellular pigments was taken from 300 nm to 550 nm at 1-nm intervals, and the absorbance units (AU) at peak wavelengths of 410 and 470 nm multiplied by the dilution ratio were used as indexes of the intracellular yellow and orange pigments concentrations (Shi et al. 2015), respectively.
Analyses of pigment compositions by HPLC
Analyses of sample compositions were performed using an Alliance e2695 HPLC system (Waters, Milford, CT, USA) equipped with a 2998 Photodiode Array (PDA) detector (Waters, Milford, CT, USA) and a Zorbax Eclipse Plus C18 column (250 × 4.6 mm, 5 μm, Agilent, Palo Alto, CA, USA). The temperature of the column oven was set at 30 °C. A mixture of H3PO4 solution (pH 2.5, phase A) and acetonitrile (phase B) were used as the mobile phase using the following gradient program: 0 min, 80% A, 20% B; 25 min, 20% A, 80% B; 35 min, 20% A, 80% B; 36 min, 80% A, 20% B; 41 min, 80% A, 20% B. The PDA was set at 200–600 nm, and the flow rate of the mobile phase was 0.8 mL/min.
Analyses of extracellular pigments by LC–MS
Liquid chromatography–mass spectrometry consisted of a HP1100 HPLC system (Agilent, Palo Alto, CA, USA) and a micro TOF-QII mass spectrometer (Bruker, Rheinstetten, Germany). The C18 column and chromatographic conditions were the same as mentioned above, except for mobile phase A (water, 0.1% formic acid).
Analysis of cell membrane fatty acid composition by GC–MS
After 8 days of fermentation, mycelia in the fermentation broth were collected. The fatty acid in cell membrane of the mycelia was extracted, purified and methylated according to the method described by Wang et al. (2013). After that, the sample dissolved in the n-hexane was collected for GC–MS analysis, using an Agilent 6890 GC (Agilent, Santa Clara, CA, USA) coupled to an Agilent 5973 mass selective detector (MSD) (Agilent, Santa Clara, CA, USA), equipped with a HP-5MS column (5% Phenyl Methyl Silox, 30 m–0.25 mm id 0.25 μm film thickness, Agilent, Santa Clara, CA, USA). The front injection was 250 °C with a split ratio of 70:1. Helium gas (purity of 99.9999%, Foshan, China) was used as the carrier gas at a flow rate of 50 mL/min. The oven temperature program was as follows: 80 °C for 2 min, then raised to 150 °C at a rate of 10 °C/min, and then further to 230 °C at a rate of 3 °C/min, keeping at 230 °C for 5 min. The electron impact energy was 70 eV, and the ion source temperature was set at 230 °C.
Gene expression analysis
The effects of high glucose stress on the expression of key genes during pigments production were investigated using real-time quantitative PCR. Mycelia were collected and stored in liquid N2 before total RNA extraction using the Plant RNA Extraction Kit (TakaRa MiniBEST). cDNA was synthesized using the PrimeScript™RT reagent Kit with gDNA Eraser (TaKaRa). Primers for the amplification of MpFasA2, MpFasB2, MpPKS5, mppR1, mppB, mppC, mppD, mppE, mppR2 (GenBank accession No. KC148521) and the actin gene (GenBank accession No.AJ417880) were listed in Additional file 1: Table S1 according to the previous study (Wang et al. 2015b) with some modifications, actin gene was used as a reference gene. Gene expression was monitored by RT-qPCR using the SYBR Premix Ex TaqII (TaKaRa). RT-qPCR was performed using a Lightcycler 96 (Roche, USA) with the following cycling program: pre-incubation at 95 °C for 30 s, followed by a two-step amplification (40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s) and dissociation curve analyses (at 95 °C for 10 s, annealing at 65 °C for 60 s, then collecting dissociation curves from 65 to 95 °C, with a final incubation at 97 °C for 1 s).
Each experiment was repeated at least in triplicate. Numerical data are presented as the mean ± SD. The differences among different treatments were analyzed using one-way ANOVA. All statistical analyses were performed by using SPSS 22.0, software. p < 0.05, p < 0.01 was considered statistically significant.
Production of Monascus pigments and lipids during high glucose stress fermentation
Pigment yield per unit LFDCW and yield increase rate under high glucose stress
Yield (AU per g LFDCW)b
Increase rate (%)c
9.326 ± 0.054
18.173 ± 0.050
17.376 ± 0.188
21.947 ± 0.111
23.688 ± 0.246
21.118 ± 0.049
Changes of fatty acids composition in cell membrane
Fatty acid composition (% total fatty acid) of cell membranes under high glucose stress
Fatty acid composition
Saturated fatty acid
Tetradecanoic acid (14:0)
0.123 ± 0.01
0.157 ± 0.02
Palmitic acid (16:0)
14.641 ± 0.22
14.431 ± 0.18
Stearic acid (18:0)
23.978 ± 0.56
17.862 ± 0.47
Eicosanoic acid (20:0)
0.293 ± 0.03
0.170 ± 0.01
Unsaturated fatty acid
Palmitoleic acid (16:1)
0.161 ± 0.02
0.395 ± 0.05
Oleic acid (18:1)
40.649 ± 0.86
45.596 ± 0.78
Linoleic acid (18:2)
17.058 ± 0.35
17.439 ± 0.27
Linolenic acid (18:3)
1.457 ± 0.08
2.730 ± 0.12
1.520 ± 0.01 a
2.028 ± 05 A
IUFA (index of unsaturated fatty acid)b
79.295 ± 0.68 b
89.055 ± 0.54 B
Expression levels of pigment biosynthetic genes
During the fermentation anaphase (after the 6th day), the expression levels of MpFasA2, MpFasB2, MpPKS5, mppD, mmpB, and mppR1 were significantly up-regulated (p < 0.01 or p < 0.05). As the genes MpFasA2, MpFasB2, MpPKS5, mppD, and mppB are structural genes for pigment biosynthesis and mppR1 is a regulatory gene (Balakrishnan et al. 2013), the polyketide chromophores and media fatty acid were still being generated during fermentation anaphase under high glucose stress. Simultaneously, the gene mppE for yellow pigment biosynthesis (Balakrishnan et al. 2017) was significantly up-regulated, while the gene mppC for orange pigment biosynthesis (Liu et al. 2014) was down-regulated in some degree. In combination with the time course of pigment production, the up-regulation of mppE was positively correlated with the production of yellow pigments in the later stages of fermentation.
Monascus pigments are mixtures with multi-components (Juzlova et al. 1996; Patakova 2013). The concentration of Monascus pigments is usually represented by the absorbance at their characteristic wavelength (Babitha et al. 2007). Thus, the pigments yield in this study was represented by the absorbance at their characteristic wavelength (350, 410, and 470 nm). Submerged fermentation of Monascus species with a low IGC in the medium resulted in the accumulation of intracellular orange Monascus pigments exhibiting a peak at 470 nm (Kang et al. 2014). In this study, high yields of both extracellular and intracellular yellow pigments were obtained using M. ruber CGMCC 10910 when the IGC were increased from 50 g/L (low) to >150 g/L (high). An interesting phenomenon was observed that the dominating intracellular pigments changed from orange to yellow pigments (Fig. 1). In the later stage of fermentation under high glucose stress, the accumulation of DCW was mostly attributable to the increased intracellular lipid weight as the LFDCW was only slightly changed when the IGC was higher than 100 g/L. When the IGC was 150 g/L, the lipid weight reached approximately 53% of the DCW, 20% higher than what was observed at a low glucose concentration (IGC = 50 g/L). It has been reported that Monascus purpureus albino strain accumulated a high content of lipids under a limited nitrogen condition (carbon to nitrogen = 80:1) (Rasheva et al. 1997). The high lipid production observed in this study was also caused by a high ratio of carbon to nitrogen in the media. Lipid droplets in living microorganisms could serve as a reservoir for intracellular Monascus pigments, and there was a positive correlation between intracellular pigments and microbial lipids (Wang et al. 2015a). The intracellular yellow pigments and lipid content all increased continuously to the 8th day under high glucose stress (Fig. 2), the reason was that the intracellular lipids act as reservoirs for intracellular yellow pigments storage. Thus, high glucose stress increased the content of Monascus mycelia mainly by increasing the lipids content of Monascus mycelia, which can improve more reservoirs for intracellular yellow pigments storage (Wang et al. 2015a), thus enhancing intracellular yellow pigments production.
Except for extractive fermentation, most of Monascus pigment studies focused on the intracellular pigments biosynthesis (Balakrishnan et al. 2013, 2014, 2017; Bijinu et al. 2014), while only a small amount of research had been done on the biosynthesis pathway of extracellular pigments (Koehler 1983; Hajjaj et al. 1997). Hajjaj et al. (1997) discovered that Monascus could produce the extracellular red pigments N-glucosylrubropunctamine and N-glucosylmonascorubramine in a chemically defined culture medium with excess glucose and monosodium glutamate (nitrogen source). Chen et al. (2017) found that the intracellular orange pigments could be converted to extracellular yellow pigments during the trans-membrane secretion process in a nonionic surfactant aqueous solution (Chen et al. 2017). So, we speculated that the extracellular water-soluble yellow pigments in this study were derivatives of intracellular pigments via the trans-membrane conversion. The pigments were further identified by means of LC–MS (Additional file 4: Figure S3). Based on their UV–Visible spectra (Additional file 2: Figure S1) and molecular weights, It could be deduced that the four pigments have not been described and reported before (Chen and Wu 2016). It needed to be confirmed by identifying the structure of four extracellular water-soluble yellow pigments further. We could also observe that the production of extracellular water-soluble yellow pigments were growth-associated and were coupled to LFDCW, while the concentration of intracellular pigments was just partially associated with cell growth (Fig. 2). A possible reason for this is that during the earlier stages of fermentation, the increased of DCW was mainly attributable to the increasing LFDCW and lower intracellular lipid accumulated, resulting in fewer reservoirs for intracellular pigment storage. The time accumulated LFDCW was extended under a high IGC (Fig. 2b), which allowed more time for the biosynthesis and secretion of derivative extracellular pigments (water-soluble yellow pigments). During the later stages of the fermentation, the increased DCW was mainly due to increased lipids (Fig. 2b), which may have served as reservoirs for accumulating intracellular pigments and caused less pigments precursors to be available for the conversion and secretion of extracellular water-soluble yellow pigments (Fig. 2d). On the other hand, the high glucose stress could also promote the biosynthesis of unsaturated fatty acids in M. ruber and make a better fluidity and permeability of the cell membrane, which would improve the trans-membrane conversion and secretion of intracellular pigments to the broth. The similar report could be found that the fumaric acid production could be improved under high glucose stress through synthesizing more unsaturated fatty acids than the saturated one to alternate the fluidity and permeability of the cell membrane with Rhizopus oryzae (Lyu et al. 2015). High glucose stress changed the permeability of Monascus mycelia, enhanced the trans-membrane conversion and secretion of intracellular pigments to the broth, and improved the production of extracellular yellow pigments.
The biosynthesis of Monascus pigments follows the polyketide pathway (Hajjaj et al. 1997; Shao et al. 2014). MpPKS5 and mppD are the structural genes of Monascus pigments and encode the polyketide synthases which are keys to the biosynthesis the polyketide chromophore of these pigments. The genes MpfasA2 and MpfasB2 (Mpfas2) encode a canonical fungal fatty acid synthase and supply the medium-chain (C8 and C10) fatty acyl moieties for Monascus pigments biosynthetic activities (Balakrishnan et al.2013, 2014). The mppB gene encodes a trichothecene 3-O-acetyltransferase (AT), which can transfer the medium-chain (C8 and C10) fatty acyl group into the polyketide chromophore to complete pigment biosynthesis. The mppR1 and mppR2 genes are regulatory genes for pigments biosynthesis (Balakrishnan et al. 2013). The genes MpPKS5, MpfasA2, MpfasB2, mppB, mppR1, and mppD were up-regulated during high glucose stress in the later stage of fermentation (Fig. 4). Furthermore, the increased glucose as the sole carbon source could offer more precursors and cofactors such as acetyl-CoA, malonyl-CoA, NADH and NADPH for the biosynthesis of Monascus pigments and lipids (Beatriz Ruiz et al. 2010). These results illustrated that the polyketide biosynthesis capacity could be enhanced by increasing the polyketide chromophores, medium-chain fatty acyl moieties and critical polyketide synthases under high glucose stress. It helped support that high glucose stress promoted the production of yellow pigments through an internal power and the promoting effect is stable.
In summary, high glucose stress improved more reservoirs for intracellular pigments storage by increasing the content of Monascus mycelia and the lipids content in Monascus mycelia. Simultaneously, high glucose stress up-regulated the expression of pigment biosynthetic genes, especially the genes involved in yellow pigments biosynthetic. Thereby, a high proportion of intracellular yellow pigments rather than orange pigments were achieved under high glucose stress. High glucose stress also improved the fluidity and permeability of the cell membrane and enhanced the trans-membrane conversion of intracellular pigments to extracellular water-soluble yellow pigments and secretion into the broth, resulted in a twofold increase of extracellular water-soluble yellow pigments compared to low IGC condition. Further studies are needed to elucidate the molecular pathways through which high glucose stress regulates yellow pigments production. Thus, submerged fermentation under high glucose stress has potential application in the production of Monascus yellow pigments.
high performance liquid chromatography
dry cell weight
lipid-free dry cell weight
real-time quantitative PCR
gas chromatograph–mass spectrometer
liquid chromatograph–mass spectrometer
TH planned and carried out the experiments, analyzed the data and wrote the manuscript; MHW, KS and GC assisted to carry out experiments; XFT reviewed the manuscript; ZQW participated in the data analysis and finalized the manuscript. All authors read and approved the final manuscript.
This study was supported by the financial support of the National Natural Science Foundation of China (No: 31271925), the Special Project on the Integration of Industry, Education and Research of Guangdong Province, China (No: 2013B090600015) and the Science and Technology Program of Guangzhou, China (No: 2014J410019).
The authors declare that they have no competing interests.
Availability of data and materials
Ethics approval and consent to participate
Not applicable. This article does not contain any studies with human participants or animals performed by any of the authors.
This study were funded by the financial support of the National Natural Science Foundation of China (No: 31271925), the Special Project on the Integration of Industry, Education and Research of Guangdong Province, China (No: 2013B090600015) and the Science and Technology Program of Guangzhou, China (No: 2014J410019).
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