Enhanced Monacolin K Production in Monascus purpureus Via Mutagenesis and Nitrogen Source Optimization

ABSTRACT

Monascus fungi are valued for their production of Monacolin K (MK), a bioactive compound with recognized lipid‐lowering and multifunctional properties. To enhance MK yield in solid‐state fermentation, this study combined atmospheric and room‐temperature plasma (ARTP) mutagenesis with L‐arginine supplementation. A mutant strain, C‐23, was obtained, and the addition of L‐arginine increased MK production by 97.3%, reaching 9.45 mg/g. Untargeted metabolomics revealed that L‐arginine reshaped metabolic pathways related to amino acids, cofactors, carbohydrates, and lipids, elucidating the mechanism behind MK overproduction. This work provides a strategic approach for developing high‐yielding Monascus strains and offers insights into the metabolic regulation of MK biosynthesis.

Abbreviations

ARTP

atmospheric and room‐temperature plasma

KEGG

Kyoto Encyclopedia of Genes and Genomes

LC–MS

liquid chromatography‐mass spectrometry

MK

monacolin K

OPLS‐DA

orthogonal partial least squares discriminant analysis

PCA

principal component analysis

RYR

red yeast rice

SSF

solid‐state fermentation

TCA

tricarboxylic acid

VIP

Variable Importance in Projection

1. Introduction

Monascus, a widespread fungal species, has diverse and unique applications in several fields [1]. Its fermentation product, red yeast rice (RYR), has been used for thousands of years [2]. Monascus species play a key role in the food industry and many other fields, such as medicine, chemicals, and agriculture [3, 4, 5, 6]. Among their secondary metabolites, monacolin K (MK), also known as lovastatin, has become a research hotspot worldwide, owing to its lipid‐lowering and anti‐inflammatory effects. These properties of MK are attributed to its effective inhibition of 3‐hydroxy‐3‐methylglutaryl coenzyme A [7].

Recently, several methods have been developed to increase MK production by Monascus species. For example, the mutant CSUFT‐1 strain exhibited a 1.67‐fold increase in MK production during solid‐state fermentation (SSF) conditions [8]. Another Monascus strain was subjected to ultraviolet irradiation and ethyl methanesulfonate mutagenesis, resulting in varying increases in MK content [9]. Another study enhanced MK production in the mutant KU4 strain by gamma‐irradiating the spore suspension [10]. Overall, these mutagenesis methods effectively increase the content of MK produced by Monascus. In addition, MK yield can be increased by optimizing fermentation, for example, by adding amino acids and other nutrients. For example, adding glutamic acid to Monascus fermentation liquid can significantly improve MK yield [11]. Moreover, adding arginine to the Monascus fermentation broth significantly increased its MK yield from 94.6 to 228.4 mg/L [12]. The addition of soy protein isolate can also significantly alter MK metabolism [13].

The present study aimed to lay a new foundation for optimizing Monascus fermentation and increasing MK production. To this end, we used the ARTP mutation technology to obtain a Monascus mutant with increased MK yield during SSF. We also assessed the effects of adding L‐arginine to Monascus SSF on MK yield. Finally, we investigated the metabolic regulatory mechanism of L‐arginine on MK production using liquid chromatography‐mass spectrometry (LC–MS), revealing the underlying pathways responsible for enhanced MK production.

2. Materials and Methods

2.1. Strains

Monascus M‐1 (CGMCC No. 18110) was isolated from Monascus samples collected from Monascus products in major Monascus‐producing areas in China. The sample has been deposited in the general microbiology center of the China microbiology preservation and management committee and our laboratory. The mutant strain C‐23 was mutated using the ARTP technique and is maintained in our laboratory. Rice was purchased from Heilongjiang (China).

2.2. ARTP Mutation and Screening

The spore suspension was prepared by washing the spores with sterile water, then centrifuging and repeating the washes twice with sterile water. Microscopically, spore concentration was determined to be 10⁶–10⁸/mL. A 10 μL aliquot of the suspension was evenly spread onto the substrate, and ARTP irradiation was performed for varying durations (0, 30, 60, 75, 90, 120, 150, and 180 s). The treatment parameters were set as follows: processing distance, 2 mm; processing power, 100 W; and gas flow rate, 10 standard liters per min [14].

After processing, the sheets were placed in Eppendorf tubes containing 1 mL of an aseptic solution using aseptic tweezers. The tubes were then placed in an oscillator and allowed to oscillate for 1 min. Microorganisms attached to the slides were eluted into an aseptic solution to form a bacterial suspension. The new bacterial suspension was diluted, and 100 μL of the dilution was spread on a Petri dish. Colonies were then counted, and the lethal rate was calculated. Mutant strains were screened using SSF, and those with high MK content were selected.

2.3. Genetic Stability Test of the C‐23 Mutant

The selected Monascus mutant strains with high MK production were inoculated into potato dextrose agar medium and transferred to five generations of solid fermentation cultures under the same conditions [15, 16, 17]. MK content was determined using high‐performance liquid chromatography to analyze the genetic stability of the mutant strains [18, 19].

2.4. Optimization of Cultivation and Fermentation Conditions

The mutant C‐23 was cultured for 7 d at 30°C on the potato dextrose agar medium. After culture, the hyphae were scraped into sterile water, and filtered it into a conical flask using sterile gauze and a funnel. And inoculated into a seed medium containing 60 g/L glucose, 25 g/L peptone, 10 g/L corn starch, 2 g/L NaNO₃, 1 g/L KH₂PO₄, and 1 g/L MgSO₄·7H₂O. The seed medium concentration was adjusted to 10⁶ CFU/mL. The seeds were incubated at 30°C for 48 h with shaking at 180 rpm. Subsequently, inoculate 15% of the seed liquid into a solid fermentation medium containing 30 g of steamed rice with an initial moisture content of 45%. The medium is placed in a 250 mL conical flask, with a loading density of 20% of the flask volume [20].

To determine the effect of L‐arginine on MK yield, different concentrations of arginine solutions are added in solution form during solid‐state fermentation. (Arg group), and a control medium (CK group) was used for comparison. The optimal arginine concentration was determined for the subsequent fermentation. Fermentation was carried out at 30°C for 72 h, followed by incubation at 25°C for 20 d [21].

2.5. Detection of MK Yield in Monascus SSFProducts

RYR samples were dried at 55°C for 8–10 h. The dried sample was ground into a fine powder, passed through a 40‐mesh screen, and any impurities or uneven particles were removed. RYR powder (0.06 g) was accurately weighed and transferred to a 10 mL colorimetric tube for analysis. To further increase the contact area of the sample with the reagent and ensure accurate control, a 70% ethanol solution was used at a constant volume of 6 mL [22]. The reaction mixture was then incubated at 55°C in a water bath for 1 h. The samples were shaken every 20 min to ensure uniform mixing. The extract was then removed, and the sample solution was filtered using a filter film (0.22 μm).

High‐performance liquid chromatography was performed using the following parameters: detection wavelength 238 nm, C18 column (150 mm × 4.6 mm × 5 μm), column temperature 28°C, mobile phase H₃PO₄ solution of aqueous solution and acetonitrile (45:55, v/v) as buffer, pH value 2.5, injection quantity 20 μL, and flow rate 1.0 mL/min [23].

2.6. LC–MS Analysis

RYR samples were collected after 23 d of SSF, and seven biological replicates were used for LC–MS analysis. For metabolite extraction, a sample (50 ± 5 mg) was accurately weighed and placed in a 2 mL centrifuge tube. One grinding bead (6 mm diameter) was added along with the extraction solution containing four internal standards (e.g., L‐2‐chlorophenylalanine (0.02 mg/mL) in a methanol: water ratio of 4:1 (v:v). The tube was placed in a frozen tissue grinder set at −10°C and 50 Hz, and the sample was ground for 6 min. Low‐temperature ultrasonic extraction was then performed for 30 min, after which the sample was allowed to stand at −20°C for 30 min. The mixture was centrifuged at 4°C for 15 min, and the supernatant was transferred to an autosampler with an insert for further analysis. Simultaneously, 20 µL of the supernatant from each sample was combined to prepare a quality‐control sample.

ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 µm; Waters, Milford, MA, USA) was used for chromatography, with an injection volume of 3 μL and a column temperature of 40°C. Mobile phase A comprised 95% water and 5% acetonitrile, and mobile phase B comprised 47.5% isopropanol and 5% water.

2.7. Statistical Analysis

All experiments were conducted in triplicate, and the results are presented as mean ± standard deviation. Data visualization was performed using Origin 9.0 (OriginLab Corporation, Northampton, MA, USA) and GraphPad Prism 9.5 (GraphPad, San Diego, CA, USA). Statistical analysis of each component was performed using a t‐test to identify significant differences between the groups. Differential metabolites were selected based on fold change ≥ 1, Variable Importance in Projection (VIP) > 1, and p‐value < 0.05.

3. Results

3.1. Screening of Monascus Mutants and Genetic Stability Experiments

ARTP mutagenesis offers advantages such as simplicity, low cost, the absence of toxic or harmful substances in the mutation process, and good genetic stability, which is why researchers favor it. As shown in Figure 1A, ARTP treatment for 30 and 90 s resulted in 79.2% lethality in the M‐1 strain, which increased to 96.7% when the treatment time was extended to 90 s. When the treatment duration was further increased to 120, 150, or 180 s, almost no colonies grew on the medium, indicating that lethality increased with increasing treatment duration. Therefore, we selected 90 s as the optimal treatment duration.

Figure 1.

(A) Fatality rate of the strain M‐1. (B) MK yield of the mutant strain and the strain M‐1. (C) Genetic stability of mutant strain C‐23. (D) Comparison of MK production in mutant strain C‐23 with and without the addition of l‐arginine. Numbers 1‐6 respectively represent: C‐23 strains with no added arginine and arginine added at levels of 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, and 0.7%.*p < 0.05, **p < 0.01,***p < 0.001.

After the initial screening, we selected mutant strains with positive mutations following ARTP treatment for 90 s: C‐2, C‐19, C‐22, C‐23, and C‐39 (Figure 1B). Notably, the MK yield of the three mutant strains C‐22, C‐23, and C‐39 is significantly higher compared to strain M‐1. the MK yield of the C‐23 mutant was the highest, at 6.92 mg/g, representing a 44.5% increase over the starting strain M‐1, which yielded 4.79 mg/g. We subjected the C‐23 mutant to five consecutive subcultures and performed SSF cultivation for each generation separately. We then evaluated genetic stability using MK content as an indicator (Figure 1C). As the MK yield of the C‐23 mutant remained stable, we selected this high‐MK‐yielding mutant for further analysis.

3.2. Effect of Arginine on MK Production by Monascus Mutant Strain C‐23

When L‐arginine was added at levels between 0.2% and 0.7% during the SSF of the mutant strain C‐23, the MK yield exhibited a trend of first increasing and then decreasing with an increase in L‐arginine addition. Specifically, when added at 0.20%–0.40%, L‐arginine increased MK yield in a dose‐dependent manner, when L‐arginine was added at levels between 0.4% and 0.6%, the MK yield of strain C‐23 was significantly increased, reaching the highest value of 9.45 mg/g at 0.4%, representing a 36.6% increase compared with that observed in the absence of L‐arginine addition (Figure 1D). However, when L‐arginine was added at levels exceeding 0.40%, the overall MK yield decreased. Therefore, we considered 0.40% L‐arginine as the optimal amount.

3.3. Addition of L‐Arginine Promotes MK Production by C‐23 During SSF

To further explore the mechanism of action of L‐arginine in promoting MK production, we used LC–MS to analyze key metabolites and affected metabolic pathways, and to detect fermentation metabolites on the 23rd day of SSF.

The ion chromatograms of all samples in both positive and negative ion modes are displayed separately (Figure 2). Under these detection conditions, the peak shapes were well‐defined, and the distribution was relatively uniform. As indicated in the principal component analysis and orthogonal partial least squares discriminant analysis (OPLS‐DA) plots of the quality control samples in both positive and negative ion modes, along with the CK and Arg group samples (Figure 3A,B), the eight replicates within each group were closely clustered, indicating good biological reproducibility. Moreover, a clear separation between the two groups was observed, suggesting significant differences in the metabolites. We further analyzed the OPLS‐DA permutation test plot (Figure 3C), which exhibited an R²X value greater than 0.9 and both R²Y and Q² values approaching 1, indicating that the OPLS‐DA model was robust and stable. Collectively, these results indicate that L‐arginine addition induces metabolic changes in cells.

Figure 2.

(A) Cation chromatogram. (B) Anion chromatogram.

Figure 3.

All samples were obtained from the L‐arginine and control groups. (A) Principal component analysis (PCA) plot. (B) Orthogonal partial least squares discriminant analysis (OPLS‐DA) plot. (C) OPLS‐DA permutation test plot.

To further investigate the metabolic changes, we identified 2208 differential metabolites using VIP > 1, fold change ≥ 1, and p value < 0.05. The number of differential metabolites was visualized using hierarchical clustering heatmaps and volcano plots (Figure 4A,B). The classification and proportion of these differential metabolites, presented as a pie chart in Figure 4C, were as follows: 27.27% peptides, 24.24% lipids, 12.12% carbohydrates, 12.12% antibiotics, 9.09% steroids, 9.09% hormones and transmitters, and 6.06% vitamins and cofactors.

Figure 4.

(A) Hierarchical clustering heatmap of the significantly different metabolites between the Arg and CK groups. (B) Volcano plot of all samples in the Arg and CK groups. (C) Classification pie charts of all samples in the Arg and CK groups. Arg, L‐arginine; CK, control.

We then subjected the differential metabolites to Kyoto Encyclopedia of Genes and Genomes enrichment analysis (Figure 5A,B). This analysis revealed that the addition of L‐arginine (Arg) resulted in significant changes in the metabolism of cofactors, vitamins, amino acids, carbohydrates, and lipids in Monascus SSF. The specific enriched pathways are shown in Figure 5B,C.

Figure 5.

(A) Statistical chart of compound classification based on the Kyoto Encyclopedia of Genes and Genomes (KEGG). (B) KEGG pathway enrichment bubble chart. (C) KEGG enrichment analysis network diagram.

4. Discussion

In amino acid metabolism, the addition of Arg upregulated the production of 3‐hydroxyphenylacetic acid, hydroquinone, N‐methyltyramine, homovanillin, prephenate, chorismate, (Z)‐but‐1‐ene‐1,2,4‐tricarboxylate, and other compounds. These compounds significantly upregulated the biosynthesis of tyrosine, tryptophan, lysine, proline, and several other amino acids. In contrast, the addition of Arg significantly decreased the levels of histidine, methionine, leucine, and isoleucine. Amino acids, which serve as nitrogen sources, provide essential nutrients for Monascus metabolism [24]. Acetyl‐CoA and malonyl‐CoA can be generated through amino acid catabolism, such as that of alanine, valine, leucine, and isoleucine. Moreover, other amino acids, including arginine and proline, participate in the tricarboxylic acid (TCA) cycle [13]. Our metabolomics analysis indicated that the addition of Arg led to the upregulation of several amino acids and the downregulation of others. This suggests that Arg may promote the growth and development of Monascus by regulating the degradation and utilization of nitrogen resources [25].

In the metabolism of cofactors and vitamins, Arg was associated with increased levels of with anolide B, chorismate, folinic acid, 10‐formyltetrahydrofolate, 5,10‐methylene‐Thf, 5,10‐methenyltetrahydrofolic acid, 3‐succinoylpyridine, beta‐d‐ribosylnicotinate, biotin, biopterin, and tetrahydrofolyl‐[Glu]. These metabolites are involved in the metabolism of nicotinic acid and nicotinamide, as well as in the biosynthesis of cofactors such as biotin, folate, porphyrin, and vitamins. These results suggest that Arg promotes the biosynthesis of secondary metabolites in Monascus via cofactors and other substances [26].

In carbohydrate metabolism, the addition of Arg resulted in the upregulation of arbutin, phenethylamine glucuronide, ibuprofen glucuronide, 15‐hydroxynorandrostene‐3, 17‐dione glucuronide, galactose, inositol, and d‐glucosaminide. This result suggests that Arg may regulate the glycolysis pathway, pentose and glucuronic acid interconversion, galactose metabolism, amino sugar and nucleotide sugar metabolism, and the TCA cycle. By modulating these pathways, Arg generates substantial energy to support microbial growth and metabolism and promotes the production of secondary metabolites [27]. Therefore, Arg, directly or indirectly, provides nutrients for MK production via glycolysis, the pentose phosphate pathway, and the TCA cycle, ultimately promoting MK production.

In lipid metabolism, Arg addition upregulated the levels of thromboxane, leukotriene, methyl epijasmonate, and 7‐epijasmonic acid. These compounds regulate arachidonic and α‐linolenic acid metabolism and the biosynthesis of unsaturated fatty acids. Notably, Monascus is rich in polyunsaturated and unsaturated fatty acids, which may enhance the cholesterol‐inhibitory effects of MK. These results suggest that Arg promotes lipid metabolism, which, in turn, promotes MK production [28].

MK is a polyketide compound synthesized via the condensation of acetyl‐CoA and malonyl‐CoA units by polyketide synthase (PKS). The Arg‐induced upregulation of glycolysis and the catabolism of specific amino acids (e.g., valine, leucine, and isoleucine) significantly expands the intracellular acetyl‐CoA and malonyl‐CoA pools, thereby increasing substrate availability for the PKS complex [29]. Furthermore, Arg stimulates the pentose phosphate pathway, boosting the supply of NADPH required by the polyketide synthase for reductive steps. Concurrent activation of energy metabolism ensures sufficient ATP.

In addition to these major metabolic pathways, Arg regulates several others, including terpenoid and polyketide metabolism, ABC transport, energy metabolism, and nucleotide metabolism. Collectively. Arg acts as a metabolic modulator, creating an optimal environment for MK synthesis by ensuring abundant substrate supply, reducing power, and cofactor availability, these results indicate that Arg regulates secondary metabolites in a multifaceted manner.

In conclusion, we successfully identified a high MK‐producing Monascus mutant (C‐23) using ARTP mutagenesis. Compared to the original strain M‐1, this strain exhibited a 44.5% increase in MK production. After five generations of SSF culture, we confirmed that the C‐23 strain stably produced MK at levels of 6.53–6.99 mg/g. We also demonstrated that an optimal L‐arginine concentration (0.40%) significantly enhanced MK yield by 36.6% (9.45 mg/g) and, compared to the parent strain M‐1, increased MK yield by 97.3%. We explored the metabolic regulation of L‐arginine on the production of secondary metabolites by Monascus using LC–MS, revealing that it regulates amino acid, cofactor, and vitamin metabolism, as well as carbohydrate and lipid metabolism in Monascus, and promotes the production of secondary metabolites by Monascus, thus directly or indirectly enhancing MK production. Overall, this study provides valuable insights into improving Monascus SSF MK production.

Conflicts of Interest

The authors declare no conflicts of interest.

Yang C., Yajing Y., and Housheng H., “Enhanced Monacolin K Production in Monascus purpureus Via Mutagenesis and Nitrogen Source Optimization,” Journal of Basic Microbiology 66 (2026): e70151, 10.1002/jobm.70151.

Data Availability Statement

The original contributions presented in this study are included in this article; further enquiries can be directed to the corresponding authors.