Inactivation of MrPigH in Monascus ruber M7 results in its Monascus pigments increase and citrinin decrease with mrpyrG selection marker

: Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries and nowadays in the whole world via Asian catering. The MPs biosynthetic pathway has been well-illustrated, however, the functions of a few genes including mrpigH in the MPs gene cluster of M. ruber M7 are still unclear. In current study, mrpigH was disrupted in Δ mrlig4 Δ mrpyrG , a highly efficient gene modification system, using mrpyrG as a selection marker, and Δ mrpigH Δ mrlig4 Δ mrpyrG :: mrpyrG and Δ mrpigH Δ mrlig4 Δ mrpyrG have been obtained. Subsequently, their morphologies, biomasses, MPs and citrinin (CIT) production were analyzed, respectively. These results have revealed that the deletion of mrpigH has significant effects on the morphology and growth of M. ruber M7. Moreover, compared with M. ruber M7, the yields of MPs and CIT were drastically increased and decreased in mrpigH mutants, respectively.


Introduction
Monascus species are famous medicinal and edible filamentous fungi used in traditional fermentation in Asian countries, such as China, Japan, and the Korean Peninsula, for nearly 2,000 years [1,2]. At present, their fermented products, such as Hongqu also called red fermented rice, red yeast rice and red mold rice, are widely used as food additives and nutraceutical supplements worldwide owing to their production of abundant beneficial secondary metabolites (SMs), such as Monascus pigments (MPs), monacolin K (MK) and γ-amino butyric acid (GABA) [1,3]. However, citrinin (CIT), a nephrotoxic mycotoxin produced by some strains of Monascus spp., restricted the application of Monascus fermented products [4].
M. ruber M7, which can produce MPs and CIT, without MK, was subjected to wholegenome sequencing analysis [5]. And the functions of most genes in the MPs gene cluster of M. ruber M7 have been investigated by gene manipulation [6]. However, there are a few genes in the MPs gene cluster of M. ruber M7, such as mrpigH and mrpigI, which have not been investigated [6,7]. In 2017, Balakrishnan et al. predicted that the mppE in M. purpureus KACC (highly homologous to mrpigH in M. ruber M7) encoded a reductase, which can decrease orange pigments (OPs) and red pigments (RPs) in the biosynthesis of MPs [8]. In 2019, Chen et al. also guessed that MrPigH might contribute to reducing the carbon double bond of the precursor compounds to typical yellow pigments (YPs) monascin and ankafiavin [7].
In this study, we firstly cloned mpigH from M. ruber M7. Subsequently, mrpigH was disrupted in the highly efficient system Δmrlig4ΔmrpyrG [9] using the mrpyrG selection marker, and 2 mrpigH deletion strains ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG have been constructed. Finally, the morphologies, biomasses, MPs and CIT production of these mrpigH mutants were assessed. The results revealed that the deletion of mrpigH led to a significant reduction in biomass accumulation. Crucially, the inactivation of MrPigH resulted in an increase of MPs and a decrease of CIT.

Materials and Methods
2.1. Fungal strains, culture media, and growth conditions M. ruber M7 (CCAM 070120, Culture Collection of State Key Laboratory of Agricultural Microbiology, Wuhan, China), which can produce MPs and CIT, but no MK, is an original strain, which was isolated from Hongqu and preserved in our laboratory [10]. Δmrlig4ΔmrpyrG, the highly efficient gene modification system for M. ruber M7, also named as the markerless disruption strain [9] was used to generate ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG. All strains used in this study are described in Table 1. Strains were cultivated in PDA (potato dextrose agar) or minimal medium (MM, 2.0 g NH4Cl, 1 g (NH4)2SO4, 0.5 g KCl, 0.5 g NaCl, 1.0 g KH2PO4, 0.5 g MgSO4·7H2O, 0.02 g FeSO4·7H2O, 20.0 g glucose, distilled water to 1 L, pH 5.5). When required, 10 mM uridine and/or 0.75 mg/mL 5-fluoroorotic acid (5-FOA) were added. For observation of colonial and microscopic morphologies, four different types of media, PDA, malt extract agar (MA), Czapek yeast extract agar (CYA) and 25 % glycerol nitrate agar (G25N) were utilized [11]. PDA was used for the analysis of MPs and CIT production. All strains were maintained on PDA slants at 28 °C.

Construction of deletion cassettes and plasmids
The genomic DNA of M. ruber M7 used for PCR was isolated as described previously [12]. The mutant strains ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG were constructed using site-directed homologous recombination. The mrpigH gene markerless deletion cassette (5'UTR-mrpyrG-5'-1UTR-3'UTR) was constructed by seamless cloning, and shown schematically in Figure 1a. The relative primer pairs were shown in Table 2.

Deletion of mrpigH in Δmrlig4ΔmrpyrG strain
The plasmid pCPGPIGH was transformed into Agrobacterium tumefaciens EHA105 using a freeze-thaw method [13]. Δmrlig4ΔmrpyrG, a markerless disruption strain, was used as a host strain to delete mrpigH with the mrpyrG recyclable marker [9]. The A. tumefaciens clones containing pCPGPIGH were incubated for transformation with Δmrlig4ΔmrpyrG to generate a mrpigH gene deletion mutant (ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG) by minimal medium without uridine/uracil. The conidia of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG were collected and spread onto PDA with 0.75 mg/mL 5-FOA and 10 mM uridine. After incubated at 28 °C for 6 days, the surviving colonies were transferred to a new PDA under the same conditions for 4 days. The final surviving colonies were selected and verified by PCR. Selected transformants were designated as ΔmrpigHΔmrlig4ΔmrpyrG.

MPs and CIT analyses
M. ruber M7 can produce MPs and CIT, but no MK. Previous researches have shown that MPs mainly accumulated in the mycelia, while CIT exists in the media [14]. Therefore, the intracellular MPs and extracellular CIT were detected. 1 mL spores suspension (10 5 cfu/mL) of each strain were inoculated on PDA plate coated with cellophane membranes and incubated at 28℃ for 11 days. 20 mg freeze-dried mycelia or media powder were suspended in 1 mL 80 % (v/v) methanol solution, and subjected to 30 min ultrasonication treatment (KQ-250B, Kunshan, China). Then, the extraction solutions were separated by centrifugation at 10,000 ×g for 15 min and filtered with a 0.22 μm filter membrane for further analysis.
The pigments groups concentration was measured using a UV−vis UV-1700 spectrophotometer (Shimadzu, Tokyo, Japan) at 380, 470 and 520 nm which are the maximal absorption of yellow, orange, and red pigments, respectively. The results were expressed as optical density (OD) units per gram of dried media multiplied by a dilution factor [15].

Detection of the relative gene expression level in MPs and CIT gene clusters by RT-qPCR
To analyze the influence of mrpigH deletion on gene expression in MPs and CIT gene cluster, ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and the wild-type strain (M. ruber M7) were selected for quantitative real-time PCR (RT-qPCR) detection. The ΔmrpigHΔmrlig4ΔmrpyrG was lacked of mrpyrG and had to supply uridine, which might have had an effect on the yields of MPs and CIT.
One milliliter freshly harvested spores (10 5 cfu/mL) of each strain were inoculated on PDA plate and incubated at 28 ℃, and samples were taken every other day from the 3rd day to the 9th day. RT-qPCR was performed according to the method described by Liu et al. [13]. Beta-actin was used as a reference gene. The primers used in these analyses were listed in Table S1.

Verification of the mrpigH deletion in Δmrlig4ΔmrpyrG
The Δmrlig4ΔmrpyrG strain is a promising host for efficient gene targeting in M. ruber M7 and analysis of biosynthesis of SMs. The deletion of mrpigH was executed in Δmrlig4ΔmrpyrG with the mrpyrG marker (Figure 1a.). After the plasmid pCPGPIGH harbouring mrpyrG was transformed into Δmrlig4ΔmrpyrG, transformants without uridine/uracil auxotrophic were obtained and verified by PCR, and 5 of 28 transformants were mrpigH-deleted strains. As shown in Figure. 1c, a 0.5-kb product was amplified when the genomic DNA of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG was used as template with primers pyrG F2-pyrG R2, while no DNA band was amplified using genome of the Δmrlig4ΔmrpyrG. A 0.58-kb fragment of the mrpigH gene could be amplified from Δmrlig4ΔmrpyrG using primers pigH F-pigH R, while no band was obtained from ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG.
Meanwhile, amplicons of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG (3.54 kb and 1.73 kb) and Δmrlig4ΔmrpyrG(2.85 kb) differed in size when primers pigHpyrG 5F-pigHpyrG 3R annealing to homologous arms were used. In the ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG strain, the mrpigH deletion cassette contains two completely homologous sequences (5'UTR and 5'-1UTR) between the mrpyrG expression fragments. If ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG was incubated on PDA plates containing 5-FOA, the strains that lost the mrpyrG expression fragments by homologous recombination should be selected. As predicted, ΔmrpigHΔmrlig4ΔmrpyrG strains without the mrpyrG fragment could be isolated from ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG after growth on PDA containing 0.75 mg/mL 5-FOA and 10 mM uridine. Total 12 putative ΔmrpigHΔmrlig4ΔmrpyrG strains with 5-FOA resistance were obtained and analyzed, and one of them was shown as follows. In PCR analysis as shown in Figure. 1e, a 0.58-kb fragment of the mrpigH gene and a 0.5-kb product of the mrpyrG gene could be amplified from M. ruber M7 using primers pigH F-pigH R and pyrG F2-pyrG R2, while nothing was obtained from ΔmrpigHΔmrlig4ΔmrpyrG, respectively. Meanwhile, amplicons of ΔmrpigHΔmrlig4ΔmrpyrG (1.73 kb) and M. ruber M7 (2.85 kb) differed in size when primers pigHpyrG 5F-pigHpyrG 3R annealing to homologous arms were used. Those results indicated that there was mrpigH markerless deletion was constructed in Δmrlig4ΔmrpyrG.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 19 November 2021
To investigate whether the mrpigH was markerlessly deleted by mrpyrG in Δmrlig4ΔmrpyrG, M. ruber M7 and its mrpigH markerless mutants were cultivated on PDA supplemented the appropriate additive (10 mM uridine for the uridine /uracil auxotrophy).The results (Figure 2a) revealed that ΔmrpigHΔmrlig4ΔmrpyrG showed no growth on PDA, but it was able to grow on PDA with 0.75mg/mL 5-FOA and 10 mM uridine, while the growth of M. ruber M7 and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG were inhibited by the addition of 0.75 mg/mL 5-FOA to PDA, but could grow on PDA. Those results indicated that the mrpigH markerless mutants were successfully constructed. To test the influences of deleting mrpigH on developmental processes, M. ruber M7 and ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG were cultivated on different media (PDA, CYA, MA and G25N) to observe their colonial and microscopic characteristics. The results showed that the colonial morphologies of mrpigH mutants (Figure 2b) were obviously different from those of M. ruber M7 on different culture plates, especially on PDA plate, the ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG showed slower growth rate and darker color. However, the microscopic morphologies, including conidia and cleistothecia, of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG was not significantly different from those of M. ruber M7 on different culture plates (Figure 2c). Moreover, the biomasses of 2 mrpigH mutants apparently decreased compared with M7 on PDA in 5-11 d (Figure 2d).

Analysis of MPs and CIT production
In order to evaluate the effect of detecting mrpigH on MPs and CITs during fermentation, the samples cultured for 3, 5, 7, 9 and 11 days were obtained and each test was repeated 3 times independently. OD values representing yellow, orange and red pigments production were determined using a spectrophotometer at 380 nm, 470 nm and 520 nm, respectively. As shown in Figure 3a-c, from the 7th day to the 11th day, M. ruber M7 produced much fewer MPs (including YPs, OPs and RPs) than 2 mrpigH mutants (ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG), whereas the ability of producing MPs among 2 mrpigH mutants showed no obvious difference. After 11 days of cultivation, the YPs, OPs and RPs production in ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and As to CIT, the UPLC has been performed to detect the production during fermentation. As shown in Figure 3d, CIT produced by M. ruber M7 and all mrpigH mutants showed an obvious difference. At the end of the 11 days of fermentation, CIT production in 2 mrpigH mutants (ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG) decreased significantly, and was 2-3 orders of magnitude less than that of M.ruber M7. Among 2 mrpigH mutants, CIT production in ΔmrpigHΔmrlig4ΔmrpyrG was higher than that of ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG. The possible cause was that the ΔmrpigHΔmrlig4ΔmrpyrG lacked mrpyrG and had to supply uridine, which might have an effect on the yields of CIT.

The genes' expression in MPs and CIT gene clusters from mrpigH mutants
The genes expression in MPs and CIT gene clusters in ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and M. ruber M7, were analyzed by RT-qPCR. As shown in Figure 4, The relative expression levels of mrpigA, mrpigB, mrpigC, mrpigD, mrpigE, mrpigF, mrpigG, mrpigJ, mrpigK, mrpigM, mrpigN, mrpigO and mrpigP in ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG were obviously higher than that of M. ruber M7 at 5th to 9th day. Therefore, the deletion of mrpigH increased the majority of MPs gene expression level, which might correspond with the enhanced MPs production.
In this current study, in order to explore the function of mrpigH, we used the Δmrlig4ΔmrpyrG as the starting strain to generate two mrpigH disruptants. The colonial and microbiological phenotypes and biomasses of the mrpigH mutants showed an obviously difference from those of M. ruber M7 (Figure 2b, d). In particular, the biomasses of mrpigH mutants accumulated more slowly than that of M ruber M7. Our laboratory previously constructed MPs gene knockout strains with the resistance selection markers (hph/neo) in M7, and found that the morphologies and biomasses of mrpigC, mrpigE, mrpigF, mrpigM and mrpigO knockouts were comparable to those of M. ruber M7, whereas the growth rates of mrpigA, mrpigJ, and mrpigK knockouts were increased, and the deletion of mrpigN resulted in a reduction in biomass accumulation compared with M. ruber M7 [22].
Moreover, deletion of mrpigH in M. ruber M7 enhanced YPs, OPs and RPs (Figure 3ac), which is mostly consistent with the results obtained in M. purpureus [8]. We also found that deletion of mrpigH decreased the production of CIT (Figure 3d), which is a promising scheme for control of CIT. CIT is a kind of mycotoxin produced via the polyketide pathway by filamentous fungi, mainly by Monascus spp. and Penicillium spp. [23][24][25]. To this end, we investigated that the changes in the expression levels of MPs and CIT genes in their gene clusters of mrpigH mutants, and found that the relative gene expression levels almost corresponded with the increase of MPs and decrease of CIT (Figure 4, 5).
So why the knockout of mrpigH can increase the pigment content while reducing the production of CIT, needs further study. In early research, both MPs and CIT were considered to be derived from polyketide pathways [26]. Lately, two hypotheses about the biosynthetic pathways of MPs and CIT were put forward. One of them is based on metabolic pathways, the MPs and CIT shared a common pathway to a certain branch [26]. The other is based on the analysis of the whole genome sequence, their biosynthetic gene clusters had been found separately, thereby, they belonged to 2 different pathways [6,18].
Previous results suggested that the yields of CIT and MPs in Monascus spp. could affect each other. For example, Xie et al. (2013) and Liu et al. (2014) separately identified that the overexpression of mrpigB and mrpigE in MPs gene cluster of M. ruber M7 resulted in a reduction of CIT production [13,27]. Meanwhile, Liang et al. (2017) obtained a mutant of a putative glyoxalase (orf6) in CIT gene cluster of M. purpureus, and found that the deletion of orf6 could improve the MPs and CIT yields at the same time [28]. Recently, Li et al. (2020) reported that MPs biosynthetic gene cluster was a composite supercluster, and the naphthoquinone (monasone) gene cluster was embedded in the MPs gene cluster, and speculated that MrPigH was essential in the biosynthesis of naphthoquinone, but it was a supplemental enzyme in the biosynthesis of MPs [29]. So, the blockage of the naphthoquinone biosynthesis pathway in mrpigH knockout might result in naphthoquinone reduction and MPs enhancement. However, why the knockout of mrpigH can reduce the CIT production of citrinin is worthy of further study.
In conclusion, the mrpigH gene pertains to the MPs biosynthetic gene cluster of M. ruber M7 and plays a remarkable role in the biosynthesis of MPs and CIT. The disruption of mrpigH had very little effect on the microscopic morphologies, while the mrpigH mutants showed slower biomass accumulation and darker color on PDA. Compared with M. ruber M7, the YPs, OPs and RPs production in the mrpigH mutants (ΔmrpigHΔmrlig4ΔmrpyrG::mrpyrG and ΔmrpigHΔmrlig4ΔmrpyrG) increased significantly. However, the CIT production of the mrpigH mutants decreased drastically. This work will make some contribution to the regulation of MPs and CIT production in M. ruber M7.