Mutational analysis of MpPhy reveals magnetoreception and photosensitivity involvement in secondary metabolites biosynthesis in Monascus purpureus.

Light or low frequency magnetic field (LF-MF) as one of the cultivation environments affects secondary metabolites (SMs) production of M. purpureus. Phytochrome (Phy) is a hybrid histidine kinase possessing dual properties of photoreceptor and kinase to sense red and far-red light. The interaction effects of LF-MF and light on SMs of M. purpureus was investigated by knocking out the Phy-like gene in M. purpureus (MpPhy) by homologous recombination. A MpPhy-deletion (ΔMpPhy) strain produced less Monascus pigments (MPs) and monacolin K (mon K) than the wild-type (WT) strain and reduced citrinin production by 78.3% on 10th day but didn't affect the biomass. These results indicated that the MpPhy gene is involved in SMs biosynthesis of M. purpureus. MPs production in WT was decreased significantly when the inoculum was exposed to white/blue/green/red light (500 Lux). But it in ΔMpPhy was no significant difference when exposed to white/red light. The colony size of ΔMpPhy was smaller on potato dextrose agar media containing 0.01% SDS. These results indicated that the deletion of MpPhy gene affected the aerial hyphae and increased sensitivity to cell membrane stress but decreased sensitivity to red light. The inoculum of both WT and ΔMpPhy was exposure to the LF-MF (50 Hz). The accumulation of WT secondary metabolites was not changed, while SMs production of ΔMpPhy was significantly enhanced under exposed to 2.0 mT LF-MF. This indicated that the decrease of SMs caused by the deletion of MpPhy gene was restored by LF-MF. It revealed that there is a crosstalk between magnetoreception and photosensitivity.

Analyses of Monascus pigment secretion and cellular morphology in non-ionic surfactant micelle aqueous solution.

Monascus pigments produced by Monascus spp. are widely used as natural food colourants. Extractive fermentation technology can facilitate the secretion of intracellular Monascus pigments into extracellular non-ionic surfactant micelle aqueous solution, so as to avoid the feedback inhibition and decomposition. In this study, behaviour of the trans-membrane secretion of Monascus pigments was investigated using morphological and spectroscopic analyses. Laser scanning confocal microscopy (LSCM) traced that pigment secretion occurred through rapid trans-membrane permeation in 4 min, with a simultaneous conversion in pigment characteristics. Approximately 50% of intracellular pigments (AU470 ) extracted to extracellular broth with 40 g l-1 Triton X-100, indicating the capacity for pigment extraction was limited by the saturation concentrations of surfactant. Scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging showed some damage in the cell wall but an intact cell membrane with a slightly increased mycelial diameter. However, the physiological properties of the cell membrane, including integrity, fluorescence intensity and permeability, were altered. A diagram was provided to demonstrate the behaviour of Monascus pigment secretion induced by Triton X-100. This study lays a foundation for the further investigation of Monascus pigment metabolism and secretion in extractive fermentation.

Enhanced production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship between fermentation conditions and mycelial morphology.

Natural yellow pigments produced by submerged fermentation of Monascus purpureus have potential economic value and application in the food industry. In the present study, the relationships among fermentation conditions (in terms of pH and shaking/agitation speed), mycelial morphology and the production of Monascus yellow pigments were investigated in both shake-flask and scale-up bioreactor experiments. In the shake-flask fermentation, the highest yield of the Monascus yellow pigments was obtained at pH 5.0 and a shaking speed of 180 rpm. Microscopic images revealed that these results were associated with the formation of freely dispersed small mycelial pellets with shorter, thicker and multi-branched hyphae. Further investigation indicated that the hyphal diameter was highly correlated with the biosynthesis of the Monascus yellow pigments. In a scaled-up fermentation experiment, the yield of yellow pigments (401 U) was obtained in a 200-L bioreactor, which is the highest yield to the best of our knowledge. The present findings can advance our knowledge on the conditions used for enhancing the production of Monascus yellow pigments in submerged fermentation and facilitate large-scale production of these natural pigments.

Effects of blue light on pigment biosynthesis of Monascus.

The influence of different illumination levels of blue light on the growth and intracellular pigment yields of Monascus strain M9 was investigated. Compared with darkness, constant exposure to blue light of 100 lux reduced the yields of six pigments, namely, rubropunctatamine (RUM), monascorubramine (MOM), rubropunctatin (RUN), monascorubrin (MON), monascin (MS), and ankaflavin (AK). However, exposure to varying levels of blue light had different effects on pigment production. Exposure to 100 lux of blue light once for 30 min/day and to 100 lux of blue light once and twice for 15 min/day could enhance RUM, MOM, MS, and AK production and reduce RUN and MON compared with non-exposure. Exposure to 100 lux twice for 30 min/day and to 200 lux once for 45 min/day decreased the RUM, MOM, MS, and AK yields and increased the RUN and MON. Meanwhile, the expression levels of pigment biosynthetic genes were analyzed by real-time quantitative PCR. Results indicated that gene MpPKS5, mppR1, mppA, mppB, mmpC, mppD, MpFasA, MpFasB, and mppF were positively correlated with the yields of RUN and MON, whereas mppE and mppR2 were associated with RUM, MOM, MS, and AK production.

High-throughput screening of high Monascus pigment-producing strain based on digital image processing.

This work proposed a new method which applied image processing and support vector machine (SVM) for screening of mold strains. Taking Monascus as example, morphological characteristics of Monascus colony were quantified by image processing. And the association between the characteristics and pigment production capability was determined by SVM. On this basis, a highly automated screening strategy was achieved. The accuracy of the proposed strategy is 80.6 %, which is compatible with the existing methods (81.1 % for microplate and 85.4 % for flask). Meanwhile, the screening of 500 colonies only takes 20-30 min, which is the highest rate among all published results. By applying this automated method, 13 strains with high-predicted production were obtained and the best one produced as 2.8-fold (226 U/mL) of pigment and 1.9-fold (51 mg/L) of lovastatin compared with the parent strain. The current study provides us with an effective and promising method for strain improvement.

Efficient gene targeting in ligase IV-deficient Monascus ruber M7 by perturbing the non-homologous end joining pathway.

Inactivating the non-homologous end joining (NHEJ) pathway is a well established method to increase gene replacement frequency (GRF) in filamentous fungi because NHEJ is predominant for the repair of DNA double strand breaks (DSBs), while gene targeting is based on homologous recombination (HR). DNA ligase IV, a component of the NHEJ system, is strictly required for the NHEJ in Saccharomyces cerevisiae and Neurospora crassa. To enhance the GRF in Monascus ruber M7, we deleted the Mrlig4 gene encoding a homolog of N. crassa DNA ligase IV. The obtained mutant (MrΔlig4) showed no apparent defects in vegetative growth, colony phenotype, microscopic morphology, spore yield, and production of Monascus pigments and citrinin compared with the wild-type strain (M. ruber M7). Gene targeting of ku70 and triA genes revealed that GRF in the MrΔlig4 strain increased four-fold compared with that in the wild-type strain, reached 68 % and 85 %, respectively. Thus, the MrΔlig4 strain is a promising host for efficient genetic manipulation. In addition, the MrΔlig4 strain is more sensitive than M. ruber M7 to a DNA-damaging agent, methyl methanesulfonate.

Retrograded maize starch used as a medium to enrich Monascus from the air in winter.

Red pigments extracted from fungus Monascus are used for food coloration in China. Wild-growing Monascus spores are usually enriched in the yeast and mold media in the air, but those media are also favorable for yeast and bacteria. In the paper, Monascus species have grown in retrograded maize starch lain in air outdoors in winter, molds, yeast or bacteria colonies have been absent. Then a medium of the retrograded maize starch for enriching Monascus in the air is explored and its physicochemical properties are determined by ordinary camera photos, NMR, SEM spectra and X-ray diffraction. The lamellar structure of frozen retrograded maize starch, whose interlamellar spacing is about 2µm, provides a favorable condition for Monascus spore to germinate and grow.

Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7.

Normally, gene targeting by homologous recombination occurs rarely during a transformation process since non-homologous recombination is predominant in filamentous fungi. In our previous researches, the average gene replacement frequency (GRF) in Monascus ruber M7 was as low as 15 %. To develop a highly efficient gene targeting system for M. ruber M7, two M. ruber M7 null mutants of ku70 (MrΔku70) and ku80 (MrΔku80) were constructed which had no apparent defects in the development including vegetative growth, colony phenotype, microscopic morphology and spore yield compared with M. ruber M7. In addition, the production of some significant secondary metabolites such as pigments and citrinin had no differences between the two disruptants and the wild-type strain. Further results revealed that the GRFs of triA (encoding a putative acetyltransferase) were 42.2 % and 61.5 % in the MrΔku70 and MrΔku80 strains, respectively, while it was only about 20 % in M. ruber M7. Furthermore, GRFs of these two disruptants at other loci (the pigE, fmdS genes in MrΔku70 and the ku70 gene in MrΔku80) were investigated, and the results indicated that GRFs in the MrΔku70 strain and the MrΔku80 strain were doubled and tripled compared with that in M. ruber M7, respectively. Therefore, the ku70 and ku80 null mutants of M. ruber M7, especially the ku80-deleted strain, will be excellent hosts for efficient gene targeting.

Export of intracellular Monascus pigments by two-stage microbial fermentation in nonionic surfactant micelle aqueous solution.

Microbial fermentation of intracellular product is usually limited by high intracellular product concentration inhibition and complex downstream product processing. Perstractive fermentation of intracellular Monascus pigments in the nonionic surfactant Triton X-100 aqueous solution was studied in the present work, in which the intracellular product was exported from the intracellular to the extracellular aqueous solution and consecutively extracted into the nonionic surfactant micelles. After the second stage perstractive fermentation in the two-stage operation mode, biomass increased from 5 to 24 g/l DCW. The corresponding extracellular concentrations of yellow, orange, and red pigments were 60, 49 and 26 AU. The increase of cell density and the final pigment concentration were difficult to occur in a conventional aqueous medium using the two-stage fermentation. This positive effect of perstractive fermentation was ascribed to low intracellular pigment density, which eliminated the product inhibition and prevented the product from further degradation. The high efficiency of perstractive fermentation was further confirmed by fed-batch operation mode, in which the final biomass reached 28 g/l DCW and the corresponding extracellular concentrations of yellow, orange, and red pigments were 130, 84 and 47 AU.

Breeding an amylolytic yeast strain for alcoholic beverage production.

A starch-utilizing, yeast-like fusant was successfully created from fused protoplasts of Schizosaccharomyces pombe and Monascus anka, and the feasibility of using this fusant as a new strain for alcoholic beverage development was reported. The new fusant utilized various carbon sources more efficiently than its parent cells did. Rice koji prepared separately by cultivating the fusant and its parental strains on rice was compared to explore the effect of yeast strain on the production of α-amylase, glucoamylase, and acid protease that are crucial in wine making using cereal grains. It was found that the fusant produced greater levels of the above-mentioned enzymes than its parental strain does. Consequently, the usage of this fusant in the alcoholic fermentation of polished rice was found to reduce approximately 50% consumption of added glucoamylase than when its parental strain was used. Besides, at the end of fermentation, the fusant yeast resulted in a mash with distribution of flavor components very different from that produced by its parental strains. Thus, the fusant can be used as a new yeast strain for creating novel alcoholic beverages.

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid.

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.

Evaluation of Epicoccum nigrum for growth, morphology and production of natural colorants in liquid media and on a solid rice medium.

Four nonpathogenic and nontoxigenic Epicoccum nigrum strains were evaluated for their growth, morphology and pigment producing ability in three complex and one defined liquid media. Epicoccum nigrum IBT 41028 produced pigments in all the four media tested with a maximum pigment of 3.68 AU at 410 nm in M1 medium (unoptimized) containing 5 g/l yeast autolysate. The color hue of the crude pigment extracts ranged from 74 to 102 exhibiting dark orange to green-yellow color. Pelleted morphology was shown to have a positive influence on the pigment production by E. nigrum strain IBT 41028 in the liquid media, and the use of Bis-tris buffer was found to diminish or reduce the pellet formation. Since Monascus is a well known pigment producer on rice. Pigment producing ability of E. nigrum IBT 41028 was tested on rice and compared to liquid media with Monascus ruber IBT 7904 as control. Though, both genera preferred rice but E. nigrum produced 4.6 folds higher pigment in the liquid unoptimized fermentation medium compared to M. ruber. Solid phase extraction and subsequently HPLC-DAD analysis of the crude pigment extracts showed qualitative as well as quantitative variation in the pigment composition under solid and liquid cultivations.

Agrobactrium tumefaciens-mediated transformation of Monascus ruber.

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to Monascus ruber. The optimum cocultivation time was 84 h with an efficiency of 900 to 1,000 transformants when 1x106 spores were used with the same volume of bacteria. The stability of transformants was over 98% after five generations. When M. ruber was transformed with A. tumefaciens YL-63 containing the green fluorescent protein gene (egfp), the green fluorescent signal was observed throughout hyphae, confirming expression of the gene. This efficient transformation and expression system of M. ruber by ATMT will facilitate the study of this fungus at a molecular genetic level.

Effect of stress on growth, pigment production and morphology of Monascus sp. in solid cultures.

The aim of the current work was to study the influence of stress on pigment production in Monascus purpureus under solid-state fermentation. Thermal stress was induced by incubating the culture at various high temperatures. For giving osmotic stress, different concentration of NaCl and glycerol were added to the solid substrate prior to autoclaving. Morphological studies were done by light microscopy and scanning electron microscopy. When the cells were exposed to high temperature, high glycerol and salt concentration, significant changes in pigment production and growth were observed. High temperature (>45 degrees C) induced the production of more yellow pigments. High concentration of NaCl induced conidiation and caused a decrease in fungal biomass (up to 50%) but red pigment production increased from 11.86 OD/gds to 20.14 OD/gds. When subjected to glycerol stress, a significant increase in aerial mycelia was observed when compared with the control conditions. The results attain significance for exploiting the fungal culture of Monascus purpureus LPB 97 for producing colors of choice--red, or yellow, or increasing the yield of red pigments considerably. Thus, these results could well impact the commercial aspect of Monascus pigments for industrial application.

Enhancement of monascus pigment production by the culture of Monascus sp. J101 at low temperature.

In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 degrees C. In a batch culture at 30 degrees C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 degrees C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 degrees C was 10 times greater than at 30 degrees C.

Light effects on cell development and secondary metabolism in Monascus.

In nature, light is one of most crucial environmental signals for developmental and physiological processes in various organisms, including filamentous fungi. We have found that both red light and blue light affect development in Monascus, influencing the processes of mycelium and spore formation, and the production of secondary metabolites such as gamma-aminobutyric acid, red pigments, monacolin K and citrinin. Additionally, we observed that the wavelength of light affects these developmental and physiological processes in different ways. These findings suggest that Monascus possesses a system for differential light response and regulation.