Production of water-soluble yellow pigments via high glucose stress fermentation of Monascus ruber CGMCC 10910

Monascus pigments are secondary metabolites of Monascus species and are mainly composed of yellow pigments, orange pigments and red pigments. In this study, a larger proportion of Monascus yellow pigments could be obtained through the selection of the carbon source. Hydrophilic yellow pigments can be largely produced extracellularly by Monascus ruber CGMCC 10910 under conditions of high glucose fermentation with low oxidoreduction potential (ORP). However, keeping high glucose levels later in the culture causes translation or a reduction of yellow pigment. We presume that the mechanism behind this phenomenon may be attributed to the redox level of the culture broth and the high glucose stress reaction of M. ruber CGMCC 10910 during high glucose fermentation. These yellow pigments were produced via high glucose bio-fermentation without citrinin. Therefore, these pigments can act as natural pigments for applications as food additives.

     

MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (?MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ?MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (?MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.     

Production of citrinin by various species ofMonascus

Summary The production of citrinin by variousMonascus species was determinated using various culture mediums and conditions. The maximal production was obtained in fermentor usingM. ruber with concentrations of 380 mg/l. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives fromMonascus sp. avoid the occurrence of citrinin; so, we argue that some nitrogen sources are unfavorable to the production of citrinin.     

Improvement of red pigment/citrinin production ratio as a function of environmental conditions by monascus ruber

The growth and metabolic behaviour of the filamentous fungus Monascus ruber were studied in submerged cultures under various aeration and agitation conditions. Improving the oxygen supply, by increasing either the air input or the agitation speed, resulted in modified metabolism: the biomass yield, the consumption of the nitrogen source (monosodium glutamate), and the production of secondary metabolites (red pigment and citrinin) all increased. However, the citrinin production increased more than that of the red pigment. In consequence, a low oxygen transfer coefficient was required to improve the red pigment/citrinin production ratio. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 497–501, 1999.     

Verticillium lecanii spore production in solid-state and liquid-state fermentations

Verticillium lecanii has been recognized as an entomopathogen with high potential in biological control of pests. Two types of cultivation methods, the solid-state fermentation (SSF) and the liquid-state fermentation (LSF), were examined for V. lecanii. In SSF, the substrate types including rice, rice bran, rice husk, and the mixtures of these components were tested. The results showed that both cooked rice with appropriate water addition and rice bran gave significantly higher spore production of 1.5 2 10⁹ spores/g substrate and 1.4 2 10⁹ spores/g substrate, respectively. In LSF, SMAY liquid medium was used as a base, and the effects of environmental conditions on the spore production of V. lecanii were investigated. From the time course study, on the 9th day the spore yield reached 1.2 2 10⁹ spores/ml of broth at 24v°C, 150 rpm for this strain. A series of medium volumes in the shaker-flask have been tested for the requirement of aeration. The largest surface aeration test, one tenth of the medium volume in the shaker-flask for cultivation, gave the highest spore count. The optimal pH value was tested and the initial pH 5 in the SMAY medium produced a high spore density. Finally, V. lecanii spores from SSF and LSF were different in size, shape, and size distribution; while mean spore length from SSF was 6.1 7m, and mean spore length from LSF was 5.0 7m.     

Solid state fermentation for cellulases and β-glucosidase production by Aspergillus niger

Production of cellulases and β-glucosidase was studied using locally-isolated Aspergillus niger on various cheap sources of cellulose like bagasse, corn corbs, computer cards and sawdust, by solid state fermentation (SSF) and by liquid state fermentation (LSF). Enzyme activities were increased about 30–80% by SSF in comparison with conventional LSF. Enzyme production was further improved by various pretreatments, making cellulosic material easily accessible. The best results were obtained with 5 M NaOH treatment.     

Effect of fermentation system on the production and properties of tannase of Aspergillus niger van Tieghem MTCC 2425

The tannase-producing efficiency of liquid-surface fermentation (LSF) and solid-state fermentation (SSF) vis-à-vis submerged fermentation (SmF) was investigated in a strain of Aspergillus niger, besides finding out if there was a change in the activity pattern of tannase in these fermentation processes. The studies on the physicochemical properties were confined to intracellular tannase as only this form of enzyme was produced by A. niger in all three fermentation processes. In LSF and SmF, the maximum production of tannase was observed by 120 h, whereas in SSF its activity peaked at 96 h of growth. SSF had the maximum efficiency of enzyme production. Tannase produced by the SmF, LSF and SSF processes had similar properties except that the one produced during SSF had a broader pH stability of 4.5-6.5 and thermostability of 20 degrees-60 degrees C.     

Optimisation of extracellular tannase production from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

The tannase production by Paecilomyces variotii was confirmed by high performance thin layer chromatography (HPTLC), and substrate specificity of the tannase was determined by zymogram analysis in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE). A clear band of activity observed after electrophoresis of culture filtrate in non-denaturing gels indicated the production of extracellular tannase by P. varoitii. HPTLC analysis revealed that gallic acid was the enzymatic degradation product of tannic acid during the fermentation process. The optimum condition for tannase production was at 72 h of incubation in shaking condition and addition of 1.5% tannic acid, 1% glucose and 0.2% sodium nitrate at temperature of 35°C and pH of 5–7. The production of extracellular tannase from Paecilomyces variotii was investigated under optimized conditions in solid-state fermentation (SSF), submerged fermentation (SmF) and liquid surface fermentation (LSF) processes. The maximum extracellular tannase production was obtained within 60 h of incubation under SSF followed by SmF and LSF.     

Biosynthetic Pathway of Citrinin in the Filamentous Fungus Monascus ruber as Revealed by 13C Nuclear Magnetic Resonance

Carbon isotope distribution of [¹³C]citrinin from Monascus ruber incubated with [¹³C]acetate revealed that the biosynthesis of the toxin originated from a tetraketide, instead of a pentaketide as has been shown for Penicillium and Aspergillus species. The production of polyketide red pigments and citrinin by M. ruber may therefore be regulated at the level of the tetraketide branch point.     

Protein enrichment of apple pomace by co-culture of cellulolytic moulds and yeasts

The co-culture of cellulolytic moulds and yeasts on apple pomace in solid-state fermentation (SSF) and liquid-state fermentation (LSF) increased the protein content of apple pomace. The co-culture of Candida utilis and Aspergillus niger was the best among several combinations and increased the protein content of dried and pectin-extracted apple pomace to 20% and 17%, respectively, under SSF conditions.The authors are with the Microbiology Research Laboratory, Department of Biosciences, Himachal Pradesh University, Shimia-171005, India.     

Isolation and screening of low-density polyethylene (LDPE) bags degrading bacteria from Addis Ababa municipal solid waste disposal site “Koshe”

Purpose

The present study aimed to explore the binding ability of acyl-CoA binding protein 2 to fatty acid acyl-CoA esters and its effect on Monascus pigment production in M. ruber CICC41233.

Methods

The Mracbp2 gene from M. ruber CICC41233 was cloned with a total DNA and cDNA as the templates through the polymerase chain reaction. The cDNA of the Mracbp2 gene fragment was ligated to expression vector pGEX-6P-1 to construct pGEX-MrACBP2, which was expressed in Escherichia coli BL21 to obtain the fusion protein GST-MrACBP2 and then measure the binding ability of fatty acid acyl-CoA esters. Additionally, the DNA of the Mracbp2 gene fragment was ligated to expression vector pNeo0380 to construct pNeo0380-MrACBP2, which was homologously over-expressed in M. ruber CICC41233 to evaluate Monascus pigment production and fatty acid.

Results

The cloned Mracbp2 gene of the DNA and cDNA sequence was 1525 bp and 1329 bp in length, respectively. The microscale thermophoresis binding assay revealed that the purified GST-MrACBP2 had the highest affinity for palmitoyl-CoA (Kd =70.57 nM). Further, the Mracbp2 gene was homologously overexpressed in M. ruber CICC41233, and a positive transformant M. ruber ACBP-E was isolated. In the Monascus pigments fermentation, the expression level of the Mracbp2 gene was increased by 1.74-fold after 2 days and 2.38-fold after 6 days. The palmitic acid content and biomass in M. ruber ACBP2-E were significantly lower than that in M. ruber CICC41233 on 2 days and 6 days. However, compared with M. ruber CICC41233, the yields of total pigment, ethanol-soluble pigment, and water-soluble pigment in M. ruber ACBP2-E increased by 63.61%, 71.61%, and 29.70%, respectively.

Conclusions

The purified fusion protein GST-MrACBP2 exhibited the highest affinity for palmitoyl-CoA. The Mracbp2 gene was overexpressed in M. ruber CICC41233, which resulted in a decrease in palmitic acid and an increase in Monascus pigments. Overall, the effect of MrACBP2 on the synthesis of fatty acid and Monascus pigment was explored. This paper explored the effect of MrACBP2 on the fatty acid synthesis and the synthesis of Monascus pigment. The results indicated the regulation of fatty acid synthesis could affect Monascus pigment synthesis, providing a novel strategy for improving the yield of Monascus pigment.

     

Medium-Chain Fatty Acids Affect Citrinin Production in the Filamentous Fungus Monascus ruber

During submerged culture in the presence of glucose and glutamate, the filamentous fungus Monascus ruber produces water-soluble red pigments together with citrinin, a mycotoxin with nephrotoxic and hepatoxic effects on animals. Analysis of the ¹³C-pigment molecules from mycelia cultivated with [1-¹³C]-, [2-¹³C]-, or [1,2-¹³C]acetate by ¹³C nuclear magnetic resonance indicated that the biosynthesis of the red pigments used both the polyketide pathway, to generate the chromophore structure, and the fatty acid synthesis pathway, to produce a medium-chain fatty acid (octanoic acid) which was then bound to the chromophore by a trans-esterification reaction. Hence, to enhance pigment production, we tried to short-circuit the de novo synthesis of medium-chain fatty acids by adding them to the culture broth. Of fatty acids with carbon chains ranging from 6 to 18 carbon atoms, only octanoic acid showed a 30 to 50% stimulation of red pigment production, by a mechanism which, in contrast to expectation, did not involve its direct trans-esterification on the chromophore backbone. However, the medium- and long-chain fatty acids tested were readily assimilated by the fungus, and in the case of fatty acids ranging from 8 to 12 carbon atoms, 30 to 40% of their initial amount transiently accumulated in the growth medium in the form of the corresponding methylketone 1 carbon unit shorter. Very interestingly, these fatty acids or their corresponding methylketones caused a strong reduction in, or even a complete inhibition of, citrinin production by M. ruber when they were added to the medium. Several data indicated that this effect could be due to the degradation of the newly synthesized citrinin (or an intermediate in the citrinin pathway) by hydrogen peroxide resulting from peroxisome proliferation induced by medium-chain fatty acids or methylketones.     

Cloning and functional analysis of the Gβ gene Mgb1 and the Gγ gene Mgg1 in Monascus ruber

The ascomycetous fungus Monascus ruber is one of the most well-known species widely used to produce Monascus-fermentation products for natural food colorants and medicine. Our previous research on the Gα subunit Mga1 and the regulator of G protein signaling MrflbA indicated that heterotrimeric G protein signaling pathways were involved in aspects of growth, sporulation and secondary metabolite production in M. ruber. To better understand the G protein signaling pathways in this fungus, a Gβ subunit gene (Mgb1) and a GΓ subunit gene (Mgg1) were cloned and investigated in the current study. The predicted Mgb1 protein consisted of 353 amino acids and Mgg1 consisted of 94 amino acids, sharing marked similarity with Aspergillus Gβ and GΓ subunits, respectively. Targeted deletion (Δ) of Mgb1 or Mgg1 resulted in phenotypic alterations similar to those resulting from ΔMga1, i.e., restricted vegetative growth, lowered asexual sporulation, impaired cleistothecial formation, and enhanced citrinin and pigment production. Moreover, deletion of Mgg1 suppressed the defects in asexual development and in biosynthesis of citrinin and pigment caused by the absence of MrflbA function. These results provide evidence that Mgb1 and Mgg1 form a functional GβΓ dimer and the dimer interacts with Mga1 to mediate signaling pathways, which are negatively controlled by MrflbA, for growth, reproduction and citrinin and pigment biosynthesis in M. ruber.     

Platotex: an innovative and fully automated device for cell growth scale-up of agar-supported solid-state fermentation

Among various factors that influence the production of microbial secondary metabolites (MSM), the method of cultivation is an important one that has not been thoroughly investigated. In order to increase microbial throughput and simplify the extraction and workup steps, we performed a study to compare liquid-state fermentation (LSF) with agar-supported solid-state fermentation (AgSF). We found that AgSF is not only more suitable for our applications but offers, for some microbial strains, a higher yield and broader diversity of secondary metabolites. The main limitation of AgSF is the lack of a system to allow production scale-up. In order to overcome this obstacle we developed Platotex, an original fermentation unit offering 2 m² of cultivation surface that combines automatic sterilization, cultivation, and drying steps. Platotex is also able to support both LSF and solid-state fermentation (SSF). Platotex conforms to international security and quality requirements and benefits from total remote automation through industrial communication and control standards.     

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.     

Characterization of a non-pigment producing Monascus purpureus mutant strain

A characterization of a non-pigment producing mutant Monascus purpureus M₁₂ compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (Y_X/C 0.2 – 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 – 0.08 U/mg protein and 0.01 – 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C₁₇, C₂₀ and C₂₂ fatty acids and did not produce citrinin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.     

Production of citrinin by Monascus ruber submerged culture in chemically defined media

Following our investigations on citrinin production by Monascus ruber in a chemically defined medium, the kinetic behaviour of this toxin in the fermenter was studied with relation to production of pigments, biomass and nutrient consumption. Showing a secondary metabolite pattern, citrinin was produced when dμ/dt and dQ_s/dt were ≤ 0, while a high specific production rate of pigments occurred when dμ/dt and dQ_s/dt were > 0. No trophophase-idiophase transition was detected during the cuitivation, and the behaviour of pigment production was similar to that of a primary metabolite.