Effect of light on growth,pigment production and culture morphology of <Emphasis Type="Italic">Monascus purpureus</Emphasis> in solid-state fermentation

The capacity to sense and respond to light is widespread in animals, plants, fungi and bacteria. The effect of light quality on growth and pigment yield of Monascus purpureus was investigated. Incubation in total darkness increased red pigment production from 14. 5 OD/g dry substrate to 22 OD/g dry substrate. In contrast, growth of the fungus in direct illumination resulted in total suppression of pigment production. It was found that both red and blue light influenced pigment yield as well as culture morphology. The authors propose the existence of a light-perception system in Monascus purpureus.     

Growth kinetics of biopigment production by Thai isolated <Emphasis Type="Italic">Monascus purpureus</Emphasis> in a stirred tank bioreactor

Monascus purpureus is a biopigment-producing fungi whose pigments can be used in many biotechnological and food industries. The growth kinetics of biopigment production were investigated in a liquid fermentation medium in a 5-l stirred tank bioreactor at 30°C, pH 7, for 8 days with 100 rpm agitation and 1.38 × 10⁵ N/m² aeration. Thai Monascus purpureus strains TISTR 3002, 3180, 3090 and 3385 were studied for color production, growth kinetics and productivity. Citrinin as a toxic metabolite was measured from the Monascus fermentation broth. The biopigment productions were detected from fermentation broth by scanning spectra of each strain produced. Results showed a mixture of yellow, orange and red pigments with absorption peaks of pigments occurring at different wavelengths for the four strains. It was found that for each pigment color, the color production from the strains increased in the order TISTR 3002, 3180, 3090, 3385 with 3385 production being approximately 10 times that of 3002. Similar results were found for growth kinetics and productivity. HPLC results showed that citrinin was not produced under the culture conditions of this study. The L*, a* and b* values of the CIELAB color system were also obtained for the yellow, orange and red pigments produced from the TISTR 3002, 3180, 3090 and 3385 strains. The colors of the pigments ranged from burnt umber to deep red.     

Expression of pigmentation genes following electroporation of albino<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)—i.e., new mutations—that resulted in pigment production.     

红曲霉T-DNA插入突变库中色素突变子的筛选

红曲霉（Monascus purpureus）是我国重要的传统食用和药用微生物,能分泌红曲色素、莫纳卡琳（Mo-nacolin K）、γ-氨基丁酸、麦角固醇等物质,被广泛用于生产食品发酵剂、食品着色剂、保健食品和药品等,在我国有上千年的应用历史。本实验以根癌农杆菌（Agrobacterriumtum efaciens）介导的红曲霉突变体库中754株突变子菌株为材料,通过乙醇提取法筛选出了9株产红曲色素发生显著变化的色素突变子,其中S50在505 nm波长处的色价降为原始菌株S的0.12倍,几乎变为白化株。300~600 nm波长下UV-VIS扫描图谱特征显示,色素突变子不仅色价发生了变化,而且吸收波长、吸收峰值也发生了不同程度的变化。最后,对色素突变子菌落形态、显微结构、潮霉素抗性、遗传稳定性以及产桔霉素能力等进行分析,为研究红曲霉产色素相关基因奠定了基础。     

Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802

The production of red pigments and citrinin by Monascus purpureus CCT3802 was investigated in submerged batch cultures performed in two phases: in the first phase, cells were grown on glucose, at pH 4.5, 5.5 or 6.5; after glucose depletion, pH was adjusted, when necessary, to 4.5, 5.5, 6.5, 7.0, 8.0 or 8.5, for a production phase. The highest total red pigments absorbance of 11.3 U was 16 times greater than the lowest absorbance and was achieved with growth at pH 5.5, followed by production at pH 8.5, which causes an immediate reduction of the intra cellular red pigments from 75% to 17% of the total absorbance. The lowest citrinin concentration, 5.5 mg L⁻¹, was verified in the same culture while the highest concentration, 55 mg L⁻¹, was verified in cultures entirely carried out at pH 5.5. An alkaline medium, besides promoting intra cellular red pigments excretion, strongly represses citrinin synthesis.     

Pigmentation and Antibacterial Activity of Fast Neutron- and X-Ray-induced Strains of Monascus purpureus Went

Seven new strains of Monascus purpureus Went were induced by neutron and x-ray irradiation. The quantity and quality of pigments produced by these strains differed. Strains N4S and N11S produced twice as much pigment as normal, while another strain, N14S, was albino. An unknown orange pigment was found in young colonies of the N11S strain. This orange pigment reacted with alcohols and malt extract medium to form red pigments. Strains N4S, N11S, X2P, and wild type inhibited the growth of certain bacteria, especially the Bacillus species. Strain N11S had more antibacterial activity than wild type. A major active compound was isolated with an ultraviolet absorption spectrum that was related to those of the red pigments found in this fungus. The active compound(s) was named monascidin.     

Development of Transformation System in Monascus Purpureus using an Autonomous Replication Vector with Aureobasidin A Resistance Gene

To enhance the variety of genetic tools and thus to promote molecular genetic study, aureobasidin A and its resistance gene were adopted as a new marker system together with the incorporation of the Gateway system to facilitate the introduction of long heterologous DNA fragments into Monascus purpureus. The minimum inhibitory concentration of aureobasidin A against Monascus was 0.05 μg/ml and a transformation efficiency of 17 colonies/μg DNA was obtained by the protoplast-PEG method with the vector pAUR316, containing the aureobasidin A resistance gene. Southern analysis of the transformants confirmed that pAUR316 exists as an independent vector, demonstrating that the AMA1 sequence acts as the autonomous replication sequence in M. purpureus. Through the use of the Gateway system, a polyketide synthase gene (7.8 kbp) responsible for citrinin biosynthesis was introduced. As a result, the transformants showed 1.5-fold higher production of citrinin than the wild-type strain. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 2 November 2005; Accepted 3 November 2005     

红曲霉繁殖相关wetM基因的克隆与生物信息学分析

红曲霉(Monascus spp.)是一类次级代谢产物极为丰富的食药用丝状真菌,其繁殖能力是大规模工业化生产的前提.以实验室保藏紫色红曲霉(Monascus purpureus)Mp-21为研究对象,通过高通量转录组(RNA-Seq)测序获得菌株转录本数据后进行注释分析,首次克隆出红曲霉繁殖相关wetM基因并进行生物信...     

红曲霉菌液相培养高产色素低产桔霉素的代谢调控研究

从8株固态发酵高产红曲色素菌株中筛选出一株适合于液相培养工艺的高产红曲色素菌株FL1,并对其液相培养过程特性及高产色素低产桔霉素的代谢调控策略进行了研究.发酵过程动力学研究结果表明,红曲色素与桔霉素作为次级代谢产物,其合成与红曲霉菌生长的关系属于非生长偶联型.本文首次采用连续补料的Fed-Batch培养技术,用于红曲霉...     

Production of pigments byMonascus purpureus using sugar-cane bagasse in roller bottle cultures

Solid-state fermentation, using sugar-cane bagasse, and submerged fermentation, using a semi-synthetic medium, were performed for pigment production byMonascus purpureus in both stationary and rotary conditions. Rotary cultures gave higher yields of crude red and yellow pigments than stationary cultures whereas twice the amount was synthesized at an earlier time (day 8) in liquid medium (1,285U yellow pigment/bottle, 1,728U red pigment/bottle). Supplementing the liquid medium with 0.6% (v/v) corn oil doubled the extracellular pigment yield but halved fungal growth.     

荧光物质(MFA、MFB)的分离、结构鉴定及金华地区高产菌株的筛选

采用硅胶柱层析及制备型液相色谱仪对红曲米中两种荧光物质进行分离纯化,使用高效液相色谱法(HPLC)检测荧光物质纯度,然后使用高分辨质谱(ESI-HRMS)对两种荧光物质进行分析,得到两种荧光物质的分子量分别为356和384,ESI-MS/MS二级质谱把两者鉴定为monasfluore A (MFA)和monasfluore B (MFB);从金华地区红曲米中分离得到10株红曲菌株,经固态发酵采用HPLC法分析,筛选获得1株高产MFA、MFB的菌株WZWZ,该菌株发酵制得红曲米中MFA含量为3.63 g/kg,MFB含量为7.29 g/kg,对WZWZ菌株进行外观形态学及显微观察、ITS基因序列测定与分析,最终将菌株WZWZ鉴定为紫色红曲霉(Monascus purpureus)。     

Genetic transformation of Monascus purpureus DSM1379

Monascus purpureus was transformed into hygromycin B resistance with hygromycin B phosphotransferase (hph) fused to Aspergillus nidulans trpC or a putative Monascus purpureus gpd1 promoter by electroporation. Among five strains, only M. purpureus DSM1397 was a competent recipient. Normal growth and sporulation on media containing up to 500 mg hygromycin B l^–1 occurred up to five generations. Upon transformation of the strain with the green fluorescent protein gene (sgfp) as a model gene and hph as a selection marker, characteristic green fluorescence was observed under fluoromicroscopy indicating successful transformation.     

A new process for red pigment production by submerged culture of Monascus purpureus

Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in submerged culture was optimized based on inoculum preparation and culture medium. A vegetative inoculum was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an initial pH?of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the specific consumption rate of glucose (q_G) was higher than the specific consumption rate of ethanol (q_E), whereas the specific growth rate was greatest with ethanol. The specific production rate of red pigment (p_OD) and pigment yield (Y_OD/s) with glucose was twice that with ethanol. A novel fermentation process was developed with M. purpureus initially grown with controlled ethanol formation, and consumption of the latter during pigment formation.     

Improvement of monacolin K, γ-aminobutyric acid and citrinin production ratio as a function of environmental conditions of<Emphasis Type="Italic"> Monascus purpureus</Emphasis> NTU 601

Monascus, a traditional Chinese fermentation fungus, is used as a natural dietary supplement. Its metabolic products monacolin K and -aminobutyric acid (GABA) have each been proven to be a cholesterol-lowering drug and a hypotensive agent. Citrinin, another secondary metabolite, is toxic to humans, thus lowering the acceptability of red mold rice to the general public. In this study, the influence of different carbon and nitrogen sources, and fatty acid or oils, on the production of monacolin K, citrinin and GABA by Monascus purpureus NTU 601 was studied. When 0.5% ethanol was added to the culture medium, the production of citrinin decreased from 813 ppb to 561 ppb while monacolin K increased from 136 mg/kg to 383 mg/kg and GABA increased from 1,060 mg/kg to 7,453 mg/kg. In addition, response surface methodology was used to optimize culture conditions for monacolin K, citrinin and GABA production, and data were collected according to a three-factor (temperature, ethanol concentration and amount of water supplemented), three-level central composite design. When 500 g rice was used as a solid substrate with 120 ml water and 0.3% ethanol, the production of monacolin K at 30°C increased from 136 mg/kg to 530 mg/kg, GABA production increased from 1,060 mg/kg to 5,004 mg/kg and citrinin decreased from 813 ppb to 460 ppb.     

Effect of several elicitors on sclerotia biomass and carotenoid yield of Penicillium sp. PT95 during solid-state fermentation of corn meal

Six kinds of heat-released soluble cell-wall fragments (elicitors) were prepared respectively from Neurospora crassa, Monascus purpureus, Sporobolomyces roseus, Rhodotorula rubra, Nocardia corallina N89 and Actinoplanes tuftoflagellus A05. When Penicillium sp. PT95 was grown on corn meal (CM) solid medium containing appropriate amounts of elicitors, both its sclerotia biomass and the amount of carotenoid accumulated in sclerotia were enhanced significantly (P < 0.01). Every one of the elicitors except that fromM. purpureus could also increase significantly the β-carotene fraction of total pigment (P < 0.01). Among elicitors tested, the elicitor (150 μg/g CM) originating from R. rubra gave a maximum value of sclerotia biomass, reaching 15.90 g/100g CM; the elicitor (100 μg/g CM) from M. purpureus gave the highest total carotenoid of 14,446 μg/100 g CM and β-carotene yield of 10,112 μg/100 g CM, which were respectively 2.76 and 2.72 times higher than that of control. Experimental results also showed that the elicitor from M. purpureus could inhibit effectively the occurrence of sectoring during solid-state fermentation of strain PT95. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of an hyperpigmenting mutant of Monascus purpureus IB1: identification of two novel pigment chemical structures

Monascus purpureus IB1 produces about 50-fold higher levels of azaphilone pigments than M. purpureus NRRL1596. Differently pigmented mutants were obtained from M. purpureus IB1 by nitrosoguanidine treatment. A highly pigmented strain, M. purpureus HP14, was found to lack the formation of the classical yellow and orange azaphilones and was found to produce only about 10% of the red azaphilone pigments. The intense color was associated with novel pigments as shown by high-performance liquid chromatography (HPLC). The addition of hexanoic acid to M. purpureus IB1 resulted in higher volumetric and specific red pigment productivity, but in a complete absence of the classical orange azaphilones, while the classical yellow and red azaphilone pigments were severely reduced; new peaks corresponding to less hydrophobic pigments were found in hexanoic-supplemented cultures by HPLC. Purification of pigments from hexanoic-supplemented cultures showed the presence of five new pigments as indicated by the absorption spectra and HPLC analysis. Two of them, R3 and Y3, were characterized by nuclear magnetic resonance as 9-hexanoyl-3-(2-hydroxypropyl)-6a-methyl-9,9a-dihydro-6H-furo[2,3-h]isochromene-6,8(6aH)-dione and 4-[2,4-dihydroxy-6-(3-hydroxybutanethioyloxy)-3-methylphenyl]-3,4-dihydroxy-3,6-dimethylheptanoic acid. These pigments were also found to be present in cultures of the high-producing mutant M. purpureus HP14. These new pigments are less hydrophobic than the classical azaphilones and may have better properties as natural colorants in the food industry.     

<Emphasis Type="Italic">Halomonas sediminis</Emphasis> sp. nov., a new halophilic bacterium isolated from salt-lake sediment in China

A novel Gram-negative, slightly halophilic, catalase- and oxidase-positive, obligately aerobic bacterium, strain YIM-C248^T, was isolated from a sediment sample collected from a salt-lake in the Qaidam Basin in Qinghai, north-west China. Cells were non-sporulating short rods, occurring singly or as doublets, motile with peritrichous flagella. Growth occurred with 1–15% (w/v) NaCl [optimum 2–4% (w/v) NaCl], at pH 6.0–10.0 (optimum pH 7.5) and at 4–35°C (optimum 25–30°C). The major cellular fatty acids were C_18:1 ω7c, C_12:0 3-OH, cyclo C_19:0 ω8c, C_16:0 and C_16:1. The predominant respiratory quinone was Q-9 and the genomic DNA G + C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YIM-C248^T should be assigned to the genus Halomonas. The sequence similarities between the isolate and the type strains of members of the genus Halomonas were in the range of 92.5–97.5%. The combination of phylogenetic analysis, DNA–DNA hybridization data, phenotypic characteristics and chemotaxonomic differences supported the view that strain YIM-C248^T represents a new species of the genus Halomonas, for which the name Halomonas sediminis sp. nov. is proposed, with YIM-C248^T (=CCTCC AA 207031 = KCTC 22167) as the type strain. The GenBank/EMBL/DBBJ accession number for the 16S rRNA gene sequence of strain YIM-C248^T is EU135707.     

<Emphasis Type="Italic">Marinobacter zhanjiangensis</Emphasis> sp. nov., a marine bacterium isolated from sea water of a tidal flat of the South China Sea

A novel Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped, aerobic bacterium, designated strain JSM 078120^T, was isolated from sea water collected from a tidal flat of Naozhou Island, South China Sea. Growth occurred with 1–15% (w/v) total salts (optimum, 2–4%), at pH 6.0–10.0 (optimum, pH 7.5) and at 4–35°C (optimum, 25–30°C). The major cellular fatty acids were C_18:1 ω9c, C_16:0, C_12:0 3-OH and C_16:1 ω7c. The predominant respiratory quinone was ubiquinone Q-9, and the genomic DNA G + C content was 60.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 078120^T should be assigned to the genus Marinobacter, being related most closely to the type strains of Marinobacter segnicrescens (sequence similarity 98.2%), Marinobacter bryozoorum (97.9%) and Marinobacter gudaonensis (97.6%). The sequence similarities between the novel isolate and the type strains of other recognized Marinobacter species ranged from 96.7 (with Marinobacter salsuginis) to 93.3% (with Marinobacter litoralis). The levels of DNA–DNA relatedness between strain JSM 078120^T and the type strains of M. segnicrescens, M. bryozoorum and M. gudaonensis were 25.3, 20.6 and 18.8%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078120^T represents a novel species of the genus Marinobacter, for which the name Marinobacter zhanjiangensis sp. nov. is proposed. The type strain is JSM 078120^T (= CCTCC AB 208029^T = DSM 21077^T = KCTC 22280^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078120^T is FJ425903.