Changes in the enzyme activity of flavinogenesis in the process of culturing the fungus Eremothecium ashbyii

The activity of enzymes involved in the beginning (GTP cyclohydrolase) and terminal steps (riboflavin synthase EC 2.5.1.9, riboflavin kinase EC 2.7.1.26 and FMN adenyltransferase EC 2.7.7.2) of flavinogenesis was studied in the mycelium of Erenmothecium ashbyii of different age. The activity of GTP cyclohydrolas, riboflavin kinase and FMN adenyltransferase was low in the young mycelium and increased in the process of growth, which was accompanied by the acceleration of flavinogenesis. The activity of riboflavin synthase was high in the young mycelium and changed only slightly in the process of subsequent cultivation of the fungus. 8-Azaadenine and 8-hydroxyquinoline added to the young culture of E. ashbyii inhibited the flavinogenesis of the mycelium and the increase of the enzyme activity.     

Ribitol and flavinogenesis in Eremothecium ashbyii

1. Supplementation of cultures of Eremothecium ashbyii with ribitol leads to a twofold increase in riboflavin formation compared with unsupplemented cultures or those supplemented with ribose or ribulose phosphate. Addition of unlabelled ribitol decreases the incorporation of [1-(14)C]ribose into riboflavin, indicating that free ribitol is preferred to ribose for incorporation into riboflavin. 2. The enzymes ribitol kinase, d-ribose reductase, d-ribose 5'-phosphatase and GMP nucleosidase were demonstrated in the cell-free extracts. Ribitol induces the formation of ribitol kinase. The enzyme is activated in vitro by the flavinogenic purines, guanine and xanthine. d-Ribose reductase shows a specific requirement for NADPH and forms free ribitol from ribose. 3. The activities of ribitol kinase, ribose 5'-phosphatase and GMP nucleosidase reach their maximal values before riboflavin formation reaches a maximum. 4. [U-(14)C]GMP is taken up intact by the culture of E. ashbyii and is incorporated into riboflavin as well as into a blue fluorescent compound. The radioactivity from this compound is incorporated into riboflavin by the cell-free extract of E. ashbyii.     

Fatty acid composition in lipid fractions lengthwise the mycelium of Mortierella isabellina and lipid production by solid state fermentation

This paper investigates the correlation between mycelial age and fatty acid biosynthesis. The correlation was investigated by analyzing the lipid composition lengthwise the mycelium of the oleaginous fungus Mortierella isabellina, a potential producer of γ-linolenic acid (GLA). Young mycelia were rich in polar lipids (glycolipids plus sphingolipids and phospholipids), while neutral lipid content increased in aged mycelia. In young mycelia, each polar lipid fraction contained almost 40% (w/w) polyunsaturated fatty acids (PUFAs), but this content decreased to less than 30% (w/w) in aged mycelia. On the other hand, PUFA content in neutral lipids fluctuated slightly with age. These results indicate that PUFA biosynthesis is favored in young, fast growing mycelia, while it decreases significantly in aged mycelia. This trend was also observed when we grew M. isabellina on pear pomace, an agro-industrial waste. Pear pomace cultures yielded significant amounts of lipid, which reached 12% (w/w) in dry fermented mass. The produced lipid was rich in GLA and the maximum GLA content in dry fermented mass was 2.9 mg/g.     

Intracellular transport of phosphatidic acid and phosphatidylcholine into lipid bodies: use of fluorescent lipids to study lipid-body formation in an oleaginous fungus

Fluorescent phosphatidic acid and phosphatidylcholine were used to characterize lipid-transport pathways into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Several characteristics of the lipid transport such as temperature dependence and ATP dependence were evaluated. The transport depicted by these fluorescent lipids was consistent with metabolism of radiolabelled lipids, indicating that fluorescent lipids are useful to study lipid-body formation in this fungus. The results dissect lipid transport of phosphatidic acid and phosphatidylcholine into lipid bodies and reveal regulatory steps for lipid-body formation in this fungus.     

The kinetics of riboflavin secretion by Eremothecium ashbyii nrrl 1363

For riboflavin production a highly flavinogenic strain Eremothecium ashbyii is used. Vitamins are classified as chemical substances that control and effect the physiological processes; Riboflavin is one among them. The deficiency of riboflavin in human beings results in the cracking of lips and corners of mouth (cheilosis); nerve tissues are affected. For riboflavin production a highly flavinogenic strain Eremothecium ashbyii NRRL 1363 was used. Investigations were conducted in shake flask using inexpensive and abundantly available raw materials. Among the stimulants, a combination of hog casings and beef extract stimulated the highest and promoted the maximum riboflavin yield followed by the combination of fish meal and beef extract. The fermented broth (an enriched, riboflavin concentrate) can be directly used as a feed grade riboflavin. To upgrade it to pharmaceutical grade further investigations are required.     

Influence of type and concentration of flavinogenic factors on production of riboflavin by Eremothecium ashbyii NRRL 1363

A study was conducted to determine the effect of various low cost organic wastes as flavinogenic factors and the various concentrations at which they induced flavinogenecity resulting in higher yields of riboflavin. A high-yielding riboflavin strain; Eremothecium ashbyii NRRL 1363 was chosen to determine the flavinogenicity. Carbon source at 50 g l(-1) (dextrose equivalents) of molasses and nitrogen source at 50 g l(-1) (weight/volume) of peanut seed cake were found to be optimal levels to yield higher riboflavin. Among the organic wastes, (beef extract, hog casings, blood meal, fish meal) hog casings in association with fish meal supported the highest yield of riboflavin. Among the different recovery processes studied, a vacuum drying process was the most efficient allowing maximum yield, followed by drying at 90 degrees C and freeze-drying. It is apparent from this study that inexpensive or waste organic materials could induce E. ashbyii to synthesize and secrete riboflavin at higher levels in the medium and this could be purified using a vacuum drying process. This bioconversion process allows us to recycle the biomaterials and produce a value-added product of economic importance.     

Lampteromyces bioluminescence—1. Identification of riboflavin as the light emitter in the mushroom L. japonicus

The bioluminescence of the luminous mushroom, Lampteromyces japonicus, was studied by using the mushroom gills and also the luminous mycelia, the latter being cultured from the isolated spores and grown in a potato sucrose medium. The luminescence intensity of the mushroom gills and the cultured mycelia was measured in an aqueous suspension under various conditions. The original intensity was enhanced by exposing the luminous cells to oxygen for several hours or to acids or bases for a short period. This enhancement enabled measurement of their bioluminescence spectra which were identical to the fluorescence spectrum of riboflavin, having a maximum at 524 nm. The green fluorescent substance was extracted with cold water from the mushroom and it was identified as riboflavin by spectroscopic and chromatographic analyses. Riboflavin was concluded to be the light emitter of this mushroom.     

低强度超声波刺激对阿氏假囊酵母细胞次生代谢的影响

一定强度的超声波能够促进细胞的生长和代谢,细胞膜是抑制微生物次生代谢产物产量的主要原因。以阿氏假囊酵母(E．ashbyii)为实验材料,选择功率6W、频率24kHz的超声加载于摇瓶发酵过程中,检测了加载对菌丝体干重和核黄素分泌的变化情况。结果表明,一定强度的超声间断加载对E．ashbyii的生长有一定的促进作用,并使核黄素的开始分泌时间从96h缩短到60h;同时发现,发酵96h后加载,能有效提高核黄素的产量至0．346mg／L,非常显地高于对照组;发酵后的104～112h是超声处理的最佳时间段,为维持较高的产率,需每隔1．5h再次超声处理。     

核黄素生物合成研究 1.微量培养中核黄菌(Eremothecium ashbyii)发育过程中的形态变化

从本试验观察到,核黄菌的生长全部过程是自配子或菌丝在培养基中开始发育成营养菌丝,部分菌丝形成配子囊形成配子,部分菌丝衰老自溶。当菌丝发育最盛时,核黄素的形成是最多,衰老时培养基核黄素逐渐丰富起来。核黄菌在有空气和缺乏空气时,它们的发育是有区别的,即是在缺乏空气(表面下生长)时,菌丝是细长不形成配子和产生少量核黄素。     

Single cell oil production from rice hulls hydrolysate

Rice hull hydrolysate was used as feedstock for microbial lipids production using the oleaginous fungus Mortierella isabellina. Kinetic experiments were conducted in C/N ratios 35, 44 and 57 and the oil accumulation into fungal biomass was 36%, 51.2% and 64.3%, respectively. A detailed mathematical model was used in order to describe the lipid accumulation process. This model was able to predict reducing sugar and nitrogen consumption, fat-free biomass synthesis and lipid accumulation. Neutral lipids constitute the predominant lipid fraction, while the major fatty acids were oleic, palmitic and linoleic acid. Fatty acids of long aliphatic chain were not detected, thus the microbial oil produced is a promising feedstock for biodiesel production.     

Lipids of filamentous fungi as a material for producing biodiesel fuel

Species of various filamentous fungus taxa were tested for ability to produce lipids suitable as a material for manufacturing biodiesel. The mucoralean fungus Cunninghamella japonica was found to be a promising lipid producer. The inexpensive medium for its growth developed in this study contained ammonium nitrate as a nitrogen source. With its use, up to 16 g/l biomass and over 7 g/l lipids was obtained. The fungal lipids were dominated by oleic acid. It constituted 50% of total fatty acids. The iodine index of the lipid fraction was 86.61. The heat of combustion of the lipids, 37.13 MJ/kg, was close to the value for rapeseed oil.     

beta-Carotene production and its role in sclerotial differentiation of Sclerotium rolfsii

The fungus Sclerotium rolfsii produces beta-carotene, the main detected carotenoid, in levels dependent upon oxidative growth conditions and upon differentiation. beta-Carotene accumulation is 5-, 6.5-, and 6.7-fold higher in undifferentiated mycelia, sclerotia, and differentiated mycelia, respectively, at high than at low oxidative stress. It accumulates more in older than in younger mycelia and is 2-fold higher in differentiated than in undifferentiated mycelia. We propose that beta-carotene is formed possibly to help the fungus reduce oxidative stress that develops during growth. This is supported by the finding that exogenous beta-carotene at non-growth-inhibiting concentrations causes a concentration-dependent reduction of oxidative stress (lipid peroxidation) of undifferentiated mycelia, which results in an equally proportional reduction of sclerotial differentiation. The data of this study support our hypothesis that sclerotial differentiation is induced by oxidative stress.     

Lipids of filamentous fungi as a material for producing biodiesel fuel

Species of various filamentous fungus taxa were tested for ability to produce lipids suitable as a material for manufacturing biodiesel. The mucoralean fungus Cunninghamella japonica was found to be a promising lipid producer. The inexpensive medium for its growth developed in this study contained ammonium nitrate as a nitrogen source. With its use, up to 16 g/l biomass and over 7 g/l lipids was obtained. The fungal lipids were dominated by oleic acid. It constituted 50% of total fatty acids. The iodine index of the lipid fraction was 86.61. The heat of combustion of the lipids, 37.13 MJ/kg, was close to the value for rapeseed oil.     

Bioproduction of riboflavin from molasses and lentils

Fermentation studies were carried out for the bioproduction of riboflavin with an agroindustrial byproduct, molasses as the carbon source and lentils as the nitrogen source using E. ashbyii strain. With the previously recommended 1.5% (w/v) molasses, lentils at a concentration of 3% (w/v) was found to be the optimum. Acidic medium was found to be favorable for riboflavin production when the molasses to lentils ratio was greater than one and neutral medium when the ratio was one. Effect of agitation on vitamin production was also studied and it was observed that 300 rpm gives a higher yield of product.     

Pyrene fluorescence probing of unsaturated lipids in Phytophthora infestans zoospores.

Pyrene rapidly penetrates into isolated zoospores of phytopathogenic fungus Phytophthora infestans localizing predominantly in lipid bodies. An analysis of steady-state monomer and excimer fluorescence spectra, as well as of vibronic structure has suggested a considerable part of the fluorescent probe to be located in a lipid environment. Pyrene partition into hydrophilic phase was observed at its high concentrations. Catalytic hydrogenation of unsaturated lipids in zoospores in situ reduced excimer production. The kinetics of changes of pyrene excimerization suggest that hydrogenation affects both the surface and the intrinsic lipids of the zoospores. The usefulness of pyrene as a fluorescent probe for unsaturated lipids in membranes and lipid bodies of intact cells, and the possible role of eicosapolyunsaturated fatty acids in induction of immune response in potato plants are discussed.     

Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence

A rapid estimation method of the intracellular lipid content in microorganisms using a fluorescent probe, Nile red, was established by optimization of the Nile red staining and data processing. The protocol was designed to be applicable to a wide range of microorganisms and culture conditions. In the optimized procedure, cells diluted with buffer were stained with 0.24-0.47 microg/ml of Nile red for 5 min, and the fluorescent emission spectra in the wavelength region of 400 to 700 nm excited at 488 nm were acquired before and after the Nile red addition. The fluorescence intensity corresponding to the intracellular lipid amount was determined at the peak of the corrected spectrum. The value showed a linear relation with the lipid content of various oleaginous fungi and yeasts measured by the conventional method. The relative intensities against the unit lipid amounts were almost similar except for one yeast. For the application to mycelia forming various types of pellets, a simple and easy pretreatment of shaking with glass beads for 5-10 min was added to the protocol. The established method was applicable to estimate the lipid content of a wide range of microorganism cultures containing 2-5000 microg-lipid/ml-broth.     

Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora.

Mortierella ramanniana var. angulispora accumulates triacylglycerol (TG) in lipid bodies. Studies on lipid transport into lipid bodies are essential for elucidating mechanisms of lipid body formation. We used fluorescent dyes and fluorescent lipid analogs to visualize lipid body formation with a confocal laser scanning microscope. Different sizes of lipid bodies were stained by Nile red, a lipid body marker - one with a diameter of about 1 micrometer and the other with a diameter of about 2-3 micrometers. Lipid bodies matured into larger ones with culture. To metabolically monitor lipid bodies, we used 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-phosphatidic acid (C5-DMB-PA), and C5-DMB-phosphatidylcholine (C5-DMB-PC). These were taken up into fungal cells and incorporated into intracellular organelles at 30 degrees C. C5-DMB-PA was quickly incorporated into lipid bodies while C5-DMB-PC was initially incorporated into internal membranes, presumably endoplasmic reticulum membranes, and fluorescence was then gradually transported into lipid bodies. The transport of fluorescent lipids accompanied their metabolism into diacylglycerol (DG) and TG, which, taken together with the fluorescence distribution, suggested that conversion to TG was not necessary for transport into lipid bodies. It is likely that the synthesized DG was mainly located in lipid bodies and the conversion to TG took place in lipid bodies. C5-DMB-PA and C5-DMB-PC were converted to DG and TG in the membrane and lipid body fractions of this fungus, which agreed with in vivo metabolism of these fluorescent lipids and in vitro enzyme activity related to PA and PC metabolism. These results indicate that transport and metabolism of C5-DMB-PA and C5-DMB-PC represent two different routes for lipid body formation in this fungus.     

Formation and degradation of lipid bodies found in the riboflavin-producing fungus Ashbya gossypii

Microdroplets in the hyphae of Ashbya gossypii were found to become stained by Nile red. Purification of the stainable substance showed that the yellow fluorescent bodies consisted of triacylglycerol. During growth on glucose as the carbon source 8%–12% of the mycelial dry weight was found to be neutral lipid. When glucose declined in the medium, the content decreased to 3%–4% and the respiration quotient shifted to 0.6 indicating a reserve function of the fat. The fatty acid composition of the storage lipid was found to be strongly influenced by the carbon source. Mycelia cultivated on glucose contained 5% linoleic acid and 20% palmitoleic acid in their neutral fat while linoleic acid made up 54% and palmitoleic acid was not detectable (< 0.1%) in soybean-oil-grown mycelia. When plant oil was given as the sole carbon source, the fatty acid composition of the storage lipid showed a high similarity to the fed fat. ¹⁴C-labelled free oleic acid added to a culture growing on soybean oil was immediately incorporated into the fungal lipid. A pulse of 0.9 g/l free palmitoleic acid, fed during growth on olive oil, increased the content of this particular fatty acid in the fungal triacylglycerol from 0.8% to 9.6%. In addition, a liberation of free fatty acid and diacylglyceride was found in the culture supernatant when pure triolein was given as the sole carbon source. Obviously, the fungus cleaved the lipid serving as the carbon source extracellularly and used the liberated fatty acids for its storage lipid formation.This paper is dedicated to Prof. Dr. F. Wagner on the occasion of his 65th birthday     

微藻细胞油脂含量的快速检测方法

以斜生栅藻(Scenedesmus obliquus JNU20)为实验材料,采用尼罗红(NR)荧光光谱法和傅里叶变换红外光谱法(FTIR)测定该藻细胞中的油脂含量。研究结果表明NR的最佳染色条件为:染色前微波处理40 s,二甲基亚砜体积分数1%, NR最终质量浓度1.5 μg/ml,染色时间5 min,染色温度40℃。比较了NR荧光光谱法、FTIR与传统重量法测定的该藻在不同时相的油脂积累情况,利用激光共聚焦显微镜观察了细胞中油体形成的动态过程。实验结果表明NR荧光光谱法、FTIR与传统重量法测定的结果显著相关(R²=0.9258, R²=0.9844),但NR荧光光谱法和FTIR更简便快速,研究结果为规模化筛选高含油量藻株及跟踪产油微藻油脂积累过程奠定了基础。