Fungus Penicillium citrinum, isolated from permafrost sediments, as a producer of ergot alkaloids and new quinoline alkaloids quinocitrinines

Quinocitrinines and ergot alkaloids are synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grows. The major part of these secondary metabolites are secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

The Fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>, Isolated from Permafrost Sediments,as a Producer of Ergot Alkaloids and New Quinoline Alkaloids Quinocitrinines

Quinocitrinines and ergot alkaloids were synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grews. The major part of these secondary metabolites was secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Clavine alkaloid biosynthesis by the fungus <Emphasis Type="Italic">Penicillium palitans</Emphasis> Westling 1911 isolated from ancient permafrost deposits

The relic strain of Penicillium palitans isolated from the ancient permafrost deposits produces clavine alkaloids such as festuclavine, fumigaclavine A, and fumigaclavine B. Alkaloid biosynthesis is concurrent with the growth. Tryptophan and zinc ion additives to the culture medium stimulate the synthesis of alkaloids.     

Growth and biosynthesis of rugulovasines in Penicillium variabile Sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

The influence of medium composition on alkaloid biosynthesis by Penicillium citrinum

The fungus P. citrinum produces secondary metabolites, clavine ergot alkaloids (EA), and quinoline alkaloids quinocitrinines (QA) in medium with various carbon and nitrogen sources and in the presence of iron, copper, and zinc additives. Mannitol and sucrose are most favorable for EA biosynthesis and mannitol is most favorable for QA. Maximum alkaloid production is observed on urea. Iron and copper additives in the medium containing zinc ions stimulated fungal growth but inhibited alkaloid biosynthesis. The production of these secondary metabolites does not depend on the physiological state of culture, probably due to the constitutive nature of the enzymes involved in biosynthesis of these substances.     

Growth and biosynthesis of rugulovasines in <Emphasis Type="Italic">Penicillium variabile</Emphasis> sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

Effect on microelements on biosynthes of secondary metabolites in the fungus Penicillium citrinum Thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondary O-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions affected but little fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

A novel high-cell-density protein expression system based on Ralstonia eutropha

We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.     

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.     

Effect of microelements on the biosynthesis of secondary metabolites by the fungusPenicillium citrinum thom VKM F-1079

Penicillium citrinum VKM F-1079 was found to produce clavine ergot alkaloids and citrinin, a secondaryO-heterocyclic metabolite. Citrinin was produced in the idiophase, whereas the production of ergot alkaloids paralleled fungal growth. The addition of manganese ions to the growth medium stimulated the biosynthesis of both citrinin and ergot alkaloids. Zinc ions stimulated only citrinin synthesis. The presence of these microelements in the growth medium influenced the proportion between the ergot alkaloids synthesized. Copper, manganese, and iron ions slightly affected fungal growth and alkaloid production. The effect of microelements on the main kinetic parameters of growth and alkaloid production was studied.     

Parasitic fungus Claviceps as a source for biotechnological production of ergot alkaloids

Ergot alkaloids produced by the fungus Claviceps parasitizing on cereals, include three major groups: clavine alkaloids, d-lysergic acid and its derivatives and ergopeptines. These alkaloids are important substances for the pharmatech industry, where they are used for production of anti-migraine drugs, uterotonics, prolactin inhibitors, anti-Parkinson agents, etc. Production of ergot alkaloids is based either on traditional field cultivation of ergot-infected rye or on submerged cultures of the fungus in industrial fermentation plants. In 2010, the total production of these alkaloids in the world was about 20,000 kg, of which field cultivation contributed about 50%. This review covers the recent advances in understanding of the genetics and regulation of biosynthesis of ergot alkaloids, focusing on possible applications of the new knowledge to improve the production yield.     

鸟苷发酵条件的优化研究

以枯草芽孢杆菌(Bacillus subtilis)TA208为供试菌株,运用单因素实验方法进行了摇瓶条件下发酵培养基以及发酵条件的优化研究,在最优发酵条件下鸟苷产量达到19．79g／L,比基本培养条件提高32．37％。     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.     

Analysis of Secondary Metabolites of Microscopic Fungi of the Genus Penicillium by Chromatographic Techniques

Combinations of various systems of thin-layer chromatography and high-performance liquid chromatography (HPLC) were efficient in analyzing 39 nitrogen-containing secondary metabolites (alkaloids) produced by 12 strains of microscopic fungi of the genus Penicillium. Chromatographic mobility of alkaloids on Silufol plates was determined in the following systems: (a) chloroform, methanol, and 25% NH₄OH (90 : 10 : 1, 90 : 10 : 0.1, or 80 : 20 : 0.2); (b) chloroform and acetone (9 : 1); and (c) ethyl acetate, methanol, and 25% NH₄OH (85 : 15 : 10); staining was performed using Ehrlich's reagent. Conditions for separation of clavine alkaloids by HPLC on Spherisorb ODS-2 and Supelcosil LC-18 columns (gradient elution) were optimized. Retention values of 22 alkaloids were compared to those of agroclavine and roquefortine.     

Quantitative changes of the alkaloid complex in a submerged culture ofClaviceps paspali

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 ± 1 °C resulted in an increased production of total alkaloids from the original value of 100–200 μg/mL to more than 2 000 μg/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites. 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. α-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80 % in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids,i.e. lysergamide and the corresponding minor isomers, increased. An erratum to this article is available at .     

酿酒酵母by1.1b产D-(-)-扁桃酸脱氢酶的发酵条件优化

对一株产D-(-)-扁桃酸对映选择性脱氢酶的酿酒酵母菌(Saccharomyces cerevisiae sp.strain by1.1b)发酵产酶条件进行了优化.研究各种碳源、氮源及无机盐对产酶的影响,应用正交试验优化发酵培养基组成,结果为:蛋白胨60 g/L,麦芽糖30 g/L,MgSO4 0.5 g/L,ZnSO4 0.01 g/L,KCl 1.0 g/L.优化后酶产量提高了7.9倍(由2.56 U/mL增至20.21 U/mL).摇瓶培养最佳条件为:装液量40%,发酵pH 6.5,接种量10%,发酵温度30℃.考察了细胞生长及产酶的时间进程,最佳培养时间为25 h.     

Vc生产菌“神舟七号”搭载育种

选取3株性状不同的Vc二步发酵生产菌搭载于"神舟七号"飞船进行空间诱变,返回地面后,经富集培养和分离,获得近12000株诱变菌株。采用试管微量培养并监测pH值法作3次初筛,获300余株优良株,再经摇瓶发酵定酸、糖法作3次复筛,选出12株优良菌株。新菌株摇瓶发酵转化率提高了5%～7%。研究了新菌株生产的最适发酵条件,调整了部分工艺过程,应用于大生产,转化率提高4%～5%。     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.     

Miso: III. Pure Culture Fermentation with Saccharomyces rouxii

Excellent miso has been prepared with soybean grits inoculated with a pure culture of Saccharomyces rouxii strain NRRL Y-2547. Pure culture inoculum of this osmophilic yeast was prepared by growing the culture in aerated flasks on a yeast extract medium with a salt concentration equal to that used in the manufacture of miso. It has also been found possible to make miso from whole beans with the above culture. The advantages of pure culture fermentation in producing miso are discussed.