Heterokaryosis and Alkaloid Production in Claviceps purpurea

Strain 275 FI of Claviceps purpurea, which produces large amounts of peptide alkaloids in submerged culture, is a heterokaryon; after several generations on agar media, it segregates the single components. These components (by us labeled V, C, and W) are stable and produce almost no alkaloids under described conditions of submerged culture. The mycelium of strain 275 FI consists of hyphae with multinucleate cells and does not produce conidia. Strains V, C, and W form numerous anastomoses when grown together on agar. By combining strains V and C, a heterokaryon similar to 275 FI in appearance has been obtained. This new strain produces amounts of alkaloids much larger than those produced by V and C separately or in associated submerged culture. We conclude, therefore, that in strain 275 FI the heterokaryotic condition is favorable to the production of alkaloids. Several conidiaproducing cultures of C. purpurea of various origins, as well as sclerotia of the same species, have been examined. The results demonstrated that the heterokaryotic condition is rare in the cultures, but it is frequent in the mycelium from sclerotia. Since it is known that the production of alkaloids is typical of the sclerotial phase in C. purpurea, it is suggested that this capacity is related to the heterokaryosis of the producing strains.     

Production of Peptide Ergot Alkaloids in Submerged Culture by Three Isolates of Claviceps purpurea

Three strains of Claviceps purpurea (Fr.) Tul., isolated from sclerotia grown on rye, produce under submerged conditions ergocryptine and ergotamine, ergocornine and ergosine, and ergocristine, respectively. All of the strains either lacked the ability to produce conidia or formed them sparingly, but they accumulated large quantities of lipids and sterols. The fermentations are typically divided into two phases. The first is characterized by the rapid utilization and exhaustion of the phosphate contained in the medium, rapid uptake of ammonium nitrogen and of citric acid, rapid growth, and low alkaloid production; the second phase is characterized by slower growth and by a marked accumulation of lipids, sterols, and alkaloids.     

Lipids of a citric-acid-producing Aspergillus niger strain grown in copper- and in manganese-supplemented media

A citric-acid-producing Aspergillus niger strain was cultivated in conditions favouring citric acid biosynthesis and in conditions hindering it. During both extreme processes, the mycelia were analysed for their lipid content, individual lipid classes, the content of sterols and free fatty acids. Since phospholipids, especially phosphatidylcholine and sterols, play an essential role in membrane permeability one can conclude that the differences observed substantially contribute to citric acid excretion into fermentation media. The difference in sterol composition was the most pronounced. Citric-acid-excreting mycelia contained lower quantities of sterols and ergosterol was the only component. A. niger mycelia grown in conditions hindering citric acid accumulation contained higher amounts of sterols with ergosterol as the main component and six other sterol components representing a minor amount.Offprint requests to: K. Jernejc     

苹果枝条内生菌longA的鉴定及其抑菌作用机理初探

【目的】鉴定一株分离自苹果健康枝条的内生菌long A,对其抑菌作用机理进行初步探究。【方法】通过生理生化性质测定和16S r RNA基因序列分析对其进行鉴定;采用平板对峙法检测long A对苹果树腐烂病菌(Valsa mali)菌丝生长的影响和对孢子萌发的抑制作用,利用透射电镜观察苹果树腐烂病菌的细胞内部变化。【结果】16S r RNA基因序列分析结果表明该菌株为解淀粉芽孢杆菌Bacillus amyloliquefaciens;菌株long A好氧,可利用葡萄糖、蔗糖、甘露醇、乳酸、柠檬酸等碳源;耐盐性差(不能耐受质量分数7%的氯化钠);接触酶反应呈阳性;可还原硝酸盐;能使明胶液化、水解淀粉;不能利用柠檬酸盐;乙酰甲基甲醇(V-P)试验为阴性。long A能显著地降低苹果树腐烂病菌分生孢子的萌发率,并导致其菌丝生长过程中分枝增多、顶端膨大及细胞质外渗。【结论】菌株long A能有效抑制苹果树腐烂病菌等,具有一定的生防潜能。     

Isolation of 2-Phenylacetamide as a Plant Growth Regulator Produced by Actinomyces

Isolation and screening tests were carried out in order to find microorganisms which were able to produce citric acid directly from blackstrap molasses. Some strains were obtained which accumulate considerable quantities of citric acid. Certain temperature changes during the course of incubation were found to increase the yield of citric acid.

The present investigation was undertaken to see if a simple method could be found to improve the yield of citric acid from blackstrap molasses, and we could obtain the yield of more than 70% from the untreated molasses using a newly isolated strain of Asp. niger.     

Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2

In previous studies, a new microbial strain ALA2 was isolated which produced many new products from linoleic acid [Gardner H.W., Hou C.T., Weisleder D. and Brown W. 2000. Lipids 35: 1055–1060; Hou C.T. 1998. 12,13,17-Trihydroxy-9(Z)-Octodecenoic acid and derivatives and microbial isolate for production of the acid. US Patent No. 5, 852, 196]. Strain ALA2 was preliminary identified as Clavibacter sp. based on its physiological and fatty acid profiles. To determine if strain ALA2 is the optimal strain for industrial applications, other related strains were screened for their abilities to convert linoleic acids. Two strains from Clavibacter and 20 type strains from the phylogenetically related genus Microbacterium were studied. Surprisingly, all of these strains tested showed very little or no activity in converting linoleic acid. On reexamination of the identification of strain ALA2, the sequence of the 16S ribosomal RNA gene of ALA2 was found to be 99% identical to that of Bacillus megaterium and the strain was also found to have 76.3% DNA homology to the B. megaterium type strain. Therefore, strain ALA2 is now reclassified as B. megaterium. Screening of 56 strains of B megaterium strains showed that many of them were able to produce reasonable amounts of hydroxyl fatty acids from linoleic acid, although strain ALA2 possessed the greatest activity.     

Surface localization of sialic acid on Actinomyces viscosus

This study reports the presence of sialic acid in Actinomyces viscosus strains T14V and T14AV. Mild acid hydrolysis of whole organisms released a compound which reacted positively in the periodate-thiobarbituric acid, direct Ehrlich's and resorcinol assays, and which co-chromatographed on paper with authentic N-acetylneuraminic acid. Strain T14V contained 10-fold greater concentrations of sialic acid than did strain T14AV. Sialic acid content was dependent upon the stage of growth of the culture, reaching a maximum in early stationary phase. Epifluorescence microscopy of fluorescein isothiocyanate (FITC)-conjugated Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, revealed a uniform distribution of bound lectin on the surfaces of strains T14V and T14AV. Additional evidence for surface localization was obtained by demonstration of whole-cell agglutination of both strains with LPA. All LPA interactions with A. viscosus were inhibited by the presence of 0.1 M-N-acetylneuraminic acid. Neuraminidases from Clostridium perfringens, Arthrobacter ureafaciens and Vibrio cholerae did not release detectable amounts of sialic acid, but the extracellular enzyme from A. viscosus cleaved amounts equivalent to those obtained by acid hydrolysis. Other laboratory strains (W1053, M100, W859, 5-5S, RC45, ATCC 19246, and 'binder') as well as recent clinical isolates of A. viscosus were agglutinated by LPA and released sialic acid upon mild acid hydrolysis. Surface-available sialic acid has been implicated in the inhibition of alternative complement pathway activation and subsequent opsonophagocytosis. Thus the occurrence of surface sialic acid in A. viscosus may represent a mechanism of pathogenesis for this oral bacterium.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

High specific rates of glucose utilisation under conditions of restricted growth are required for citric acid accumulation by Yarrowia lipolytica IMK 2

Yarrowia lipolytica strain IMK 2, a yeast capable of producing and excreting citric acid, only accumulated citric acid when cell growth was restricted by specific nutrient limitations. Effective citrate accumulation was linked with the ability of cells to maintain a high rate of glucose utilisation when growth was limited. Cells limited by nitrogen (N), sulphur (S), magnesium (Mg) or phosphorus (P) accumulated between 50 and 220 mm citric acid after 168 h, with N and S limitation resulting in the highest specific rates of production. In contrast, potassium (K)-limited cells accumulated 6 mm citric acid in the same time period. Cells limited by K or lower levels of Mg or P had rates of glucose utilisation that were less than 50% of those measured in cells limited by N or S. Although limitation of strain IMK 2 by Mg or P led to citrate production, significant accumulation occured only when the threshold concentration of the limiting nutrient was exceeded. There was no large accumulation of other tricarboxylic acid (TCA) cycle acids, acetate, pyruvate, lactate or ethanol, although K-limited cells produced quantities of intracellular glycogen. Mannitol was accumulated under N, S and P limitation (up to 40 mm), as were small quantities of 2-oxoglutarate, which preceded the accumulation of citrate and all of the other TCA cycle acids measured. A clear difference was evident between the rates of glucose utilisation under N limitation between two citrate-accumulating strains (IMK 2 and Candida guilliermondii IMK 1) and a citrate non-accumulating strain, Y. lipolytica YB 423. Although these strains had similar rates of glucose utilisation during exponential growth, both strains IMK 1 and IMK 2 had specific rates of glucose utilisation under N limitation more than double that of strain YB 423.Correspondence to: J. D. Brooks 2     

The capacity to produce cholera exotoxin in natural strains of Vibrio cholerae

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.     

Biochemical Mechanisms of Temperature Adaptation in the (+) and (-) Strains of Blakeslea trispora

Evidence obtained with industrial β-carotene-superproducing (+)T and (-)T strains, which fail to form zygotes, suggests that the lipids in the mycelium of the (-) strain of Blakeslea trispora lack linolenic acid. This circumstance apparently accounts for the fact that the (+) and (-) strains of B. trispora use different adaptive mechanisms to cope with an increase or decrease in cultivation temperature. In the (+) strain, temperature adaptation is based on changes in the ratio between linoleic and linolenic acyls and, also, involves shortening of acyl chains. In addition, the (+) strain contains a larger amount of protective carbohydrates, such as arabitol and trehalose. This strain is characterized by the presence of glycerol, a cryothermoprotector that protects fungal cells at low temperatures. The (-) strain lacks these biochemical mechanisms, but its neutral lipids contain a comparatively high amount of sterols and their esters. These facts enable us to interpret the enhanced thermotolerance of the (-) strain and its capacity to grow at high temperatures in terms of biochemical adaptation. In the light of the data obtained with wild-type and industrial strains, it is suggested that the lack of linolenic acid in the lipids should be considered an essential sex-specific property of the heterothallic strains of Blakeslea trispora.     

Effect of cadmium on lipid composition of Aureobasidium pullulans grown with added extracellular polysaccharides

Effect of cadmium on the growth and lipid composition in three Aureobasidium pullulans strains grown in the presence and absence of extracellular polysaccharides was studied. Strains tolerated up to 1 mM Cd²⁺ in medium, but addition of either pectin or pullulan to medium increased tolerance to up to 2 mM Cd²⁺. While cadmium enhanced lipid accumulation notably only in strains of CCY 27-1-98 and CCY 27-1-111, strain CCY 27-1-115 displayed considerably high levels of sterols. Cadmium increased fatty acid unsaturation rapidly in phosphatidylethanolamine in all strains, owing to elevated levels of linoleic acid in this lipid fraction. On the other hand, Cd²⁺ ions significantly lowered conversion of oleic acid to linoleic acid in phosphatidylcholine only in the strain CCY 27-1-115. Nevertheless, addition of either pullulan or pectin amplified the ratio of C18:C16 fatty acids and the degree of fatty acid unsaturation in all membrane lipids as a result of induced biotransformation of stearate to oleate and linoleate. It was concluded that more saturated membranes with preferred C18 fatty acyl chains and high sterol amounts in the strain CCY 27-1-115 might be connected with its lower capacity to form pullulan as a protective metabolite against heavy metals compared with the other two strains.     

一株产甘露糖赤藓糖醇脂菌株的筛选及其产物分析

【目的】解决石油长链烃类物质引起的环境污染问题,筛选可以高效降解石油烃的产糖脂类生物表面活性剂菌株。【方法】采用血平板、油平板法,从葡萄皮表面分离到6株产糖脂类的真菌,比较各菌株的排油性能,通过PCR扩增合成糖脂类表面活性剂的关键基因,筛选到一株具有emtl序列的真菌K6。经形态学、生理生化测定和分子系统发育分析(5.8S,ITS1,ITS2)对菌株进行鉴定,而且通过TLC和HPLC分析该菌株的代谢产物。【结果】经鉴定,该菌为Pseudozyma churashimaensis,可产甘露糖赤藓糖醇脂。石油烃降解实验表明,菌株K6具有很强的乳化性能和降解石油烃的能力,其石油烃降解率可达70.17%。【结论】菌株K6具有产生物表面活性剂和降解长链石油烃类的能力,其对石油污染环境的生物修复具有重要的现实意义。     

Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effects on cryopreservation

To understand the mechanisms of sucrose‐induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem cultures of different banana varieties. The four banana varieties that were selected show different post‐thaw shoot regeneration rates (0–53.4%). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2‐week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose‐pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the post‐thaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrose‐pretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post‐thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process.     

Organic Acid Production by Basidiomycetes: I. Screening of Acid-Producing Strains

Sixty-seven strains belonging to 47 species of Basidiomycetes were examined for their acid-producing abilities in glucose media, in both the presence and absence of CaCO₃, in stationary and shake cultures. Some strains were found to produce large quantities of oxalic acid. The oxalic acid-producing strains could be separated into two groups. Strains of one group (mostly brown-rot fungi) were able to produce oxalic acid, regardless of whether CaCO₃ was present in the medium. Strains of the other group (mostly white-rot fungi) were characterized by their ability to produce oxalic acid only when CaCO₃ was added to the medium. With the latter group, shake-culturing was generally more effective than stationary culturing in respect to acid production. In the CaCO₃-containing media, Schizophyllum commune, Merulius tremellosus, and Porodisculus pendulus were found to produce substantial amounts of L-malic acid as a main metabolic product, along with small quantities of oxalic and other acids in shake cultures. Especially, S. commune and M. tremellosus may be employed as malic acid-producing species.     

Biochemical mechanisms of temperature adaptation in the (+) and (-) strains of Blakeslea trispora

Evidence obtained with industrial beta-carotene-superproducing (+)T and (-)T strains, which fail to form zygotes, suggests that the lipids in the mycelium of the (-) strain of Blakeslea trispora lack linolenic acid. This circumstance apparently accounts for the fact that the (+) and (-) strains of B. trispora use different adaptive mechanisms to cope with an increase or decrease in cultivation temperature. In the (+) strain, temperature adaptation is based on changes in the ratio between linoleic and linolenic acyls and, also, involves shortening of acyl chains. In addition, the (+) strain contains a larger amount of protective carbohydrates, such as arabitol and trehalose. This strain is characterized by the presence of glycerol, a cryothermoprotector that protects fungal cells at low temperatures. The (-) strain lacks these biochemical mechanisms, but its neutral lipids contain a comparatively high amount of sterols and their esters. These facts enable us to interpret the enhanced thermotolerance of the (-) strain and its capacity to grow at high temperatures in terms of biochemical adaptation. In the light of the data obtained with wild-type and industrial strains, it is suggested that the lack of linolenic acid in the lipids should be considered an essential sex-specific property of the heterothallic strains of Blakeslea trispora.     

The influence of carbon source on the growth and development of asexual embryos of Theobroma cacao

Asexual embryos of Theobroma cacao L. cultured in a liquid medium on a rotary drum apparatus were induced to produce storage lipids, anthocyanin, and alkaloids by increasing concentration of sucrose but not glucose, and only slightly by fructose or fructose + glucose at the same osmolarity. Glucose combined with sucrose or alternating with sucrose reduced lipid concentration as compared to sucrose alone. The concomitant development of lipids, anthocyanin, and alkaloids suggests that sucrose at high concentration regulates the initiation of embryo development.     

Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae

Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain. This strain had previously been deregulated by overexpressing a truncated HMG-CoA reductase (tHMG1) in the main bottleneck of the early ergosterol pathway. The overexpression of the gene ERG1 (squalene epoxidase) induced a significant decrease of the direct substrate squalene, a high increase of lanosterol, and a small increase of later sterols. The overexpression of the ERG11 gene encoding the sterol-14alpha-demethylase resulted in a decrease of lanosterol and an increase of downstream sterols. When these two genes were simultaneously overexpressed, later sterols from zymosterol to ergosterol accumulated and the content of squalene was decreased about three-fold, indicating that these steps had limited the transformation of squalene into sterols. The total sterol content in this strain was three-fold higher than in a wild-type strain.     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。脂肪酸组成分析结果表明,菌油富含饱和及低度不饱和长链脂肪酸,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

Biosynthesis of citric acid

Summary TheA. niger strain 363 is better in its citric acid producing capacity than other strains studied. The pH value of the culture media differs from strain to strain for the good yield of citric acid. When there is good yield of citric acid the sugar consumption is less and mycelial growth is also less.