Sphaerolone and dihydrosphaerolone, two bisnaphthyl-pigments from the fungus Sphaeropsidales sp. F-24'707

Two new bisnaphthalene compounds, sphaerolone (1) and dihydrosphaerolone (2), together with 2-hydroxyjuglone (9), were isolated from the culture broth of a Sphaeropsidales sp. (strain F-24'707) after inhibition of the regular proceeding 1,8-dihydroxynaphthalene (DHN) biosynthesis with tricyclazole. The structures of 1 and 2 were established by detailed spectroscopic analysis and present novel bisnaphthalenes. The biosynthetic origin of 1 and 2 as dimerization products of 1,3,8-trihydroxynaphthalene, an intermediate of the DHN biosynthesis, is discussed.     

光滑球拟酵母新霉素抗性株加速葡萄糖代谢

为进一步提高光滑球拟酵母发酵生产丙酮酸的生产强度,在能量代谢分析的基础上提出了降低ATP合成酶活性、但不影响NADH氧化的育种策略。通过亚硝基胍诱变,获得一株新霉素抗性突变株N07,该菌株F1ATPase活性降低65%、丙酮酸产量高于48gL且单位细胞消耗葡萄糖能力提高38%。添加双环己基碳二亚胺(DCCD)、叠氮钠(NaN3)、新霉素显著降低出发株F1ATPase活性但不影响突变株F1ATPase活性。突变菌株胞内ATP含量下降23.7%导致生长速率和最终菌体浓度(为出发菌株的76%)均低于出发菌株,但葡萄糖消耗速度和丙酮酸生产速度分别提高34%和42.9%,发酵周期缩短12h。进一步研究发现,突变株糖酵解途径中关键酶磷酸果糖激酶、丙酮酸激酶和磷酸甘油醛激酶的活性提高了63.7%、28.8%和14.4%,电子传递链关键酶活性提高10%。结果表明降低真核微生物F1ATPase活性有效地提高了糖酵解关键酶活性而加速葡萄糖代谢。     

Comparision between Bacillus subtilis RP24 and its antibiotic-defective mutants

Bacillus subtilis RP24, a promising plant growth-promoting rhizobacterium and a potent biocontrol agent isolated from pigeonpea rhizosphere was mutagenized with ethyl methanesulphonate to study the possible mechanism/s involved in the potential antagonistic properties of the strain. Over 10,000 mutants were screened against the phytopathogenic fungus Macrophomina phaseolina on potato dextrose agar plates to select ten mutants showing partial antagonism as compared to the parent strain and one negative mutant showing no antagonism. The parent strain RP24 was compared with its mutants for the presence of different possible mechanisms behind antagonism. Production of hydrogen cyanide, ammonia, siderophores, and hydrolytic enzymes like lipase, amylase, and protease were detected in all the mutants as well as the parent strain, whereas fungal cell-wall-degrading enzymes, β-1, 3-glucanase and chitosanase were not detected in any of the mutants and the parent strain, indicating that none of these mechanisms was involved in the antagonistic trait of the strain. Two possible mechanisms detected behind the antifungal trait of the strain RP24 were production of antifungal volatiles and extra-cellular diffusible antibiotics. An attempt was made for extraction, partial characterization of the extra-cellular diffusible antifungal metabolite/s by thin layer chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The extracellular, methanol soluble, hydrophobic, ninhydrin-negative, thermostable and pH-stable antifungal metabolites were characterized as cyclic lipopeptides belonging to the iturin group of peptide antibiotics.     

Streptomyces spheroides mutant deprepressed for exoprotease biosynthesis

The effect of carbon, nitrogen and sulfur sources on the biosynthesis of exoproteases was studied with the parent Streptomyces spheroides strain 35 and its mutant M8-2. Addition of a carbon, nitrogen and sulfur source to the medium deficient in one of these elements did not repress the synthesis of exoproteases by the washed mycelium of the mutant as compared to the parent strain. Protein as a sole source of carbon, nitrogen and sulfur had no effect on the biosynthesis of exoproteases by the mutant. In contrast to the parent strain, the biosynthesis of exoproteases in the mutant was not controlled by metabolite repression.     

The use of cellulases from a beta-glucosidase-hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw

Conidia of the cellulolytic strain Trichoderma reesei F522 were mutagenized with UV irradiation and N-methyl|-N'-nitro-N-nitrosoguanidine (NTG). A visual agar plate detection system was developed, using esculin and ferric ions, to identify mutants of T. reesei with increased beta-glucosidase activity. Selected mutants were tested for production of extracellular cellulases in shake flasks on autohydrolyzed wheat straw as carbon source. The most active mutant V-7 showed about 6-times higher activity of beta-glucosidase than the parent strain F-522, whereas the filter paper degrading and endo-1,4-beta-D-glucanase activities increased by 45% and by almost 31%, respectively. Cellulase preparations obtained from the parent and mutant strains were then used along with Kluyveromyces fragilis cells for ethanol production from ethanol-alkali pulped straw in the simultaneous saccharification and fermentation (SSF) process. From 10% (w/v) of straw pulp (dry matter), 2.5% (w/v) ethanol was obtained at 43 degrees C after 48 h using cellulase derived from the parent strain of T. reesei. When the beta-glucosidase-hyperproducing mutant V-7 was employed, the ethanol yield in the SSF process increased to 3.4% (w/v), the reaction time was shortened to 24 h and no cellobiose was detected in straw hydrolyzates.     

Regulatory factors affecting alpha-amylase production in bacillus licheniformis.

Possible factors regulating alpha-mylase synthesis in wild-type Bacillus licheniformis and in mutants producing elevated levels of the enzyme were studied in terms of catabolite repression, apparent temperature-sensitive repression, induction, and culture age. The synthesis of alpha-amylase in the parent strain occurred long after the culture reached the stationary phase of growth as a result of de novo protein synthesis, occurred only at high temperature around 50 C and not below 45 C, appeared to be induced in the presence of oligosaccharides with some linkage of alpha-1,4-, beta-1,4, beta-1,6-glucosyl glucose, or alpha-1,6-galactosyl glucose, and was repressed by the addition of exogenous glucose or low-molecular-weight metabolites. The addition of cyclic adenosine 3',5'-monophosphate stimulated alpha-amylase accumulation in growing cultures of the parent strain, but neither shortened the long lap period prior to the start of alpha-amylase synthesis nor mitigated the repressive effect of glucose. Mutant strains derived from the parent strain showed variation in the pattern of alpha-amylase synthesis, and some of them such as F-12s and F-14 produced alpha-amylase constitutively and without sensitivity to catabolite repression or transient repression from the moment of cell growth. These results are discussed in relation to possible regulatory mechanisms that might account for the observed characteristics of alpha-amylase synthesis in this facultative thermophilic microorganism.     

Selection of ergot alkaloid producers by induced mutagenesis

Using the induced mutagenesis technique, A series of genetically modified Claviceps sp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus.

Aflatoxins are extremely potent carcinogens produced by Aspergillus flavus and Aspergillus parasiticus. Cloning of genes in the aflatoxin pathway provides a specific approach to understanding the regulation of aflatoxin biosynthesis and, subsequently, to the control of aflatoxin contamination of food and feed. This paper reports the isolation of a gene involved in aflatoxin biosynthesis by complementation of an aflatoxin-nonproducing mutant with a wild-type genomic cosmid library of A. flavus. Strain 650-33, blocked in aflatoxin biosynthesis at the afl-2 allele, was complemented by a 32-kb cosmid clone (B9), resulting in the production of aflatoxin. The onset and profile of aflatoxin accumulation was similar for the transformed strain and the wild-type strain (NRRL 3357) of the fungus, indicating that the integrated gene is under the same control as in wild-type strains. Complementation analyses with DNA fragments from B9 indicated that the gene resides within a 2.2-kb fragment. Because this gene complements the mutated afl-2 allele, it was designated afl-2. Genetic evidence obtained from a double mutant showed that afl-2 is involved in aflatoxin biosynthesis before the formation of norsolorinic acid, the first stable intermediate identified in the pathway. Further, metabolite feeding studies with the mutant, transformed, and wild-type cultures and enzymatic activity measurements in cell extracts of these cultures suggest that afl-2 regulates gene expression or the activity of other aflatoxin pathway enzymes. This is the first reported isolation of a gene for aflatoxin biosynthesis in A. flavus.     

Selection of Ergot Alkaloid Producers by Induced Mutagenesis

Using the induced mutagenesis technique, a series of genetically modified Clavicepssp. VKM F-2609 strains that display high levels of agroclavine and elymoclavine synthesis were selected by induced mutagenesis. Compared to the parent strain, c106 displayed a 40-fold higher level of agroclavine synthesis, and c66 displayed an eightfold higher level of elymoclavine synthesis. The levels of synthesis of other alkaloids were decreased in these strains. The effects of various carbohydrates on the strain growth and ergot alkaloid biosynthesis was then investigated in both the parent strain and c106. The largest amount of agroclavine was synthesized by c106 strain growing on a medium with maltose.     

Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds.     

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.     

龟裂链霉菌zwf2基因阻断提高土霉素生物合成

葡萄糖-6-磷酸脱氢酶(G6PDH)是链霉菌磷酸戊糖途径中第一个酶("看家"酶),也是形成NADPH的关键酶,由zwf1和zwf2基因编码.以温敏型质粒pKC1139为基础构建了用于阻断龟裂链霉菌zwf2的重组质粒pKC1139-zwf2',通过大肠杆菌GM2929去甲基化pKC1139-zwf2'后电转至原始龟裂链霉菌M4018感受态细胞,筛选得到转化子.转化子进一步通过PCR鉴定和点杂交印迹分析鉴定,证明是zwf2基因阻断的阳性突变子命名为M4018-△zwf2.以原始菌株为对照,突变子摇瓶发酵结果表明:突变子的葡萄糖-6-磷酸脱氢酶酶活是原始菌的50%左右,但土霉素生物合成水平则提高了27%;在细胞生长方面,二者均在第4d进入生长稳定期而开始大量合成土霉素,发酵结束时细胞菌体浓度基本相同,但突变子的单位菌丝体土霉素生物合成能力则提高了31%.因此,zwf2的阻断有利于土霉素的生物合成,而对细胞生长没有明显影响.     

Itaconate biosynthesis in Aspergillus terreus.

Itaconate biosynthesis was studied in intact cells of high-yield (RC4') and low-yield (CM85J) strains of the fungus Aspergillus terreus by methods (tracers, nuclear magnetic resonance spectroscopy, and mass spectroscopy) that did not interfere with metabolism. Itaconate formation in RC4' required de novo protein biosynthesis. Krebs cycle intermediates increased in both strains during the production of itaconic acid. The Embden-Meyerhof-Parnas pathway and the Krebs cycle were shown to be involved in this biosynthesis by using 14C- and 13C-labelled substrates and nuclear magnetic resonance spectroscopy. A metabolic pathway for itaconate formation from glucose in A. terreus is proposed.     

甾醇C-24甲基转移酶和甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成中的调控作用

摘要：【目的】研究ERG6基因编码的甾醇C-24甲基转移酶和ERG2基因编码的甾醇C-8异构酶在酿酒酵母麦角甾醇生物合成代谢中的调控作用。【方法】通过PCR扩增克隆到酿酒酵母甾醇C-8异构酶的编码序列及其终止子序列,以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体,以磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG2;同时,在本实验室已构建的ERG6表达质粒pPERG6的基础上,构建了ERG2和ERG6共表达的重组质粒pPERG6-2。将表达质粒转化酿酒酵母单倍体菌株YS58,依据营养缺陷互补筛选到重组菌株YS58(pPERG2)和YS58(pPERG6-2)。通过紫外分光光度法和气相色谱法分析重组菌株甾醇组分和含量。【结果】在ERG6高表达的重组酵母菌中,甾醇中间体和终产物麦角甾醇的含量均比对照菌高;而在ERG2高表达的酵母菌株中,无论甾醇中间体,还是麦角甾醇的含量均明显降低。ERG6和ERG2共表达重组菌株YS58(pPERG6-2)的麦角甾醇含量是对照菌株YS58(YEp352)的1.41倍,是ERG2单独高表达菌株YS58(pPERG2)的1.92倍,是ERG6单独高表达菌株YS58(pPERG6)的1.12倍。【结论】本研究首次证明甾醇C-24甲基转移酶催化的反应是酿酒酵母麦角甾醇合成代谢途径中的一个重要的限速步骤,该酶活性提高不但补偿了ERG2高表达对甾醇合成的负效应,而且使麦角甾醇含量进一步提高,为构建麦角甾醇高产酵母工程菌株提供了实验依据。     

Chemical Characterization of Ceramide and Cerebroside in Soybean Leaves

During experiments on protoplast fusion of complementary auxotrophic mutants (194 and 11M-21) of Streptomyces antibioticus for strain improvement, the clones (typified by F-40) regenerated on minimal regeneration medium (MRM) were found to be prototrophs, and to produce an antibiotic different from those produced by the parent strain. The protoplast regeneration of each parent was examined as a negative control experiment.

In the regenerated clones of 194, half of them produced actinomycins similar to those produced by the original mutant 194, but others (typified by R-20) seemed to produce antibiotics similar to those produced by F-40. In the taxonomic characterization of morphological, cultural, and physiological properties of each strain, F-40, R-20, and the parent mutant 194 had no significant differences with a few exceptions. The problem here is whether the antibiotic of R-20 is the same as that of F-40, which was first isolated and found to be a peptide antibiotic different from actinomycins, with activity against Gram-negative and Gram-positive bacteria.     

Metabolomics profiling reveals the mechanism of increased pneumocandin B<Subscript>0</Subscript> production by comparing mutant and parent strains

Metabolic profiling was used to discover mechanisms of increased pneumocandin B₀ production in a high-yield strain by comparing it with its parent strain. Initially, 79 intracellular metabolites were identified, and the levels of 15 metabolites involved in six pathways were found to be directly correlated with pneumocandin B₀ biosynthesis. Then by combining the analysis of key enzymes, acetyl-CoA and NADPH were identified as the main factors limiting pneumocandin B₀ biosynthesis. Other metabolites, such as pyruvate, α-ketoglutaric acid, lactate, unsaturated fatty acids and previously unreported metabolite γ-aminobutyric acid were shown to play important roles in pneumocandin B₀ biosynthesis and cell growth. Finally, the overall metabolic mechanism hypothesis was formulated and a rational feeding strategy was implemented that increased the pneumocandin B₀ yield from 1821 to 2768 mg/L. These results provide practical and theoretical guidance for strain selection, medium optimization, and genetic engineering for pneumocandin B₀ production.     

Targeted inactivation of the mecB gene, encoding cystathionine-gamma-lyase, shows that the reverse transsulfuration pathway is required for high-level cephalosporin biosynthesis in Acremonium chrysogenum C10 but not for methionine induction of the cephalosporin genes

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-gamma-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH(2)) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-gamma-lyase.     

Influence of Iron Regulation on the Metabolome of Cryptococcus neoformans

Iron is an essential nutrient for virtually all organisms and acts as a cofactor for many key enzymes of major metabolic pathways. Furthermore, iron plays a critical role in pathogen-host interactions. In this study, we analyzed metabolomic changes associated with iron availability and the iron regulatory protein Cir1 in a human fungal pathogen Cryptococcus neoformans. Our metabolite analysis revealed that Cir1 influences the glycolytic pathway, ergosterol biosynthesis and inositol metabolism, which require numerous iron-dependent enzymes and play important roles in pathogenesis and antifungal sensitivity of the fungus. Moreover, we demonstrated that increased cellular iron content and altered gene expression in the cir1 mutant contributed to metabolite changes. Our study provides a new insight into iron regulation and the role of Cir1 in metabolome of C. neoformans.     

盐水球菌Salinicoccus ventosaetal B2-3-5的代谢产物分析及其抑制酪氨酸酶活性的机制

[背景] 酪氨酸酶是黑色素合成过程中的关键酶,也是引起人体色素障碍性疾病和产生果蔬酶促褐变的主要原因。目前,酪氨酸酶抑制剂的开发已引起广泛关注,但一些酪氨酸酶抑制剂如熊果苷、曲酸等均存在一定的安全隐患。微生物资源丰富且具有许多优点,从微生物中寻找特异性强、高效的酪氨酸酶抑制剂已成为该领域研究的热点。[目的] 通过测定分离自新疆乌鲁木齐达坂城盐湖的盐水球菌Salinicoccus ventosaetal B2-3-5和B6-1-4代谢物提取物对酪氨酸酶活性的影响,比较2株菌发酵过程中代谢物的差异,了解所筛选菌株B2-3-5抑制酪氨酸酶活性的机制。[方法] 以曲酸为阳性对照分别测定B2-3-5和B6-1-4这2个菌株发酵产生的代谢物提取物对蘑菇酪氨酸酶的抑制活性;应用LC-MS代谢组学方法检测2株菌在相同条件下产生的所有代谢物质;采用单变量、多元变量、正交偏最小二乘判别分析（Orthogonal Partial Least Squares-Discrimination Analysis,OPLS-DA）法识别差异代谢物;利用层次聚类分析（Hierarchial Cluster Analysis,HCA）法对识别的差异物进行聚类分析;通过Kyoto Encyclopedia of Genes and Genomes （KEGG）代谢通路对比法分析这些差异代谢物主要参与的代谢途径。[结果] 菌株B2-3-5代谢物提取物对蘑菇酪氨酸酶二酚酶活性的抑制率为67%,其IC₅₀为0.277 mg/mL,同属菌株B6-1-4代谢物提取物则对酪氨酸酶无抑制活性。采用代谢组学的检测方法从2株菌的代谢物中筛选出63个差异代谢物,其中氨基酸类化合物、维生素类化合物和羧酸类化合物的种类及相对含量均是B2-3-5菌株明显高于B6-1-4菌株。通过代谢途径分析发现这些差异代谢物主要参与15个代谢通路,其中维生素B6生物合成通路的影响较为显著。[结论] 推测B2-3-5菌株可能是通过增加一些氨基酸类、维生素类及羧酸类等小分子化合物的含量来抑制酪氨酸酶活性。维生素B6代谢途径的上调也表明菌体细胞可通过产生维生素B6与酪氨酸酶中的必需氨基作用或清除酶催化循环过程中产生的活性氧自由基（reactive oxygen species,ROS）来抑制酪氨酸酶活性。