嗜热毁丝霉高通量培养技术及其应用

【背景】丝状真菌是一类重要的工业发酵生产宿主菌,如何进行高通量纯菌培养和高效检测筛选性能优异的菌株是工业丝状真菌研究的重要方向。【目的】研究建立丝状真菌的高通量培养技术并测试应用效果。【方法】通过对丝状真菌培养过程中的制种、接种、培养和检测研究,建立基于孔板的高通量培养技术,并以嗜热毁丝霉为例对该技术进行验证。【结果】与传统的平板制种和摇瓶接种培养方式相比,高通量孔板的培养方式将制种通量提高24倍,单位面积产孢子能力提高350%,液体培养转接效率提高10-40倍,并建立96孔板测定乙醇含量的高通量检测技术。【结论】将丝状真菌的培养和检测通量提高1-2个数量级,为快速检测丝状真菌改造过程产生的大量性状不同菌株并获得目标菌株奠定基础,为丝状真菌高通量筛选研究提供应用指导价值。     

同源过表达mhr2基因可提高嗜热毁丝霉纤维素酶活性

为寻找新型的与纤维素酶相关转录调控因子,以嗜热毁丝霉(Myceliophthora thermophila ATCC42464)为研究材料,通过克隆嗜热毁丝霉mhr2基因序列,构建重组过表达载体,转化并筛选到转化子Mt O24中mhr2基因表达量比野生型菌株高204倍。蛋白浓度及酶活测定的结果显示,诱导培养72 h,转化子胞外蛋白浓度和滤纸酶活分别是野生菌的1.58和1.30倍;非诱导培养144 h,转化子胞外蛋白浓度和滤纸酶活分别是野生菌的1.87和1.49倍。实时荧光定量PCR的结果表明,转化子中主要纤维素酶基因egl1、egl3和cbh1、cbh2的表达量均有显著提高。研究初步证实了mhr2基因具有调控纤维素酶基因表达的功能。     

Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstenedione respectively) or through 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.Analysis of transformation progress of studied substrates as function of time indicates that the 17β-side chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the enzyme catalyzing cleavage at 17β-acetyl chain was observed, whereas DHEA and androstenedione induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3β-alcohol was a more effective inducer that the respective 4-en-3-ketone.     

溶磷真菌ATMT转化条件优化及其对矮牵牛的促生效果评价

[目的]建立和优化一株溶磷真菌咖啡果小蠹青霉菌Penicillium brocae的转化体系,并利用分子标记观察其在根部的定殖;检测接种咖啡果小蠹青霉菌对植物的促生作用,为菌肥的研发奠定基础.[方法]利用农杆菌介导的转化体系(Agrobacterium tumefaciens-mediated transformati...     

Connection of Gnomonia intermedia to Discula betulina and its relationship to other taxa in Gnomoniaceae

Discula betulina is a foliar pathogen on birch (Betula) and Gnomonia intermedia is found on overwintered birch leaves. Perithecia of G. intermedia developed in vitro on colonies of D. betulina isolated from birch tissues in late summer, and single ascospores of G. intermedia consistently developed into colonies similar to D. betulina, producing typical D. betulina conidia. Isolates of D. betulina could be grouped into two mating types, which produced fertile perithecia of G. intermedia when mated with each other. Mycelia from single-ascospore and single-conidial isolates were inoculated onto shoots of downy birch, causing lesions and die-back from which D. betulina was consistently isolated. ITS region ribosomal DNA sequence analysis confirmed the results of the morphological and mating studies, and found that the closest known relatives of G. intermedia/D. betulina are Gnomoniella nana and Sirococcus clavigignenti-juglandacearum. The conclusion from these studies is that D. betulina is the anamorph of G. intermedia.     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Oxidation of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids by Acetobacter and Serratia strains

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110g 2-furoic acid l^-1 from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2 biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86--91% molar yields.Revisions requested 21 July 2004; Revisions received 7 September 2004     

An evolutionarily conserved nested gene pair — Mab21 and Lrba/Nbea in metazoan

The embedding of one gene in another as a nested gene pair is a unique phenomenon of gene clustering in the metazoan genome. A gene-centric paralogous genomic sequence comparison strategy was used in this study to align these paralogous nested pairs, Mab21l2-Lrba and Mab21l1-Nbea, to identify the associated paralogous non-coding elements (pNEs) they shared. A majority of these pNEs in the Mab21l2-Lrba locus display tissue-specific enhancer activities recapitulating the expression profiles of Mab21l2 and Mab21l1. Since these enhancers are spread into the introns of Lrba, dissociation of the two genes will likely disrupt the function of at least one of them. Phylogenetic analysis of this complex locus in different species suggests that Mab21 was probably locked in the Lrba/Nbea intron in the ancestral metazoan species, in which the cis-elements uncovered in this study may act as a selective force to prevent the dissociation of this gene pair in vertebrates.     

Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β- -glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] and β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β- -glucanase (cellulase), filter paper-degrading and β- -glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β- -glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β- -glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

转录因子PaPho与丝状真菌Podospora anserina中磷元素的吸收和利用研究

作为生物体必需的营养元素之一,磷在物质代谢、信号传导和能量储存中起着关键作用。【目的】研究丝状真菌Podospora anserina中调控磷酸盐代谢相关转录因子的作用,可进一步阐明真核微生物中磷元素吸收的调控机制。【方法】利用同源重组的方法定点敲除P.anserina中2个磷代谢相关转录因子PaPho1和PaPho2,遗传杂交构建双重突变体ΔPaPho1ΔPaPho2;通过表型分析、无机磷含量测定和酸性磷酸酶活性测定分析各突变菌株的变化;利用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)分析磷代谢相关基因的表达情况。【结果】在无机磷作为唯一磷来源的培养基上,ΔPaPho1ΔPaPho2无法生长;在添加有机磷的培养基中,ΔPaPho1ΔPaPho2和野生型菌株生长无显著性差异。在同时添加有机磷和无机磷的培养基中,ΔPaPho1ΔPaPho2的无机磷含量和酸性磷酸酶活性比野生型菌株的分别下降了25.0%和61.9%,ΔPaPho1ΔPaPho2中无机磷酸盐转运蛋白基因的表达水平显著降低。【结论】在P...     

嗜热丁烷氧化古菌Candidatus Syntrophoarchaeum烷基转移酶MtaA催化丁基辅酶M的分子机制研究

【背景】嗜热古菌Candidatus Syntrophoarchaeum可以与硫酸盐还原细菌共生,通过逆转产甲烷途径进行正丁烷的氧化,但在该过程中负责催化丁基辅酶M氧化的酶尚未确定。【目的】利用分子动力学模拟证明Ca.Syntrophoarchaeum中mta A基因编码的蛋白可以特异性催化丁基辅酶M中丁基的转移,并非转移甲基。【方法】使用Methanosarcina mazei辅酶M甲基转移酶Mta A的晶体结构(PDB ID:4ay8)作为模板,对Mta A_1 (Gen Bank登录号OFV65993.1)和Mta A_2 (Gen Bank登录号OFV65678.1)进行同源建模。使用分子对接得到两者分别结合CH_3-Co M和C_4H_9-Co M时的结构,并用AMBER18进行分子动力学模拟。【结果】当Mta A_1和Mta A_2分别结合C_4H_9-Co M时,表现出与4ay8晶体结构类似的TIM-Barrel折叠三维结构,但在活性中心形状、Zn~(2+)与底物距离以及活性位点附近氨基酸配位方式等方面存在差异,这可能是导致Ca.Syntrophoarchaeum中mta A基因编码的蛋白催化丁基辅酶M氧化的原因。其中Mta A_2与4ay8结构更相似,活性中心氨基酸配位更完整,暗示其更可能具备催化活性。然而当Mta A_1和Mta A_2分别结合CH_3-Co M时,整体结构不合实际,活性中心Zn~(2+)与底物距离过远,表明底物几乎不可能与酶结合。【结论】Ca.Syntrophoarchaeum中的Mta A_1和Mta A_2很可能是特异性的丁基转移酶,而非催化甲基的转移,其中Mta A_2具备活性的可能性更高。     

Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: Differential expression during the miracidium-to-sporocyst transformation

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail–schistosome interactions. Interestingly, Fucα2Fucα3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.     

Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov.

The genus Absidia comprises ubiquitously distributed soil fungi inhabiting different growth temperature optima ranging from 20–42 °C. Some of the mesophilic species are important biotechnologically in the biotransformation of steroids or as producers of rennin-like components, whereas species with higher growth temperature optima are of clinical relevance as opportunistic human pathogens. The aim of this study was to investigate the phylogenetic relationships between these species and to establish a revision of their systematics. For this purpose single and combined genealogies based on distance, MP, ML, and Bayesian analyses of aligned nucleotide sequences of the nuclear-encoded genes for actin (act) and for the 5.8S ribosomal RNA flanked by the ITS regions 1 and 2 (comprising 807 and 828 characters, respectively) of 16 Absidia species were reconstructed. The phylogenetic reconstructions suggest a trichotomy of the Absidia genus consisting of a mesophilic, a fast-growing thermotolerant, and a slowly-growing mycoparasitic Absidia group. The trichotomous phylogenetic grouping is concordant with the morphology of the zygospores, which are zygotes resulting from sexual conjugation between two compatible mating partners. Whereas the mesophilic group comprises the majority of absidiaceaeous species forming sterile hair-like, mycelial appendages on the suspensors of their zygospores, the thermotolerant group is characterised by the formation of smooth-walled zygospores, and the mycoparasitic group, namely Absidia parricida and A. zychae, by Mucor-like rough-walled zygospores. Based on the phylogenetic coherence of mesophilic and thermotolerant Absidia species, we propose that the two groups are separated into two distinct genera, Absidia for the mesophilic Absidia species resembling the Absidiaceae and Mycocladus for the thermotolerant species A. corymbifera, A. blakesleeana and A. hyalospora. Because Mycocladus is physiologically, phylogenetically, and morphologically distinct from the Absidiaceae s. str. we suggest that they are classified as a separate family, Mycocladiaceae fam. nov., which comprises the three species M. corymbifer, M. blakesleeanus and M. hyalospora.     

Detection of the nematophagous fungus Hirsutella rhossiliensis in soil by real-time PCR and parasitism bioassay

Hirsutella rhossiliensis, a nematophagous fungus, has shown potential in biocontrol of plant-parasitic nematodes. Monitoring the population dynamics of a biocontrol agent in soil requires comprehensive techniques and is essential to understand how it works. Bioassay based on the fungal parasitism on the juveniles of soybean cyst nematode, Heterodera glycines, can be used to evaluate the activity of the fungus but fails to quantify fungal biomass in soil. A real-time polymerase chain reaction (PCR) assay was developed to quantify the fungal population density in soil. The assay detected as little as 100 fg of fungal genomic DNA and 40 conidia g⁻¹ soil, respectively. The parasitism bioassay and the real-time PCR assay were carried out to investigate the presence, abundance and activity of H. rhossiliensis in soil after application of different inoculum levels. Both of the percentage of assay nematodes parasitized by H. rhossiliensis based on the parasitism bioassay and the DNA yield of the fungus quantified by real-time PCR increased significantly with the increase of the inoculum levels. The DNA yield of the fungus was positively correlated with the percentage of assay nematodes parasitized by H. rhossiliensis. The combination of the two is useful for monitoring fungal biomass and activity in soil.     

丝状真菌Podospora anserina中光敏色素基因的鉴定及功能分析

光敏色素在细菌和植物发育中起着关键作用,但它们在真菌中的生物学功能尚不完全清楚。【目的】探究光敏色素基因PaPhy1和PaPhy2在Podospora anserina有性生殖和无性发育中的作用及其调控机制。【方法】利用同源重组方法对P.anserina中2个光敏色素基因PaPhy1和PaPhy2进行定点敲除,获得光敏色素基因缺失菌株ΔPaPhy1和ΔPaPhy2,并通过遗传杂交构建双重突变体ΔPaPhy1ΔPaPhy2;分析突变型菌株和野生型菌株在不同光照下有性生殖、无性发育、生长速率和活性氧代谢等方面的差异,明确光敏色素基因在P.anserina中的主要功能。【结果】白光和蓝光诱导P.anserina子实体的形成,ΔPaPhy在光照下产生子实体的数量减少,ΔPaPhy的生命周期延长。【结论】光敏色素基因与P.anserina有性生殖密切相关;ΔPaPhy的衰老延迟和活性氧代谢有关。本研究的结果为进一步探索光照对丝状真菌繁殖调控机制以及抗衰老研究提供了新的思路。     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

The genus Setulipes (Marasmiaceae) in Madagascar and the Mascarenes, including a key to other African taxa

The authors report first records for the genus Setulipes in Madagascar, with the presence of Setulipes cf. hakgalensis and two new species, Setulipes funaliformis and S. moreaui, as well as a new species from Mauritius: S. mauritiensis. A key to these taxa, as well as to other African species, is supplied.