Determining the environmental fate of a filamentous fungus, Trichoderma reesei, in laboratory-contained intact soil-core microcosms using competitive PCR and viability plating

Trichoderma spp. are used extensively in industry and are routinely disposed of in landfill sites as spent biomass from fermentation plants. However, little is known regarding the environmental fate of this biomass. We tracked the survival of T. reesei strain QM6A#4 (a derivative of strain QM6A marked with a recombinant construct) over a 6-month period in laboratory-contained, intact soil-core microcosms incubated in a growth chamber. Survival was tested in 3 different soils and the effect of a plant rhizosphere (bush lima beans, Phaseolus limensis) was investigated. Levels and viability of the fungus were determined, respectively, by quantitative competitive polymerase chain reaction analysis of total soil DNA extracts and dilution-plating of soil on a semiselective growth medium. Whereas chemically killed QM6A#4 became undetectable within 3 d, QM6A#4 added as a live inoculum decreased approximately 4- to approximately 160-fold over the first 1-3 months and then reached a steady state. After 4 months, soil cores were subjected to a 1.5-month simulated winter period, which did not significantly affect QM6A#4 levels. Throughout the experiment, QM6A#4 remained viable. These results indicate that, following release into the environment, live T. reesei will persist in soil for at least 2 seasons.     

敲除组蛋白去乙酰化酶基因hdac对里氏木霉纤维素酶表达的调控作用

[背景]里氏木霉(Trichoderma reesei)是木霉属中产纤维素酶最具代表性的真菌之一,表观遗传调控是不涉及DNA序列变化的可遗传变化,组蛋白去乙酰化是其中一种。组蛋白去乙酰化酶(histone deacetylase,HDAC)负责脱乙酰化,敲除去乙酰化酶基因可引起菌株孢子、菌丝及纤维素酶活性等的一系列改变。[目的]通过敲除里氏木霉组蛋白去乙酰化酶基因(histone deacetylase,hdac)建立了里氏木霉hdac缺失突变株(T.reesei△hdac),以研究对纤维素酶基因表达的调控作用。[方法]利用Split-Maker技术构建了组蛋白去乙酰化酶基因敲除表达盒,并转化了里氏木霉T.reesei QM9414。经PCR及Southern blotting验证正确后,对突变体T.reesei△hdac连续7 d检测滤纸酶活(filter paper activity,AFP)、羧甲基纤维素钠酶活(carboxymethyl cellulase activity,CMCA),利用RT-qPCR检测纤维素酶及其相关基因cbh1、egl1和xyr1的表达。[结果]突变体T.reesei△hdac两种酶活力均显著高于出发菌株,分别高出8.00、30.00 IU/mL。突变体T.reesei△hdac纤维素酶及其相关基因cbh1、egl1和xyr1的转录水平分别为出发菌株T.reesei QM9414的6.50、6.01和4.51倍。[结论]里氏木霉中纤维素酶的基因表达明显受到组蛋白去乙酰化酶基因(hdac)的调控,这为研究里氏木霉表观遗传调控对纤维素酶的影响提供了新的证据。     

Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei

Bioenergy, particularly biofuel, from lignocellulosic biomass has been considered as one of the most promising renewable and sustainable energies. The industrial productivity and efficiency of microbial lignocellulolytic enzymes for cellulosic biofuel applications are significantly affected by pH of culture condition. This study established and compared hydrolytic protein expression profiles of Trichoderma reesei QM6a, QM9414, RUT C30 and QM9414MG5 strains at different pH in cellulosic culture media. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of secretome of T. reesei cultured from pH 3.0-9.0 revealed significantly higher hydrolytic protein expressions at acidic pH. The Bray-Curtis similarity indices, clustering, and Shannon diversity index elucidated differences in protein secretion at different pHs in individuals and among the strains. This study demonstrated a comparative lignocellulolytic enzyme secretion profile of T. reesei and its mutants at different pHs and provides pH sensitive and resistance enzyme targets for industrial lignocellulose hydrolysis.     

Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccharomyces pombe class of the enzyme

Dolichol phosphate mannose (DPM) synthase activity, which is required in N:-glycosylation, O-mannosylation, and glycosylphosphatidylinositol membrane anchoring of protein, has been postulated to regulate the Trichoderma reesei secretory pathway. We have cloned a T.reesei cDNA that encodes a 243 amino acid protein whose amino acid sequence shows 67% and 65% identity, respectively, to the Schizosaccharomyces pombe and human DPM synthases, and which lacks the COOH-terminal hydrophobic domain characteristic of the Saccharomyces cerevisiae class of synthase. The Trichoderma dpm1 (Trdpm1) gene complements a lethal null mutation in the S.pombe dpm1(+) gene, but neither restores viability of a S.cerevisiae dpm1-disruptant nor complements the temperature-sensitivity of the S. cerevisiae dpm1-6 mutant. The T.reesei DPM synthase is therefore a member of the "human" class of enzyme. Overexpression of Trdpm1 in a dpm1(+)::his7/dpm1(+) S.pombe diploid resulted in a 4-fold increase in specific DPM synthase activity. However, neither the wild type T. reesei DPM synthase, nor a chimera consisting of this protein and the hydrophobic COOH terminus of the S.cerevisiae DPM synthase, complemented an S.cerevisiae dpm1 null mutant or gave active enzyme when expressed in E.coli. The level of the Trdpm1 mRNA in T.reesei QM9414 strain was dependent on the composition of the culture medium. Expression levels of Trdpm1 were directly correlated with the protein secretory capacity of the fungus.     

Reidentification of cellulolytic enzyme-producing Trichoderma strains W-10 and G-39

Strain W-10, originally identified as Trichoderma koningii, and its supposed mutant G-39, published for production and gene expression of cellulase and xylanase, demonstrated morphological characteristics distinct from those of T. koningii, respectively. To clarify the identification derived from morphological characteristics, several methods were used, including electrophoretic karyotyping, internal transcribed spacer (ITS) analysis of rDNA, and polymerase chain reaction (PCR) fingerprinting using the universal primer L45. All the molecular characteristics showed that strains G-39 and W-10 were identical to T. reesei and T. longibrachiatum, respectively. The results strongly supported that T. koningii G-39 and W-10 should be reassigned as T. reesei and T. longibrachiatum, respectively. Strain G-39 should be considered a mutant from T. reesei QM9414 whose spores were contaminated with those of strain W-10 during a laboratory operation. According to this, we declare that T. koningii G-39 and W-10 must be renamed as T. reesei and T. longibrachiatum, respectively.     

Improvement of cellulose-degrading ability of a yeast strain displaying Trichoderma reesei endoglucanase II by recombination of cellulose-binding domains

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose.     

Increased endoplasmic reticulum content of a mutant of Trichoderma reesei (RUT-C30) in relation to cellulase synthesis

The structure and content of endoplasmic reticulum (ER) were studied in the shake cultures of two strains of Trichoderma reesei, one wild type (QM6a) and the other a cellulase (EC 3.2.1.4) hyper-producing and catabolite repression resistant mutant (RUT-C30). The results of quantitative electron microscopic and biochemical assays were correlated. At the stage of growth when cellulase secretion was high the ER in RUT-C30 cells was highly developed and more abundant than the ER in QM6a cells. A process regulating ER biogenesis may have been deactivated by mutation in RUT-C30 cells and thus the potential for extracellular enzyme synthesis and secretion has been enhanced.     

Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions

Response surface methodology (RSM) was used to evaluate the effects of fermentation parameters for cellulase production by Trichoderma reesei QM9414 and T. reesei MCG77 in solid-state fermentation using rice bran as substrate. Initial pH, moisture content and temperature were optimized using filter paper activity (FPA) as response. Statistical analysis of the results for T. reesei QM9414 showed that only moisture content had significant effect on cellulase activity and had a linear effect on enzyme activity (maximum enzyme activities were obtained at 70% moisture content). The results for T. reesei MCG77 showed that temperature and moisture content were the most significant parameters for cellulase activity. The optimum cellulase production was in the temperature range of 25-30 degrees C and moisture content between 55% and 70%. After the optimization, the FPA in T. reesei MCG77 was increased by 2.5 folds compared to that of T. reesei QM9414.     

A high cellulase-producing mutant strain of Trichoderma reesei

A mutant strain with increased production of cellulolytic enzymes was induced from the good cellulase producer Trichoderma reesei QM 9414. Cellulase activities of the mutant in fermenter cultivations were increased two- to three-fold and β-glucosidase activity up to six-fold when compared to the corresponding activities produced by QM 9414.     

Mutants of Trichoderma reesei resistant to 2-deoxy-d-glucose with altered sugar transport

Abstract Transport mutants resistant to 2-deoxy- d -glucose were isolated by UV-mutagenesis from a double auxotrophic strain of Trichoderma reesei QM9414. It was proved by protoplast fusion that they were altered at the same locus. The mutations were recessive and resulted in a defect of a general sugar transporting system with wide substrate specificity.     

用Overlap-PCR法从Trichoderma reesei QM9414基因组DNA中克隆并表达木聚糖酶Ⅲ

禾本科植物木聚糖酶在其成熟过程中需在细胞进入程序性死亡后,经蛋白水解酶多次剪切方显活性,常规蛋白质克隆表达系统无法表达这类酶。通过GenBank搜索获得一与之同族、结构相似的来源于T．reesei QM9414(ATCC26921)突变种PC—3—7菌株的xynⅢ。但该酶在T．reesei QM9414中不表达,而在基因组中存在。通过overlap-PCR法将4个外显子分别克隆、测序,再连接测序,最终获得该基因的全长cDNA序列。将该基因连接到表达载体pETBlue—2上,并转化到Tuner DE3表达菌株中,常规条件下可表达并有木聚糖酶活性显示。低温(15℃)64h诱导显著提高可溶性xynⅢ酶活。     

Chromosomal inversion discovered in C3H/HeJ mice

Mice of the inbred mouse strain C3H/HeJ have been shown to be homozygous for a chromosomal inversion on Chromosome (Chr) 6. The inversion encompasses about 20% of the chromosome from approximately 73 Mb to approximately 116 Mb. The importance of this finding is that linkage crosses using C3H/HeJ will show no recombination in this region of Chr 6. The inversion has no apparent effect on the phenotype of C3H/HeJ mice and its presence should not affect biological studies; however, use of C3H/HeJ mice for genetic analysis of Chr 6 should be avoided or the results interpreted with the inversion in mind. The inversion has been named In(6)1J (inversion Chr 6, Jackson 1).     

Construction of cellulase hyperproducing strains derived from polyploids of Trichoderma reesei

The mycelial mat of Trichoderma reesei strain QM 6a was treated with 0.1% (w/v) colchicine solution for 14 days and designated M14. The cellulase productivity of strain M14 was not much higher than that of the original strain. When conidia of M14 were treated with ethylmethane sulphonate (EMS) solution, the cellulase hyperproducers, M14-1 and M14-2, were isolated using a selection medium containing Avicel. The DNA content of M14-1 and M14-2 was higher than that of the original strain. Cellulase productivity per mycelium of these strains increased and was higher than that of the original strain. The cellulase productivity did not change through ten generations when these strains were cultivated successively on a medium containing Avicel. It was concluded that cellulase hyperproducers, whose cellulase productivity per mycelium increased, could be obtained when the conidia of strain M14 were treated with EMS.     

Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression

Toward a better understanding of the biochemical events that lead to biocontrol of plant pathogenic fungi by Hypocrea/Trichoderma spp., we investigated the importance of carbon catabolite (de)repression and cellulase formation in the antagonization of Pythium ultimum by Hypocrea jecorina (Trichoderma reesei) on agar plates and in planta. Hypocrea jecorina QM9414 could antagonize and overgrow P. ultimum but not Rhizoctonia solani in plate confrontation tests, and provided significant protection of zucchini plants against P. ultimum blight in planta. A carbon catabolite derepressed cre1 mutant of H. jecorina antagonized P. ultimum on plates more actively and increased the survival rates of P. ultimum-inoculated zucchini plants in comparison with strain QM9414. A H. jecorina mutant impaired in cellulase induction could also antagonize P. ultimum on plates and provided the same level of protection of zucchini plants against P. ultimum as strain QM9414 did. We conclude that cellulase formation is dispensable for biocontrol of P. ultimum, whereas carbon catabolite derepression increases the antagonistic ability by apparently acting on other target genes.     

里氏木霉及其纤维素酶高产菌株的研究进展

随着纤维素在能源、材料及化工等领域的广泛开发和应用,里氏木霉作为一种重要的产纤维素酶工业用菌种,越来越受到人们的广泛关注.为了提高其酶活,人们做了大量的工作,获得了一些相当好的突变株.对里氏木霉及其突变株的基因组进行研究,有助于人们理解其高效产酶的机制,同时也有利于构建其基因工程菌.介绍里氏木霉Trichoderma reesei 的背景及其部分高产纤维素酶突变株,并阐述近些年来对其突变株的基因组的研究进展.     

Production of cellulase on sugar cane bagasse by fungal mixed culture solid substrate fermentation

Trichoderma reesei LM-UC4 and its mutant LM-UC4E1 were co-cultured with Aspergillus phoenicis QM329 for cellu-lase production on bagasse by mixed culture solid substrate fermentation. A mutual synergism was observed between the parent Trichoderma strain and the Aspergillus, resulting in enhanced combined biomass production and corresponding increased in cellulase, endoglucanase and b-glucosidase activities. Such synergism was absent with the mutant Trichoderma strain suggesting that in the hypermutation the ability for cooperative interaction with other microbes was lost.     

瑞氏木霉表达黑曲霉葡萄糖氧化酶

利用高表达分泌纤维素酶的真菌瑞氏木霉表达重组的黑曲霉葡萄糖氧化酶。在大肠杆菌DH5α中构建瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子pUC19(命名为pCBHGOD)质粒,线性化后用瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子(命名为CBHGOD)核酸片段转化瑞氏木霉QM9414原生质体。用PCR扩增方法筛选出同源重组葡萄糖氧化酶基因的瑞士木霉突变株。用麦杆诱导瑞氏木霉突变株,生产黑曲霉葡萄糖氧化酶,Westernblot分析重组的葡萄糖氧化酶分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶一致,生产的重组酶活性25umL,相当于Sigma公司葡萄糖氧化酶标准品的产量为0.5gL。瑞氏木霉可用于生产黑曲霉葡萄糖氧化酶。