Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2

The Trichoderma reesei xln2 gene coding for the pI 9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed by targeting a construct composed of the xln2 gene, including its promoter, to the endogenous cbh1 locus. Highest overall production levels of xylanase were obtained using T. reesei ALK02721, a genetically engineered strain, as a host. Integration into the cbh1 locus was not required for enhanced expression under control of the xln2 promoter.     

SARS-CoV-2中和纳米抗体在里氏木霉中的重组表达北大核心CSCD

基于骆驼科动物单链抗体VHH结构域的纳米抗体具有分子量小、结构简单、溶解性好、稳定性强等多种优势,可通过吸入给药,在呼吸道病毒的防控中具有重要应用价值。里氏木霉是食品级的蛋白质生产宿主,其纤维素酶分泌量可达到80 g/L以上,有望用于药物蛋白的低成本生产。文中在密码子优化的基础上,使用组成型强启动子Pcdna1,实现了SARS-CoV-2中和纳米抗体Nb20在里氏木霉中的重组表达。将Nb20与里氏木霉纤维二糖水解酶CBHⅠ的N端片段融合表达,并在二者间引入胞内KEX2蛋白酶切位点,于葡萄糖培养基中摇瓶发酵48 h可生产出浓度为47.4 mg/L的Nb20蛋白。重组表达的纳米抗体能够与SARS-CoV-2刺突蛋白的受体结合区相结合,有望用于新型冠状病毒的中和。以上结果显示,里氏木霉在纳米抗体的重组表达中具有一定的应用潜力。     

Enzymatic hydrolysis and simultaneous saccharification and fermentation of steam-pretreated spruce using crude Trichoderma reesei and Trichoderma atroviride enzymes

The aim of this study was to compare the performance of the enzymes produced by Trichoderma reesei Rut C30 and the good extracellular β-glucosidase-producing mutant Trichoderma atroviride TUB F-1663 to that of commercial preparations in the enzymatic hydrolysis and the simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce (SPS).The concentrated TUB F-1663 enzyme was found to be the most efficient in the hydrolysis of washed SPS at 50 g/L water-insoluble solids (WIS) in terms of the glucose produced (18.5 g/L), even in comparison with commercial cellulases (14.1–16.7 g/L). The enzyme preparations were studied at low enzyme loadings (5 FPU/g WIS) in SSF to produce ethanol from SPS. The enzyme supernatant and whole fermentation broth of T. atroviride as well as the whole broth of T. reesei proved to be as efficient in SSF as the commercial cellulase mixtures (ethanol yields of 61–76% of the theoretical were achieved), while low ethanol yields (<40%) were obtained with the β-glucosidase-deficient T. reesei supernatant.Therefore, it seems, that instead of using commercial cellulases, the TUB F-1663 enzymes and the whole broth of Rut C30 may be produced on-site, using a process stream as carbon source, and employed directly in the biomass-to-bioethanol process.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

里氏木霉VPS13基因缺失对菌丝分支、生孢和纤维素酶产量的影响

【目的】丝状真菌里氏木霉是纤维素酶生产的主要工业真菌。纤维素酶分泌过程中的蛋白运输途径是控制大量纤维素酶成功输出的重要环节,因此,研究蛋白分泌途径的特定靶标基因功能将有助于鉴定纤维素酶运输分泌过程的关键调控因子。本研究借助基因敲除方法将里氏木霉液泡蛋白分选相关基因VPS13缺失,分析了该基因缺失对菌株生长、生孢尤其是纤维素酶分泌的影响。【方法】利用Double-joint PCR技术和同源重组策略构建里氏木霉VPS13基因缺失突变株,通过菌丝培养、显微观察、生孢检测、蛋白与酶活测定,系统比较VPS13基因敲除前后菌株的生长特征、菌丝形态、孢子形成、蛋白分泌以及纤维素酶活等。【结果】成功获得两株VPS13基因缺失株。与出发菌株相比,该基因突变后菌丝蔓延速率明显减慢,但菌体生物量在对数生长期后显著增多。通过显微观察,发现该基因缺失株菌丝更加密集,分支明显增多。此外,该基因缺失也导致菌株生孢延迟。纤维素底物平板分析发现VPS13基因缺失株菌落周围透明圈更加清晰,且透明圈圈径比是出发菌株的4倍,说明降解纤维素的能力有明显提高。进一步的液体发酵实验结果显示,该基因缺失导致蛋白产量及纤维素酶活力分别提高16.4%和21.9%。【结论】里氏木霉VPS13基因在菌丝生长、生孢、蛋白分泌等不同生物学过程中具有功能多样性,且该基因在菌种改良上可以作为提高纤维素酶产量的重要靶点。     

丝状真菌里氏木霉中shRNA沉默红色荧光基因

报道了在里氏木霉中建立的一种以红色荧光蛋白(DsRed)为报告基因的RNA干扰方法。首先,将构建的表达DsRed质粒p ANRed1转化里氏木霉QM9414,得到抗潮霉素B抗性并能稳定表达DsRed的菌株DsRed-T.reesei。其次,以丙酮酸脱氢酶启动子Ppdc和纤维二糖水解酶I终止子cbh I为原件,克隆到载体p PHL上构建质粒p PHL-Ppdc-Tcbh1。根据DsRed基因序列设计特定的siRNA干扰序列和另一条无同源序列的siRNA作为阴性对照,克隆到载体p PHL-Ppdc-Tcbh1得到重组质粒。将其转化到DsRed-T.reesei中,用含有100μg/m L潮霉素B和250μg/m L腐草霉素的PDA平板筛选转化子。结果表明,约79%的转化子出现红色荧光沉默现象,其中一些转化子DsRed的表达几乎完全被抑制。荧光定量PCR和Western印迹分析显示DsRed基因的表达受到不同程度的下调。以上结果提示,在里氏木霉中可用此方法研究基因表达调控。     

里氏木霉翻转酶DRS2对木质纤维素降解酶基因表达及分泌的影响

[目的]本研究以工业生产菌株里氏木霉为研究对象,鉴定翻转酶基因drs2对其纤维素酶表达及分泌的影响.[方法]首先通过BLAST序列比对,从里氏木霉中鉴定出翻转酶基因drs2,并通过同源重组的方法在里氏木霉中构建了drs2基因的敲除菌株△drs2.对△drs2菌株及其对照株在不同碳源上的生长发育、蛋白分泌、纤维素酶及半纤...     

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS^® HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.     

绿色木霉组蛋白去乙酰化酶编码基因TvRpd3在木霉生物防治作用中的功能分析

【目的】本文借助基因编辑技术在具有生物防治潜力的绿色木霉(Trichoderma viride)中敲除组蛋白去乙酰化酶编码基因TvRpd3,来研究TvRpd3基因及其编码蛋白在提高木霉病原菌拮抗能力中的作用。【方法】利用融合PCR和同源重组策略构建了TvRpd3基因缺失的突变菌株,通过对峙培养、表型观察、免疫组化检测、代谢组学分析等系统比较TvRpd3基因敲除前后菌株的组蛋白乙酰化修饰水平、次级代谢产物合成、病原菌拮抗能力以及田间防治效果等。【结果】与野生型菌株相比,缺失TvRpd3基因的木霉工程菌(?TvRpd3)对多种病原菌表现出了更强的对峙抑制效果,其所产的发酵液对小麦白粉病、烟草黑胫病和番茄枯萎病的防治效果分别提高了62.27%、57.45%和70.71%。同时,敲除TvRpd3基因也显著改变了木霉工程菌所产次级代谢产物的种类和产量,抗生性物质的产量大幅提高。【结论】绿色木霉TvRpd3基因及其介导的组蛋白乙酰化修饰在提高绿色木霉生物防治中起着重要作用。     

Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD)

Summary An Aspergillus nidulans gene library was constructed in a high-frequency transformation vector, pDJB3, based on the Neurospora crassa pyr4 gene. This gene library was used to isolate the structural gene for isocitrate lyase (acuD) by complementation of a deficiency mutation following transformation of A. nidulans. Plasmids rescued in Escherichia coli were able to transform five different A. nidulans acuD mutants. Transformation using plasmids containing the cloned fragment resulted in integration at the acuD locus in six of nine transformants.     

High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37–63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.     

In vitro evaluation of combination of Trichoderma harzianum and chitosan for the control of sapstain fungi

In vitro assays were undertaken to evaluate the control of two sapstain fungi, Leptographium procerum and Sphaeropsis sapinea by a combination of chitosan or chitosan oligomer and an albino strain of Trichoderma harzianum. Spore germination and hyphal growth of the test fungi were assessed on media amended with chitosan or chitosan oligomer with and without T. harzianum using either simultaneous inoculation with test fungus or inoculation 1, 2, or 3 days after pre-infection with test fungus.There was no mycelial growth of the test fungi regardless of chitosan concentrations used when either L. procerum or S. sapinea was simultaneously inoculated with T. harzianum. However, the dose–response of chitosan or chitosan oligomer on the test fungi was apparent when T. harzianum was not simultaneously inoculated with test fungus but introduced later. There was a greater growth reduction at higher concentrations (0.075–0.1% v/v) of chitosan, and overall chitosan oligomer was more effective than chitosan aqueous solution.Chitosan alone was able to restrict or delay the germination of spores but the combination of chitosan and T. harzianum inhibited spore germination and hence colony formation of test fungi regardless of time delay.     

Characterization of CaGSP1, the Candida albicans RAN/GSP1 homologue

Gsp1p is a small nuclear-located GTP binding protein from the yeast Saccharomyces cerevisiae. It is highly conserved among eucaryotic cells and is involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules. To learn more about the GSP1 structure/function, we have characterized its Candida albicans homologue. CaGsp1p is 214 amino acids long and displays 91% identity to the ScGsp1p. There is functional complementation in S. cerevisiae, and its mRNA is constitutively expressed in the diploid C. albicans grown under various physiological conditions. Disruption of both alleles was not possible, suggesting that it could be an essential gene, but heterozygous mutants exhibited genomic instability.     

多靶向沉默里氏木霉碳代谢阻遏物对纤维素酶活性和表达的调控研究

【目的】构建多靶向siRNA表达载体对里氏木霉碳阻遏抑制因子CRE1、CRE2、CRE3和CRE4进行同时多靶向siRNA干扰,以研究其对里氏木霉纤维素酶基因表达的调控作用。【方法】根据此前研究筛选出沉默cre1、cre2、cre3和cre4基因的4个最佳siRNA序列,设计并构建了A多靶向表达载体,另根据cre1、cre2、cre3和cre4基因中所含有的5个共有序列设计并构建了B多靶向表达载体,将两者转化至里氏木霉QM9414。经筛选后分别在48 h和120 h对各转化子进行纤维素酶酶活力测试(CMC活力测试和滤纸酶酶活力测试)及利用qPCR检测相关基因的表达。【结果】通过RT-qPCR测定结果表明,两种表达载体均可同时抑制里氏木霉的分解代谢物阻遏基因cre1、cre2、cre3和cre4的表达,纤维素酶活力比出发菌株明显升高,多靶向抑制菌株的CMC酶活和滤纸酶活比出发菌株平均提高了1.95倍和2.66倍。纤维素酶基因cbh1和egl1的表达水平比出发菌株也有明显提升,平均提高了3.83倍和3.95倍。纤维素酶相关基因xyr1的表达水平与出发菌株相比也明显上升,平均提高了2.78倍。【结论】多靶向沉默里氏木霉的碳代谢阻遏蛋白有利于解除葡萄糖效应,提高非还原糖的利用,从而提高纤维素酶的产量,使纤维素酶的表达得到更大的提升,为里氏木霉表达纤维素酶在分解代谢物阻遏基因调控方面提供了实验依据和新的技术思路。     

敲除aceE基因对猪链球菌丙酮酸代谢的影响

【目的】探究缺失编码丙酮酸脱氢酶蛋白的aceE基因对猪链球菌生长特性、三羧酸循环和丙酮酸代谢的影响。【方法】通过测量菌液的OD600值,绘制野生型菌株与aceE基因缺失突变株的生长曲线;利用试剂盒测定三羧酸循环和丙酮酸代谢旁路中乙酰CoA、琥珀酸CoA、延胡索酸、草酰乙酸、丙酮酸、乳酸和ATP的含量,通过荧光定量qRT-PCR确定柠檬酸合酶基因、苹果酸脱氢酶基因、琥珀酸脱氢酶基因、异柠檬酸脱氢酶基因、丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因的表达水平。【结果】与野生株相比,菌株ΔaceE在平台期OD600值下降;添加1g/L乙酸盐能够显著提升菌株ΔaceE平台期OD600值。菌株ΔaceE的丙酮酸含量上升,ATP含量下降;三羧酸循环代谢中乙酰CoA、琥珀酸CoA、延胡索酸含量降低;柠檬酸合酶基因和苹果酸脱氢酶基因表达水平上升,琥珀酸脱氢酶基因和异柠檬酸脱氢酶基因表达水平下调;在丙酮酸代谢旁路中丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因表达水平上升。【结论】结果显示,菌株ΔaceE三羧酸循环活性降低,虽然能够通过PDH旁路将部分丙酮酸分解为乙...     

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.     

里氏木霉Set2通过调控转录因子Ypr1的表达进而控制sorbicillinoids的生物合成

【目的】Sorbicillinoids是里氏木霉合成的一类重要的天然活性物质,具有抗肿瘤、抗氧化及抗病毒等多种生物活性。本研究主要是为了阐明TrSet2在里氏木霉sorbicillinoids合成调控过程中的生物学功能及其作用机制。【方法】基于生物信息分析技术鉴定里氏木霉TrSet2编码基因。采用基因敲除和过表达手段,分别构建Trset2基因敲除和过表达菌株并评估其sorbicillinoids合成能力。同时在Trset2基因敲除菌株中,过表达转录激活因子Ypr1,明确TrSet2和Ypr1之间的调控关系。【结果】Trset2基因敲除菌株完全丧失了合成sorbicillinoids的能力。相反,过表达Trset2导致sorbicillinoids合成的水平显著增加。进一步研究发现,在Trset2基因敲除菌株中过表达转录激活因子Ypr1逆转了其不能合成sorbicillinoids的表型。【结论】本研究明确了TrSet2在里氏木霉合成sorbicillinoids过程中的正向调控作用,其作用机制是通过控制转录因子Ypr1的表达水平实现的。这为基于调控机理控制里氏木霉发酵过程中sorbic...     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.