里氏木霉中纤维素酶的合成诱导及调控*

随着绿色化学的兴起,天然纤维素原料转化和利用的研究受到了高度重视和广泛应用。利用纤维素酶降解纤维素为燃料乙醇、生物柴油的生产铺设了道路。但纤维素酶的生产成本较高,限制了纤维素酶产业化应用。里氏木霉生产的纤维素酶组分丰富,是纤维素酶高产菌株,深入研究里氏木霉的纤维素酶诱导及表达调控机制,有助于提高其纤维素酶产率。近年来人们对里氏木霉的纤维素酶诱导过程和调控机制有了一定研究进展,综述了里氏木霉纤维素酶诱导和基因表达调控,首先介绍了纤维素、纤维二糖、槐糖、乳糖等几种诱导物及诱导物的转运蛋白,进一步综述了几种转录因子的调控作用,同时介绍了染色体调控、信号通路和光条件对纤维素酶诱导的影响。最后展望了未来里氏木霉纤维素酶诱导表达的研究方向,包括探明诱导物的本质及其具体过程、揭示转录因子之间的联系及转录调控网络、寻找信号转导关键功能蛋白及研究环境因素对纤维素酶的诱导作用等。     

草酸青霉纤维素酶基因和木聚糖酶基因的调控基因POX05145的鉴定

草酸青霉能产生完整的纤维素酶和木聚糖酶酶系,其纤维素酶基因的表达主要受转录因子的调控。前期工作中,通过对草酸青霉菌株HP7-1在不同碳源培养基培养条件下转录组的比较分析,获得了调控纤维素酶和木聚糖酶产量的候选调控基因集。本研究以草酸青霉ΔPoxKu70为出发菌株,通过同源重组法,构建并获得了其中一个候选调控基因POX05145的缺失突变株ΔPOX05145。在微结晶纤维素Avicel诱导培养条件下,与出发菌株ΔPoxKu70相比,ΔPOX05145的纤维素酶产量和木聚糖酶产量发生了显著改变。其中,在诱导第2天时,ΔPOX05145对硝基苯-β-D-纤维二糖苷酶产量和木聚糖酶产量分别上升43.4%和164.7%,对硝基苯-β-D-半乳糖吡喃葡萄糖苷酶产量下降92.8%,但是,滤纸酶产量和羧甲基纤维素酶产量没有显著变化。然而,在诱导第4天时,所有纤维素酶产量和木聚糖酶产量上升100.4%~294.0%。实时荧光定量PCR检测表明POX05145在不同的时间不同程度的调控主要的纤维素酶基因和木聚糖酶基因的表达。序列分析表明POX05145含有一个GAL4类锌指结构的DNA结合功能域和一个保守的真菌特有的转录因子结构域(Fungal_TF_MHR)。     

HrpNCSDS001基因克隆及其表达产物诱导拟南芥基因表达谱变化的研究

Erwinia carotovora subsp.carotovora CSDS001菌株具有可直接诱导烟草过敏反应特征,从构建的CSDS001菌株基因组文库,鉴定、克隆到hrpNCSDS001基因,GenBank登录号AY939927;构建的重组hrpNCSDS001基因工程菌株经IPTG诱导培养,获得的高效表达HarpinCSDS001蛋白,可诱导烟草发生过敏反应。30μg/mLHarpinCSDS001蛋白喷施拟南芥后,分析第3h、12h、24h、36h和48h拟南芥全基因谱表达动态变化,结果显示发生显著表达差异(logratio≤?1或≥1)的基因数分别为912、1787、2393、1833和1755。对被诱导发生显著表达差异的转录因子基因分析表明,有13个转录因子家族:ZIM、BES1、TCP、C2C2、AP2/EREBP、WRKY、bHLH、bZIP、GARP、MYB、NAC、HB、C2H2与HarpinCSDS001蛋白作用相关,这些转录因子家族主要参与调控植物抗性、光合作用、生长发育、开花等相关功能基因表达。     

球毛壳菌(Chaetomium globosum)NK102 纤维素酶基因及其表达条件

[目的]生物质的利用是当前生物技术研究的一个热点.本小组分离到一株高效降解纤维素球毛壳菌(Chaetomium globosum)NK102,本文拟探索研究此菌的纤维素酶表达系统并寻找影响酶基因表达的关键因素.[方法]通过对NK102测序,本文界定了球毛壳菌NK102的主要纤维素酶编码基因,使用数字基因表达谱升级版(RNA-Seq)的方法得到纤维素酶基因的表达差异,然后观察了营养、物理条件下纤维素酶基因表达和酶活性变化的情况.[结果]发现随着培养时间的延长,纤维素酶基因整体上表达量升高.在所选基因中,外切葡聚糖酶、纤维二糖脱氢酶和内切葡聚糖酶基因(cbh1,cdh和egl1)的表达量最高.糖代谢的负调控因子ACE I和CreA的随时间表达量均降低,而Hap2/3/5复合体的表达量反而升高.之后检测了不同碳源培养基对纤维素酶基因表达量和酶活性的影响,发现葡萄糖为强阻遏因子,纤维二糖为其诱导物,而山梨醇没有影响.特别是,我们发现光照也影响纤维素酶基因的表达,黑暗条件明显抑制酶基因的表达.[结论]转录组学的方法可以初步探索纤维素酶表达的规律,酶基因的表达受到营养、物理条件的影响.本研究为揭示球毛壳菌降解纤维素分子机理和阐释生物质糖代谢途径提供了有用参考.     

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、纤维素酶及半纤维素酶的表达水平等进行比较分析,并对DSR2蛋白进行了亚细胞定位。【结果】与对照菌株Cpyr4相比,△drs2菌株在葡萄糖、乳糖、微晶纤维素(avicel)等条件下生长速率都明显降低。△drs2菌株在纤维素诱导条件下的总蛋白分泌量、纤维素酶活力、半纤维素酶活力均显著提高,但drs2基因的缺失并不影响关键纤维素酶基因的转录水平。DRS2蛋白位于里氏木霉菌丝近顶端的顶体位置。【结论】在纤维素为唯一碳源的条件下,drs2基因的缺失导致纤维素酶的产量显著提高,drs2基因不调控纤维素酶基因的转录,而是在其分泌过程中发挥作用。     

人工锌指蛋白介导调控的里氏木霉纤维素酶生产

里氏木霉Trichoderma reesei Rut-C30是目前研究最广泛的纤维素酶生产菌,选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T.reeseiRut-C30,筛选获得了两株高产纤维素酶的突变株T. reesei M1和M2,与出发菌株比较,突变株M1和M2滤纸酶活分别提高100%和53%,且M1突变株外泌蛋白量提高69%,M2内切纤维素酶活提高64%。实时定量PCR分析结果表明,与对照菌株相比,突变株M1和M2中主要纤维素酶基因转录均上调,但不同酶基因在两株菌中有不同的变化特征。此外,纤维素酶抑制转录因子基因ace1在两株突变株中都转录下调,而纤维素酶正调控转录因子基因xyr1仅在M1突变株中上调。以上结果表明,不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析,为进一步深入探究里氏木霉纤维素酶合成调控的机理,以及利用代谢工程选育更高效的产酶菌株提供了基础。     

里氏木霉有性生殖的分子机理及其在遗传育种中的应用

将木质纤维素转化为可发酵糖用于生产生物燃料以及生物基化学品是实现碳中和的有效途径之一.木质纤维素降解酶在这一过程中发挥着重要作用.里氏木霉是应用最为广泛的纤维素酶、半纤维素酶工业生产菌株.长期以来,里氏木霉一直被认为是红褐肉座菌的无性型,只能进行无性繁殖,菌种改良以经典诱变、基因育种等为主.直到近些年才证实里氏木霉可以...     

CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

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。     

黄色黏细菌转录因子FruA对自身基因的表达不具自调节作用

 粘细菌是研究多细胞结构形态发生机制的良好模型.FruA是粘细菌发育所必需的一种 关键性转录因子, 调节一系列发育相关基因的表达,本文研究FruA对自身基因是否存在反馈调节从而导致发育后期fruA表达水平的下调.以野生型粘细菌模式菌株DK1622为基础构建fruA基因敲除突变株DK1622ΔfruA,再将fruA-lacZ转录融合载体pMF1A整合入fruA突变株染色体attB, 获得重组菌株DK1622ΔfruA/pMF1A,通过检测β-半乳糖苷酶活性来确认FruA对自身基因的表达水平是否有影响. 结果表明fruA调控序列完整的fruA-lacZ转录融合体β-半乳糖苷酶活性在DK1622/pMF1A和DK1622ΔfruA/pMF1A之间无明显差异, 即fruA表达产物作为一种转录因子对自身基因的转录没有调节作用,黏细菌发育后期fruA表达水平的下降存在其它调节机制.     

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.     

The Hypocrea jecorina gal10 (uridine 5'-diphosphate-glucose 4-epimerase-encoding) gene differs from yeast homologues in structure,genomic organization and expression

As part of a comprehensive study on lactose metabolism in Hypocrea jecorina (anamorph: Trichoderma reesei), a genomic clone of the gal10 gene encoding H. jecorina uridine 5'-diphosphate (UDP)-glucose 4-epimerase has been cloned and sequenced. It contains an open reading frame of 1548-base pair, interrupted by three introns, and encoding a 370-amino acids protein with similarity to pro- and eukaryotic UDP-glucose-4-epimerases. H. jecorina Gal10 does not contain the C-terminal mutarotase domain which is present in yeast Gal10 proteins but is able to functionally complement a corresponding Saccharomyces cerevisiae gal10 mutant. gal10 is not clustered with other H. jecorina gal genes (gal7, gene encoding galactose-1-phosphate uridylyltransferase and gal1, gene encoding galactokinase). The genomic location of H. jecorina gal10 and gal7 was syntenic with that in Neurospora crassa and colinear over an area of 6 and 3.5-kilobase. gal10 is constitutively expressed, and--unlike H. jecorina gal7--not further stimulated by D-galactose or L-arabinose or its corresponding polyols.     

The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1

In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H. jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.     

The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element

The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.     

Direct identification of hydrophobins and their processing in Trichoderma using intact-cell MALDI-TOF MS

Intact-cell MS (ICMS) was applied for the direct detection of hydrophobins in various species and strains of Hypocrea/Trichoderma. In both mycelia and spores, dominating peaks were identified as hydrophobins by detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis of knockout mutants, and comparison with protein databases. Strain-specific processing was observed in the case of Hypocrea jecorina (anamorph Trichoderma reesei). An analysis of 32 strains comprising 29 different species of Trichoderma and Hypocrea showed hydrophobin patterns that were specific at both at the species and isolate (subspecies) levels. The method therefore permits rapid and direct detection of hydrophobin class II compositions and may also provide a means to identify Trichoderma (and other fungal) species and strains from microgram amounts of biomass without prior cultivation.     

Three-dimensional structure of an intact glycoside hydrolase family 15 glucoamylase from Hypocrea jecorina

The three-dimensional structure of a complete Hypocrea jecorina glucoamylase has been determined at 1.8 A resolution. The presented structure model includes the catalytic and starch binding domains and traces the course of the 37-residue linker segment. While the structures of other fungal and yeast glucoamylase catalytic and starch binding domains have been determined separately, this is the first intact structure that allows visualization of the juxtaposition of the starch binding domain relative to the catalytic domain. The detailed interactions we see between the catalytic and starch binding domains are confirmed in a second independent structure determination of the enzyme in a second crystal form. This second structure model exhibits an identical conformation compared to the first structure model, which suggests that the H. jecorina glucoamylase structure we report is independent of crystal lattice contact restraints and represents the three-dimensional structure found in solution. The proposed starch binding regions for the starch binding domain are aligned with the catalytic domain in the three-dimensional structure in a manner that supports the hypothesis that the starch binding domain serves to target the glucoamylase at sites where the starch granular matrix is disrupted and where the enzyme might most effectively function.