东方肉座菌EU7-22纤维素酶基因的克隆及序列分析

东方肉座菌EU7-22与XC-9、里氏木霉、康宁木霉、黑曲霉、斜卧青霉进行产纤维素酶比较,结果表明菌株EU7-22具有较高的产纤维素酶能力及完整的纤维素酶系.根据里氏木霉和绿色木霉的外切葡聚糖酶,内切葡聚糖酶及β-葡萄糖苷酶相关基因序列,设计引物PCR扩增出菌株EU7-22 cbhⅠ、cbhⅡ、egⅠ、egⅡ及bgl Ⅰ.基因序列经NCBI Blast分析表明,cbhⅠ与绿色木霉cbh1基因(FJ871063)同源性最高达99％;cbhⅡ与康宁木霉cbh2基因(DQ504304)同源性最高达99％;eg Ⅰ与长枝木霉egl基因(GU144298)同源性最高达99％;egⅡ与绿色木霉eg2基因(EF602036)同源性最高达99％;bglⅠ与菌株Trichoderma sp.SSL bgl基因(FJ040193)同源性最高达100％.5种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高.对上述纤维素酶基因编码的相应蛋白的分子量、等电点、N-糖基化位点、信号肽序列进行分析;对纤维素结合区及糖基水解酶家族特征结构区进行了定位;用SWISS-Model模拟了酶蛋白的三级结构.     

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。     

长梗木霉纤维素酶基因的克隆及序列分析

从富含纤维素环境筛选获得一株纤维素降解菌株FU05,通过形态学特征及ITS序列分析确定其为长梗木霉(Trichoderma longibrachiatum)。PCR扩增获得该菌株的bgl2、cbh2和eg1。序列分析表明,这3种纤维素酶基因与GenBank上其他木霉同种纤维素酶基因具有较高同源性:bgl2基因与里氏木霉bgl2基因(AB003110)同源性达91%;cbh2基因与康宁木霉cbh2基因(DQ504304)同源性达99%;eg1基因与长梗木霉eg1基因(X60652)同源性达95%。3种纤维素酶基因编码的相应氨基酸序列与其他木霉纤维素酶的氨基酸序列相似性也非常高。对上述纤维素酶基因编码的相应蛋白进行PROSITE motif search,对其N端糖基化位点、纤维素结合区、糖基水解酶家族特征结构区等进行了定位。     

沉默里氏木霉组蛋白赖氨酸甲基转移酶基因对纤维素酶表达的影响

【背景】里氏木霉(Trichoderma reesei)是一种比其他真菌小很多的多细胞真核微生物,在工业上受到广泛应用,而里氏木霉QM9414是目前研究最多基因产纤维素酶丰富的突变菌株。【目的】构建里氏木霉中组蛋白赖氨酸甲基化酶(Histone Lysine Methyltransferase)基因hkmt的siRNA沉默载体和过表达载体来降低或者增强hkmt在里氏木霉QM9414中的表达量,以分析其对里氏木霉纤维素代谢的调控作用。【方法】根据里氏木霉hkmt序列设计siRNA沉默片段并用反转录的方法获得过表达hkmt片段。将沉默片段和过表达片段克隆至里氏木霉组成型表达载体中,构建沉默hkmt的载体和过表达hkmt的载体,并将其转化里氏木霉QM9414。通过荧光显微镜观察重组菌的菌丝生长情况,此外对各重组菌进行纤维素酶的滤纸酶活性(Filter Paper Enzyme Activity,FPA)和羧甲基纤维素钠酶活性(Carboxymethyl Cellulose Enzyme Activity,CMCA)的测试;利用荧光定量PCR的方法检测hkmt、纤维素酶基因cbh1、egl1及木聚糖酶激活因子xyr1的表达量变化。【结果】通过使用荧光显微镜观察,发现沉默、过表达hkmt重组菌的菌丝形态均与出发菌株无明显差异。荧光定量PCR测定结果表明,沉默载体和过表达载体可以分别沉默和促进hkmt的表达。沉默hkmt重组菌株中FPA和CMC酶活力相比出发菌平均升高2.5倍。此外,纤维素酶相关基因和激活因子在沉默hkmt重组菌中的表达量均有所增加,但是在过表达hkmt重组菌株中以上相应指标均呈现相反的趋势。【结论】组蛋白赖氨酸甲基转移酶基因表达产物负调控里氏木霉产纤维素酶基因的表达,这为提高里氏木霉产纤维素酶水平提供了参考,并为里氏木霉产纤维素酶的表观遗传调控研究提供了新的证据。     

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。     

里氏木霉组成型表达siRNA干扰cre1基因对纤维素酶表达的调控作用

使用组成型siRNA干扰载体对里氏木霉碳阻遏抑制因子CRE1进行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。根据里氏木霉cre1基因序列设计siRNA干扰片段。利用里氏木霉组成型表达载体将干扰片段分别构建至里氏木霉cre1干扰载体并将其转化里氏木霉QM9414。分别在48和144 h对各转化子进行纤维素酶酶活力测试(CMC酶活力测试和滤纸酶活力测试)及利用qPCR检测相关基因的表达。在诱导144 h时转化子的两种酶活力平均约比出发菌株高出1倍。qPCR检测cre1基因的表达结果表明,转化子的cre1表达量比出发菌株平均降低约50%,而ace1基因表达量变化不大。其他纤维素酶相关基因的表达水平也均高于出发菌株。通过组成型表达siRNA干扰里氏木霉cre1基因可以明显调控纤维素酶基因的表达,为研究纤维素酶的基因表达与调控提供参考。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

敲除里氏木霉hkmt基因可增强纤维素酶的酶活性和表达水平

表观遗传是不涉及DNA序列变化的可遗传变化,包括DNA甲基化、组蛋白修饰和miRNA调控等。在组蛋白甲基化修饰中,主要是组蛋白赖氨酸甲基转移酶（histone lysine methyltransferase,HKMT）参与调控。有文献报道,HKMT蛋白的催化核心为SET结构域,它具有促进或抑制基因表达的作用。在里氏木霉(Trichoderma reesei)中,HKMT对纤维素酶基因的表达调控的机制尚不明确。本文阐述了以里氏木霉为研究对象,利用Split-Maker技术构建了组蛋白赖氨酸甲基转移酶基因敲除表达盒,并转化了里氏木霉T. reesei QM9414。经PCR及Southern印迹验证正确后,显微镜观察到T.reesei Δhkmt菌株菌丝较长,分支较多。检测到突变体菌株连续7d滤纸酶活（filter paper enzyme activity,AFP）和羧甲基纤维素钠酶活 (carboxymethyl cellulose sodium enzyme activity,CMCA)。结果分别比野生型菌株高出5.00 IU·mL^-1、15.00 IU·mL^-1。利用RT-qPCR检测到突变菌株纤维素酶及其相关基因cbh1、egl1和xyr1的表达分别高出野生型4.51、3.87和2.51倍。通过对野生型菌株和突变菌株形态特征、纤维素酶酶活性、纤维素酶相关基因表达量的探索,为进一步研究里氏木霉表观遗传调控对纤维素酶表达的影响提供了新思路和实验资料。     

红色荧光蛋白在丝状真菌里氏木梅中的表达

目的：建立红色荧光蛋白在里氏木霉中的表达方法,为深入研究里氏木霉中纤维素酶的合成机理打下基础。方法：采用PCR方法分离了里氏木霉纤维二糖水解酶Ⅰ（CBHI）的启动子（Pcbh1）和终止序列（Tcbh1）,将这两个片段与红色荧光蛋白（DsRed）的基因连接,得到Pcbh1-DsRed-Tcbh1表达盒。用此表达盒和质粒pAN7—1对里氏木霉QM9414的原生质体进行共转化,并用含100μg/ml潮霉素B的选择性平板进行筛选。结果：经筛选得到20个抗性转化子,在乳糖的诱导下有5个转化子可以表达红色荧光蛋白。对插入片段进行了扩增和序列测定,结果表明DsRed通过同源重组整合到了转化子的基因组DNA上,并处于cbh1启动子的下游。结论：通过cbh1启动子可以实现红色荧光蛋白在里氏木霉细胞内的稳定表达。     

启动子cbh1的改造与里氏木霉通用质粒载体的构建

里氏木霉具有优异的表达及分泌异源蛋白的能力。本研究为了提高里氏木霉在工业生产中异源蛋白表达产量,对cbh1启动子中四处葡萄糖反馈抑制位点进行突变,并且成功构建两株定点插入型表达质粒pG与pH。在后续的报告基因红色荧光蛋白DsRed表达过程中,pG比pH表现出更优异的表达能力,并且异源蛋白基因被定点插入了cbh1基因位点而对该基因进行了敲除。pG质粒具有工业应用前景。     

High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain.     

绿色木霉纤维素酶CBHII基因的分子克隆

本文以噬菌体lambda EMBL3 DNA为载体,通过克隆绿色木酶(Trichoderma viride)高分子量基因组DNA的部分酶解片段,并将重组分子进行体外包装后侵染Escherichia.coli K802,由此构建了绿色木霉基因文库。以李氏木霉(Trichoderma reesei)纤维素酶CBHII基因的末端片段为探针,用轮迥噬菌斑原位杂交从文库中筛选出CBHII基因的阳性克隆5个,随机取其中3个克隆用上述探针作斑点杂交,结果进一步证明克隆了全长或近全长的绿色木霉CBHII基因,用李氏木霉CBHI基因的末端片段探针作斑点杂交,结果提示CBHI与CBHII基因的末端序列之间无同源性存在。从斑点杂交的阳性克隆中提取DNA,酶切鉴定插入片段的长度,并克隆于质粒pUC19,Southern杂交结果证明获得了含绿色木霉CBHII基因的重组质粒pCBHII-14。     

Novel cellulase profile of Trichoderma reesei strains constructed by cbh1 gene replacement with eg3 gene expression cassette

To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbhl gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transforments denoted as L 13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbhl promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.     

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).     

里氏木霉纤维二糖水解酶Ⅱ在毕赤酵母中的高效表达

本工作采用巴氏毕赤酵母Pichiapastoris表达系统进行了里氏木霉Trichodermareesei纤维二糖水解酶Ⅱ(CellobiohydrolaseII)的表达。用RT-PCR的方法从经稻草粉诱导的里氏木霉培养物中分离出纤维二糖水解酶Ⅱ的基因,将其插入到巴氏毕赤酵母的表达载体pPICZαA中,并使之处于α-因子信号肽序列的下游,得到重组质粒pPICZαA-cbh2。通过电穿孔的方法用线性化的pPICZαA-cbh2转化巴氏毕赤酵母GS115菌株,经过大量筛选后得到可以高效表达纤维二糖水解酶的毕赤酵母菌株P.pastorisCBHⅡ1。在甲醇诱导的条件下培养P.pastorisCBHⅡ1,培养液中的CMC活性可达到3.82U/mL,SDS-PAGE分析结果表明纤维二糖水解酶在P.pastorisCBHⅡ1中的表达量远远高于里氏木霉。对表达产物进行了LC-MS分析,结果表明所表达的蛋白为里氏木霉的纤维二糖水解酶。     

Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.     

ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression

We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Deltaace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Deltaace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.     

Novel Cellulase Profile of Trichoderma reesei Strains Constructed by cbh1 Gene Replacement with eg3 Gene Expression Cassette

The filamentous fungus Trichoderma reesei is consi-dered to be the most efficient cellulase producer, and hasa long history in the production of hydrolytic enzymes,which was widely used in the food and feed industriesand recently also used in the textile,…     

Genetic engineering of Trichoderma to produce strains with novel cellulase profiles.

Genetic engineering has been used to modify the proportion of different cellulases produced by a hypercellulolytic Trichoderma reesei mutant strain. A general expression vector, pAMH110, containing the promoter and terminator sequences of the strongly expressed main cellobiohydrolase 1 (cbh1) gene was used to overexpress a cDNA coding for EGI, the major endoglucanase (1,4,beta-D-glucan glucanohydrolase, EC 3.2.1.4). An in vitro modified cbh1 cDNA, incapable of coding for active enzyme, was used to inactivate the major cellobiohydrolase (1,4-beta-D-glucan cellobiohydrolase, EC 3.2.1.91) gene. In this way, new strains producing elevated amounts of the specific endoglucanase 1 (EGI) and/or lacking the major cellobiohydrolase (CBHI) were produced, and these have been further characterized.