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。     

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。     

里氏木霉不同糖苷水解酶基因转录水平比较分析

以前期里氏木霉RNA-seq中发现的7个糖苷水解酶基因为对象,分析其不同条件下的表达特性,以期为寻找新的纤维素降解功能酶提供证据。运用生物信息学方法,分析了7个基因可能的编码产物和结构特征。以不同的产纤维素酶菌株（QM 9414、RUT C30）为材料,采用实时荧光定量PCR,对7个糖苷水解酶基因（编号4–10）在各种碳源条件下转录情况与主要的3个纤维素酶基因cbh1,cbh2,egl1（编号1–3）进行了比较分析。信息学分析表明,7个基因编码蛋白分属于GH47（4号、5号）,GH92（6–8号）,GH16（9号）,GH31（10号）糖苷水解酶家族,具有典型的信号肽序列。cbh1,cbh2,egl1基因在纤维素酶诱导条件下,转录水平均表现显著的增加,上调倍数以QM 9414菌株表现的最高。QM 9414菌株中,cbh1,cbh2,egl1基因在纤维素条件下的上调倍数显著高于乳糖,3个基因在RUT C30菌株中的转录水平则显示乳糖条件下上调幅度更大。7个糖苷水解酶基因也存在类似的情况,而且编码α-甘露糖苷酶和内切β-葡聚糖酶的8号、9号基因上调倍数在纤维素酶诱导条件下仅次于纤维素酶基因,而以甘油为碳源条件下,8号、9号基因上调倍数高于纤维素酶基因。4号基因在上述碳源条件下,转录水平变化不大。结果表明：4号基因可能是组成型表达。基因5、6、7、8、9、10的表达呈现明显的菌株和碳源依赖性,且在纤维素酶诱导条件下基本上是和3个纤维素酶基因共转录的。     

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。     

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.     

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...     

里氏木霉绿色荧光蛋白表达载体的构建及功能鉴定

为了后续研究里氏木霉(Trichoderma reesei)纤维素酶基因的表达与调控,利用overlap PCR及分子克隆技术构建了含有Col E1原核复制起始位点、氨苄青霉素抗性、里氏木霉的丙酮酸脱羧酶启动子、丙酮酸脱羧酶终止子、潮霉素B抗性的筛选标记并能表达增强型绿色荧光蛋白(Zs Green)的表达载体p LXT-Zs Green。将该载体转化里氏木霉QM9414原生质细胞,使用潮霉素B筛选平板得到阳性转化子,随后使用荧光显微镜在488 nm激发光下观察菌丝,并随机挑取4个转化菌株进行Western-blot验证。结果显示,里氏木霉菌丝体可发出明亮的绿色荧光,而且Western-blot验证了该载体能够在里氏木霉中有效地表达增强型绿色荧光蛋白。上述研究表明,载体p LXT-Zs Green在里氏木霉中能够稳定高效地表达外源基因,为研究里氏木霉的基因表达调控奠定了实验基础。     

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.     

里氏木霉GH61家族糖苷酶的高效表达及酶学特性研究

【目的】GH61家族糖苷水解酶具有葡聚糖氧化酶活性,通过对葡聚糖链的随机氧化而破坏木质纤维素的结晶结构,从而使木质纤维素容易被纤维素酶降解。重组表达、纯化获得里氏木霉的GH61家族糖苷水解酶(TrGH61,原名为EGⅣ),并研究其在纤维素酶水解木质纤维素中的作用。【方法】通过Overlap PCR将里氏木霉丙酮酸脱羧酶的启动子、纤维二糖水解酶cbh1的信号肽、EGⅣ基因和PDC终止子依次连接构建了里氏木霉的表达盒,通过该表达盒使TrGH61蛋白基因整合到里氏木霉的基因组DNA上进行同源表达。研究表达产物TrGH61的水解活性、与纤维素酶水解协同效应,以及TrGH61作为金属氧化酶的特性研究。【结果】在PDC启动子的作用下,TrGH61得到高效表达,摇瓶培养的表达量达到2.33 g/L。TrGH61有微弱的内切葡萄糖苷酶活性,比活力为0.02 IU/mg,但能显著提高纤维素酶水解稻草粉的活性,协同度最高可达1.998。低浓度的金属离子Cu2+、Co2+和还原性电子供体还原型谷胱甘肽、L-抗坏血酸、焦性没食子酸均能显著促进其水解效应。TrGH61能够降低稻草粉纤维素聚合度和结晶度。【结论】通过PDC启动子可以实现TrGH61蛋白高效组成型表达,TrGH61作为纤维素酶活性促进因子,通过破坏纤维素结晶结构作用机制协同增强纤维素酶水解木质纤维素。     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Screening of highly cellulolytic fungi and the action of their cellulase enzyme systems

Over 100 strains of wood-rotting fungi were compared for their ability to degrade wood blocks. Some of these strains were then assayed for extracellular cellulase [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] activity using a variety of different solid media containing carboxymethyl cellulose or acid swollen cellulose. The diameter of clearing on these plates gave an approximate indication of the order of cellulase activities obtained from culture filtrates of these strains. Trichoderma strains grown on Vogels medium gave the highest cellulase yields. The cellulase enzyme production of T. reesei C30 and QM9414 was compared with that of eight other Trichoderma strains. Trichoderma strain E58 had comparable endoglucanase and filter paper activities with the mutant strains while the β- -glucosidase [β- -glucoside glucohydrolase, EC 3.2.1.21] activity was approximately six to nine times greater.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Action of Trichoderma reesei QM9414 and C30 cellulase on substrates with varying crystallinity

The action of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations from Trichoderma reesei QM9414 and C30 has been compared on Sigmacell, Solka Floc and alkali-treated bagasse in the presence and absence of added d-glucose and cellobiose. On the basis of equal filter paper activity the two preparations acted similarly on the two cellulosic substrates, while in the case of alkali-treated bagasse the C30 preparation gave greater d-glucose release. The relative levels of cellobiose produced from alkali-treated bagasse suggests that the non-cellobiose route was more important in d-glucose release by the C30 preparation compared to the QM9414 preparation.     

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.