Trichoderma reesei acetyl esterase catalyzes transesterification in water

Partially purified Trichoderma reesei RUT-C30 acetyl esterase preparation was found to catalyze acyl transfer reactions in organic solvents, mixtures of organic solvents with water and even in water. Using different acyl donors, the best results for acetyl transfer in water were obtained using vinyl acetate. As acetyl acceptors, a variety of hydroxyl bearing compounds in aqueous solutions were used. Degree of conversion and the number of newly formed acetates varied according to the acceptor used. Conversions over 50% were observed for the majority of several common monosaccharides, their methyl and deoxy derivatives and oligosaccharides. In several cases, the transesterification reaction exhibited strict regioselectivity, leading to only one acetyl derivative. Preparative potential of the transesterification in water was demonstrated by acetylation of methyl β- -glucopyranoside, 4-nitrophenyl β- -glucopyranoside and kojic acid, yielding 56.4% of methyl 3-O-acetyl β- -glucopyranoside, 70.2% of 4-nitrophenyl 3-O-acetyl β- -glucopyranoside and 30.9% of 7-O-acetyl-kojic acid as the only reaction products.

This enzymatically catalyzed transacetylation in water, which is applied to transformation of saccharides for the first time, opens a new area in chemoenzymatic synthesis. Its major advantages are simplicity, highly regioselective esterification of polar compounds, high yields, low enzyme consumption and elimination of the need to use toxic organic solvents.     

里氏木霉产纤维素酶研究进展

木质纤维素类生物质被认为是重要且可持续的可再生能源,其主要组成部分是纤维素.纤维素酶是一种能将纤维素分解为葡萄糖的复合酶,能有效地降解木质纤维素生物质.真菌、细菌、放线菌、酵母等多种微生物均可以产生纤维素酶,其中里氏木霉具有完整的纤维素酶系结构,常作为生物技术领域中一个重要菌株,广泛应用于纤维素酶的商业生产.介绍了纤维...     

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。     

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。     

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。     

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.     

Action of acetylxylan esterase from Trichoderma reesei on acetylated methyl glycosides

Substrate specificity of purified acetylxylan esterase (AcXE) from Trichoderma reesei was investigated on partially and fully acetylated methyl glycopyranosides. Methyl 2,3,4-tri-O-acetyl-β-

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-xylopyranoside was deacetylated at positions 2 and 3, yielding methyl 4-O-acetyl-β-

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-xylopyranoside in almost 90% yield. Methyl 2,3-di-O-acetyl β-

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-xylopyranoside was deacetylated at a rate similar to the fully acetylated derivative. The other two diacetates (2,4- and 3,4-), which have a free hydroxyl group at either position 3 or 2, were deacetylated one order of magnitude more rapidly. Thus the second acetyl group is rapidly released from position 3 or 2 after the first acetyl group is removed from position 2 or 3. The results strongly imply that in degradation of partially acetylated β-1,4-linked xylans, the enzyme deacetylates monoacetylated xylopyranosyl residues more readily than di-O-acetylated residues. The T. reesei AcXE attacked acetylated methyl β-

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-glucopyranosides and β-

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-mannopyranosides in a manner similar to the xylopyranosides.     

Novel inducers derived from starch for cellulase production by Trichoderma reesei

Soluble inducers derived from starch were investigated for cellulase production by T. reesei. Various methods of starch treatment were compared. Acid-hydrolysed starch was found to be most effective. When 1·0 g starch was employed for cellulase production, the cellulase so produced after 6 days of fermentation, with supplementation of 1% (0·01 ml/g paper) β-glucosidase, saccharified more than 15 g shredded waste office paper in 9·34% suspension, resulting in 10 g fermentable sugars, 72% of which was glucose. The effectiveness of the starch-derived inducers was compared with that of lactose, pure cellulose, CMC, xylan and prehydrolysate of pine wood. The effects of calcium chloride and sorbose addition on cellulase production, and the kinetics of cellulase production by the starch-derived inducers are presented.     

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.     

Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency

Three DNA fragments, trs1, 2 and 3, were isolated from the Trichoderma reesei genome on the basis of their ability to promote autonomous replication of plasmids in Saccharomyces cerevisiae. Each trs element bound specifically to the isolated T. reesei nuclear matrix in vitro, and two of them bound in vivo, indicating that they are matrix attachment regions (MARs). A similar sequence previously isolated from Aspergillus nidulans (ans1) was also shown to bind specifically to the T. reesei nuclear matrix in vitro. The T. reesei MARs are AT-rich sequences containing 70%, 86% and 73% A+T over 2.9, 0.8 and 3.7 kb, respectively for trs1, 2 and 3. They exhibited no significant sequence homology, but were shown to contain a number of sequence motifs that occur frequently in many MARs identified in other eukaryotes. However, these motifs occurred as frequently in the trs elements as in randomly generated sequences with the same A+T content. trs1 and 3 were shown to be present as single copies in the T. reesei genome. The presence of the trs elements in transforming plasmids enhanced the frequency of integrative transformation of T. reesei up to five fold over plasmids without a trs. No evidence was obtained to suggest that the trs elements promoted efficient replication of plasmids in T. reseei. A mechanism for the enhancement of transformation frequency by the trs elements is proposed. Received: 1 March 1997 / Accepted: 13 May 1997     

利用红色荧光蛋白分析里氏木霉合成纤维素酶的机理

以红色荧光蛋白作为报告蛋白研究了里氏木霉的纤维素酶合成机理。构建了里氏木霉的表达盒,通过该表达盒使红色荧光蛋白的基因整合到里氏木霉的基因组DNA上,并受纤维二糖水解酶基因启动子的调控,得到重组菌株T.reeseiTR2。在不同的条件下培养T.reeseiTR2,红色荧光蛋白的表达情况可以反映在不同条件下里氏木霉合成纤维素酶的情况。在诱导的情况下,红色荧光蛋白随时间变化的情况与培养液中纤维素酶活性的变化相似,培养至36h后可以观察到荧光,并且不断增强,到菌丝自溶时荧光减弱。另一方面,诱导后里氏木霉菌丝的各个部位均可以观察到荧光,而且分布均匀,表明菌丝的各个部位在纤维素酶合成过程中所起的作用相同。在非诱导的情况下,培养时间较长时也可以观察到较弱的荧光,表明在此条件下里氏木霉仍可以合成少量的纤维素酶,这一结果为解释纤维素诱导里氏木霉合成纤维素酶的机理提供了另一个试验依据。     

Production of cellulase by freely suspended and immobilized cells of Trichoderma reesei

Trichoderma reesei (QM 9123) was immobilized within the open porous network of reticulated polyurethane foam matrices, and the growth pattern, glucose consumption and cellulase production were compared with those of freely suspended cells. It was found that the method of immobilization was simple and had no detrimental effect on cell activity. Various production media, to be used after the cultivation of T. reesei were tried. It was found that a nitrogen source-free production medium gave the highest enzyme titers of 1.5 × 10³ FPA U l⁻¹. Similar results were obtained with both freely suspended and immobilized cells.     

Cellobiohydrolase I (Cel7A) from Trichoderma reesei has chitosanase activity

Cellobiohydrolase CBH I (Cel7A) from the filamentous fungus Trichoderma reesei (TrCBHI), which is a member of glycoside hydrolase family (GHF) 7, was expressed in Aspergillus oryzae. We found that the recombinant enzyme showed significant chitosanase activity, as well as cellulase activity, and acted in an endo-type manner on soluble polymeric substrate. Furthermore, another GHF7 CBH I from Aspergillus aculeatus (AaCBHI) expressed in A. oryzae also had chitosanase activity, while endoglucanase EG I (Cel7B) from T. reesei had no activity towards chitosan. To our knowledge, this is the first report of GHF7 enzymes possessing chitosanase activity.     

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...     

Growth of Trichoderma reesei RUT C-30 in stirred tank and reciprocating plate bioreactors

A series of fed-batch experiments at different agitation speeds were performed using the industrially important strain Trichoderma reesei RUT C-30 in two different bioreactors to understand the close relationship that exists between the shear field within a bioreactor, the morphology of the microorganism, the rheology of cultivation broth, and the process performance. The two bioreactors, stirred tank bioreactor (STB) and reciprocating plate bioreactor (RPB), are characterized by a significantly different shear field to which microorganisms are exposed. Highest biomass concentration (ca. 15 g l⁻¹) was obtained at higher agitation rates in both bioreactors due to better oxygen supply. However, better filter paper activities per mg of protein were obtained at lower agitation in both bioreactors. In both bioreactors, young and healthier fungi in the batch phase were not affected by shear even at higher agitation rates. However, during the fed-batch phase, higher degree of fragmentation of clump morphology at high agitation intensity was confirmed by image analysis. Also, the rheological analysis showed an increase in apparent viscosity during the batch phase and early fed-batch phase due to the increase in the biomass concentration. During the late stages of cultivation, the apparent viscosity decreased due to cell lysis and spore formation.     

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.     

N-糖链加工影响里氏木霉形态发生并提高木质纤维素降解能力

[背景] 烟曲霉α-1,2-甘露糖苷酶MsdS在高尔基体中将N-糖链Man₈GlcNAc₂加工为成熟分泌糖蛋白的糖型Man₆GlcNAc₂,有研究表明MsdS与烟曲霉的形态发生、细胞壁合成及蛋白质分泌密切相关;与烟曲霉不同的是,里氏木霉的成熟分泌糖蛋白上的N-糖链结构为Man₈GlcNAc₂,细胞却能正常生长,说明丝状真菌N-糖链的加工具有物种特异性,但其生物学意义不明。[目的] 为研究N-糖链加工对里氏木霉细胞生长及蛋白质分泌的影响,本研究将烟曲霉MsdS转入里氏木霉中以改变其成熟分泌糖蛋白的糖型。[方法] 构建带有烟曲霉msdS基因的重组质粒并转入里氏木霉中,获得msdS表达菌株Tr-MsdS,分析Tr-MsdS菌株的生长表型、N-糖组、蛋白质分泌途径和纤维素酶活性的变化。[结果] 在里氏木霉msdS表达菌株Tr-MsdS中,分泌糖蛋白的主要糖型由出发株的Man₈GlcNAc₂转变为Man₆GlcNAc₂,细胞壁组分发生变化,但细胞壁完整性未受影响;与出发株相比,Tr-MsdS菌株产孢、出芽及分枝增多;另外,MsdS的表达还影响蛋白质分泌,在50℃时降解纤维素和β-葡聚糖的能力分别提高9.9%和32.2%。[结论] 研究结果表明,N-糖链的加工可影响里氏木霉蛋白质,尤其是纤维素酶的分泌,干扰N-糖链加工可能是提高里氏木酶纤维素酶产量的新策略。     

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...