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).     

Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei.

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.     

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.     

Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism

The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase activity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences in the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observed in greenhouse experiments.     

Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene （cbh1） promoter optimization

To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene （cbhl） promoters was obtained. The region from -677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from -620 to -820 of the modified cbhl promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbhl promoter, obtaining promoters with copy numbers 2, 4, and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase （gus） reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.     

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.     

丝状真菌瑞氏木霉生产重组蛋白的分子生物学研究进展

瑞氏木霉是自然界中普遍存在并有重要经济意义的一种丝状真菌,作为工业生产菌株生产多种水解酶类已有多年历史。本文报道了用基因工程手段对瑞氏木霉进行遗传改造,构造具新性状的重组菌株,用以过量产生同源和异源蛋白类物质的分子生物学研究进展。包括利用ＣＢＨＩ基因的强启动子在瑞氏木霉中过量表达瑞氏木霉内切葡聚糖酶、小牛凝乳蛋白酶、人抗体片段、哈茨木霉几丁质酶、Ｈｏｒｍｏｃｏｎｉｓｒｅｓｉｎａｅ葡萄糖淀粉酶等同源和异源蛋白以及利用在葡萄糖上强表达的启动子生产纤维素酶等遗传工程进展情况。     

里氏木霉双基因同步缺失菌株的构建与甘露聚糖酶表达研究

在里氏木霉中建立了一个快速的双基因位点同步同源重组新方法,较好解决了里氏木霉基因逐个敲除周期长等问题。研究以里氏木霉自身甘露聚糖酶基因（man5A）为重组表达的报告基因,通过一步转化,将该基因定点整合入纤维二糖水解酶Ⅰ（cbh1）基因位点,同时缺失主要的两个纤维素酶基因（cbh1、cbh2）,得到重组工程菌Man12。将重组工程菌Man12与出发菌株Tu6Δku70进行摇瓶发酵,结果显示,重组菌株的甘露聚糖酶产量比出发菌株提高10倍,而纤维素酶产量降低了60%,胞外总蛋白分泌水平降低了40%。Real-time PCR检测甘露聚糖酶基因（man5A）的转录水平,发现重组菌株较出发菌株提高了25倍。在里氏木霉中首次报道了通过一步转化实现两个基因同步定点整合的方法,对利用基因工程手段构建高效表达重组蛋白的里氏木霉工程菌株具有一定的指导意义。     

康氏木霉纤维素酶转录因子ACEII与外切纤维二糖水解酶基因 I（cbh1）启动子的结合分析

ACEI、ACEII和Xyr1是康氏木霉中调控纤维素酶基因表达的转录因子。体外实验已证实ACEI和Xyr1可与cbh1启动子上的287bp序列（-304bp~-18bp）结合从而调控cbh1基因转录,但ACEII是否可与此序列结合仍未清楚。为进一步研究ACEII调控纤维素酶基因表达的机制,利用PCR技术扩增康氏木霉ACEII DNA结合区的基因序列,并使其在大肠杆菌中表达。凝胶迁移率移动试验表明ACEII DNA结合区不能与cbh1启动子的287bp序列结合。提示了康氏木霉cbh1基因在诱导表达时起调控作用的主要是Xyr1,而不是ACEII。这对阐明真菌纤维素酶基因表达调控的分子机制具有重要的意义。     

康氏木霉中调控纤维素酶形成的两个调控因子的克隆及功能性分析

锌指蛋白ACEI和Xyrl是里氏木霉中调控纤维素酶和木聚糖酶形成的两个调控因子,它们能竞争性结合木聚糖酶基因xynl的启动子.为进一步研究ACEI和Xyr1调控纤维素酶基因表达的机制,本研究利用PCR技术扩增康氏木霉ACEI和Xyr1 DNA结合区的基因序列,并使其在大肠杆菌中得到表达.凝胶迁移率移动试验表明ACEI和Xyrl的DNA结合区均可与纤维素酶基因cbh1启动子的287 bp序列特异性结合.提示ACEI与Xyr1不仅能竞争性结合xynl启动子,也能竞争性结合cbhl启动子.     

Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host.

For monitoring chitinase expression during mycoparasitism of Trichoderma harzianum in situ, we constructed strains containing fusions of green fluorescent protein (GFP) to the 5'-regulatory sequences of the T. harzianum nag1 (N-acetyl-beta-d-glucosaminidase-encoding) and ech42 (42-kDa endochitinase-encoding) genes. Confronting these strains with Rhizoctonia solani led to induction of gene expression before (ech42) or after (nag1) physical contact. A 12-kDa cut-off membrane separating the two fungi abolished ech42 expression, indicating that macromolecules are involved in its precontact activation. No ech42 expression was triggered by culture filtrates of R. solani or by placing T. harzianum onto plates previously colonized by R. solani. Instead, high expression occurred upon incubation of T. harzianum with the supernatant of R. solani cell walls digested with culture filtrates or purified endochitinase 42 (CHIT42, encoded by ech42) from T. harzianum. The chitinase inhibitor allosamidin blocked ech42 expression and reduced inhibition of R. solani growth during confrontation. The results indicate that ech42 is expressed before contact of T. harzianum with R. solani and its induction is triggered by soluble chitooligosaccharides produced by constitutive activity of CHIT42 and/or other chitinolytic enzymes.     

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.     

Expression of Barley Endopeptidase B in Trichoderma reesei

The gene for barley endopeptidase B (EPB) has been expressed in the filamentous fungus Trichoderma reesei from the cbh1 promoter. The EPB signal sequence allowed secretion of over 90% of the recombinant protein. Yields reached about 500 mg of immunoreactive protein per liter and exceeded values for any other protein derived from a higher eukaryotic organism produced in T. reesei.     

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.     

Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum

Mycoparasitism of fungal plant pathogens by Trichoderma species is a complex process that involves the production and coordinated secretion of cell-wall degrading enzymes. Genes implicated in mycoparasitism by Trichoderma atroviride contain motifs in the promoter region, designated MYRE1-MYRE4, that are proposed to act as binding sites for a global inducer of the mycoparasitic response. The aim of our study was to establish whether these motifs also were present in Trichoderma hamatum and whether the presence of these motifs could predict co-expression when T. hamatum was confronted by a pathogen. Using a combination of targeted, degenerate and inverse PCR, homologues of the mycoparasitism-related genes ech42 (chit42), prb1 and lam1.3 (xbg1.3-110), which encode an endochitinase, proteinase, and β-1,3-glucanase, respectively, were cloned and sequenced from T. hamatum. Alignment of the promoter regions of the three genes revealed identical regions in the chit42 and prb1 promoters, which were 6-9 base pairs in length and conserved in position. Specifically, the regulator y motifs MYRE1-MYRE4 were fully conserved, together with a fifth motif, identified by this research. A substrate assay designed to investigate the response of these genes from T. harzianum and T. hamatum to a simple carbon source (glycerol) showed that, in contrast to chit42 and prb1, xbg1.3-110 was not expressed. Further comparison of the expression patterns of these three genes between T. harzianum and T. hamatum using the glycerol substrate assay showed that no chit42 or prb1 expression could be detected in T. harzianum when it was grown under the same conditions as T. hamatum. This showed that the response of these genes to glycerol was species specific and that a single expression pattern for these genes was not common to all Trichoderma species. Confrontation assays were used to investigate the response of the three T. hamatum genes to the more complex substrate posed by the fungal pathogen Sclerotinia sclerotiorum. Once again gene expression analysis showed that both chit42 and prb1 were co-expressed and moderately induced during confrontation against Sclerotinia sclerotiorum. Although xbg1.3-110 previously had been implicated in mycoparasitism by T. harzianum, this study detected no xbg1.3-110 expression during confrontation between T. hamatum and S. sclerotiorum. These findings show that the MYRE1-MYRE4 together with MYRE5 are present in two species of Trichoderma, T. atroviride and T. hamatum and that the presence of these motifs could predict co-expression in response to two carbon sources.