Gene cloning and heterologous expression of a novel endoglucanase, swollenin, from Trichoderma pseudokoningii S38

The coding sequence of a novel cellulolytic factor, swollenin, was isolated from the cellulolytic fungus Trichoderma pseudokoningii S38. The full-length swo2 gene encodes a protein of 494 amino acids with a calculated molecular mass of 51,393 Da, which includes a putative 22-amino-acid signal peptide. Sequence analysis revealed significant identity between isolated swollenin and that from Trichoderma reesei. The swollenin gene was further expressed and purified in T. reesei QM9414. The expressed swollenin protein was consequently purified by two-step ion exchange chromatography. The purified swollenin had subtle hydrolytic activities on xylan and yeast cell wall glucan, while no apparent activities on carboxymethy cellulose, cotton fiber, filter paper, or cellulose powder CF11 were observed. These results indicate that although swollenin maintains unidentified glycohydrolytic activities, it is inactive against beta-1,4-glycosidic bonds in cellulose. Its exact role in lignocellulose hydrolysis calls for further analysis.     

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.     

Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization

Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction.     

Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts

Bulk production of xylanases from thermophilic microorganisms is a prerequisite for their use in industrial processes. As effective secretors of gene products, fungal expression systems provide a promising, industrially relevant alternative to bacteria for heterologous enzyme production. We are currently developing the yeast Kluyveromyces lactis and the filamentous fungus Trichoderma reesei for the extracellular production of thermophilic enzymes for the pulp and paper industry. The K. lactis system has been tested with two thermophilic xylanases and secretes gram amounts of largely pure xylanase A from Dictyoglomus thermophilum in chemostat culture. The T. reesei expression system involves the use of the cellobiohydrolase I (CBHI) promoter and gene fusions for the secretion of heterologous thermostable xylanases of both bacterial and fungal origin. We have reconstructed the AT-rich xynB gene of Dictyoglomus thermophilum according to Trichoderma codon preferences and demonstrated a dramatic increase in expression. A heterologous fungal gene, Humicola grisea xyn2, could be expressed without codon modification. Initial amounts of the XYN2 protein were of a gram per liter range in shake-flask cultivations, and the gene product was correctly processed by the heterologous host. Comparison of the expression of three thermophilic heterologous microbial xylanases in T. reesei demonstrates the need for addressing each case individually.     

Fibril formation from cellulose by a novel protein from Trichoderma reesei: A non-hydrolytic cellulolytic component?

A low molecular weight protein, named fibril-forming protein (FFP), was isolated from the culture supernatant of Avicel-grown Trichoderma reesei. The protein was purified to homogeneity and it exhibited a molecular weight of 11,400Da. Low amounts of this protein caused apparently non-hydrolytic disruption of filter paper, releasing fibrils without any detectable release of reducing sugars. It displayed no hydrolytic activity on carboxymethylcellulose (CMC), p-nitrophenyl--d-glucoside (pNPG) or 4-methylumbelliferyl cellobioside. The pH optimum of the protein was between 4 and 5. The temperature optimum was 40°C and the computed activation energy (E_a) for the filter paper disruption process was 4.18kcal/mol, suggesting disruption of non-covalent bonds. It had no immunological cross reactivity with reported cellulase components of T. reesei.     

Glycoprotein hypersecretion alters the cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.     

Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei.     

Expanisns

Biochemical dissection of the “acid-growth” process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril-matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit, meristerms and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.     

瑞氏木霉表达黑曲霉葡萄糖氧化酶

利用高表达分泌纤维素酶的真菌瑞氏木霉表达重组的黑曲霉葡萄糖氧化酶。在大肠杆菌DH5α中构建瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子pUC19(命名为pCBHGOD)质粒,线性化后用瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子(命名为CBHGOD)核酸片段转化瑞氏木霉QM9414原生质体。用PCR扩增方法筛选出同源重组葡萄糖氧化酶基因的瑞士木霉突变株。用麦杆诱导瑞氏木霉突变株,生产黑曲霉葡萄糖氧化酶,Westernblot分析重组的葡萄糖氧化酶分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶一致,生产的重组酶活性25umL,相当于Sigma公司葡萄糖氧化酶标准品的产量为0.5gL。瑞氏木霉可用于生产黑曲霉葡萄糖氧化酶。     

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS‐MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono‐oxygenases (AA9), carbohydrate‐binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two‐fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327–336, 2016     

Cellular and extracellular localization of endocellulase in Trichoderma reesei

Abstract An endocellulase (1,4-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4) was purified by preparative isoelectric focusing from culture fluids of Trichoderma reesei QM 9414 grown on cellulose. Its properties were studied by affinity titration curves and immunoelectrophoresis. FITC-labeled protein A-antibody was used to document its occurrence in cellulose and in fungal cell walls. Immunogold electron microscopy served to detect endocellulose sites within the outer exopolysaccharide layer of the fungal cell wall.     

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism.

The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly.     

The protein disulphide isomerase gene of the fungus Trichoderma reesei is induced by endoplasmic reticulum stress and regulated by the carbon source

The gene pdi1 encoding protein disulphide isomerase was isolated from the filamentous fungus Trichoderma reesei by degenerate PCR based on a consensus PDI active-site sequence. It was shown that the Trichoderma pdi1 cDNA is able to complement a yeast mutant with a disrupted PDI1 gene. The putative T. reesei PD1I protein has a predicted 20-amino acid N-terminal signal sequence and the C-terminal fungal consensus ER retention signal HDEL. The mature protein shows strong conservation relative to other fungal protein disulphide isomerases. The T. reesei pdi1 promoter has two possible unfolded protein response (UPR) elements and it was shown by treatments with dithiothreitol and tunicamycin that the gene is under the control of the UPR pathway. Expression of a heterologous protein, an IgG antibody Fab fragment, in Trichoderma increases pdi1 expression, probably by inducing the UPR. The level of T. reesei pdi1 mRNA is also regulated by the carbon source, being lowest in glucose-containing media and highest on carbon sources that induce the genes encoding extracellular enzymes. The mechanism of this regulation was studied by examining pdi1 mRNA levels under conditions where the extracellular enzymes are induced by sophorose, as well as in the strain RutC-30, which is mutant for the glucose repressor gene cre1. The results suggest that neither sophorose induction nor glucose repression by the CREI protein affect the pdi1 promoter directly. Received: 4 May 1998 / Accepted: 23 April 1999     

Quantitative investigation of non-hydrolytic disruptive activity on crystalline cellulose and application to recombinant swollenin

For the efficient degradation and bioconversion of cellulosic biomass, it is important to efficiently disrupt and convert crystalline regions of cellulose into easily hydrolyzable regions than to simply hydrolyze cellulose. Expansin-like proteins such as swollenins have disruptive functions on lignocellulose, including crystalline cellulose, via non-hydrolytic mechanisms. In this work, we produced the swollenin from Trichoderma asperellum in Escherichia coli. The recombinant protein was then refolded into the bioactive form with simultaneous purification via a novel cellulose-assisted process. We devised a novel, simple, and efficient method to quantitatively determine the non-hydrolytic disruptive activity of swollenin on crystalline cellulose. This method is based on the synergism of the swollenin and the endoglucanase FnCel5A from Fervidobacterium nodosum. The change from crystalline regions into easily hydrolyzable forms, due to non-hydrolytic disruption, might be slight and not easily be observed. However, disrupted regions of cellulose could be hydrolyzed by FnCel5A, and reducing sugars were formed by the synergism. The disruptive function of the swollenin was quantitatively characterized by measuring the release of reducing sugars. These methods and processes will be useful for further research on non-hydrolytic disruptive bioactivities and provide novel approaches for the efficient and economical bioconversion of cellulosic biomass.     

Purification and antifungal activity of recombinant chitinase from Escherichia coli carrying the family 19 chitinase gene of Streptomyces sp. J-13-3

A recombinant chitinase was purified from the cell extract of Escherichia coli JM109 transformed by plasmid pUC19 carrying the gene encoding family 19 chitinase of Streptomyces sp. J-13-3 by column chromatography on DEAE-Sepharose, CM-Sepharose, and Bio-Gel P-100. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 32,000. The recombinant chitinase hydrolyzed the trimer to hexamer of N-acetylglucosamine and had the identical N-terminal amino acid sequence of the mature protein, indicating removal of the signal sequence by E. coli signal peptidase. The fungal growth in well (200 microl of medium) of microplate by measurement of absorbance at 595 nm indicated that the chitinase (10 microg) completely and half inhibited growth of Trichoderma reesei and Aspergillus niger respectively.