K. M. Helena Nevalainen

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.

Electrophoretic karyotyping of wild-type and mutant Trichoderma longibrachiatum (reesei) strains

SummaryAn electrophoretic karyotype of Trichoderma longibrachiatum (reesei) was obtained using contourclamped homogeneous electric field (CHEF) gel electrophoresis. Seven chromosomal DNA bands were separated in the wild-type T. longibrachiatum strain QM6a. The sizes of the chromosomal DNA bands ranged from 2.8 to 6.9 Mb, giving an estimated total genome size of about 33 Mb. The electrophoretic karyotype of the strain QM6a was compared to three hyper-cellulolytic mutant strains, QM9414, RutC30 and VTT-D-79125. The chromosome pattern of the mutant QM9414 was quite similar to that of the wild-type QM6a except that the smallest chromosome differed somewhat in size. The VTT-D-79125 and RutC30 strains, which have undergone several mutagenesis steps, showed striking differences in their karyotype compared to the initial parent. The chromosomal DNA bands were identified using the previously characterized T. longibrachiatum genes (egl1, egl2, cbh1, cbh2, pgk1, rDNA) and random clones isolated from a genomic library. In all strains the cellulase genes cbh1, cbh2 and egl2 were located in the same linkage group (chromosome II in the wild-type), while the main endoglucanase, egl1, hybridized to another chromosomal DNA band (chromosome VI in the wild-type).

Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei

A cDNA encoding for the glucoamylase P enzyme (GAMP) of the fungus Hormoconis resinae was introduced into the cellulolytic filamentous fungus Trichoderma reesei under the control of the promoter of the major cellulase gene (cbh1) of Trichoderma. The transforming vector plasmid used was found to be integrated into the genome of T. reesei at various locations and in multiple copies. The size of the GAMP secreted by Trichoderma varied because of different glycosylation patterns. The best transformant strains secreted about 700 mg/l of active GAMP, which is 20-fold more than obtained with H. resinae.

Production of Recombinant Bleaching Enzymes from Thermophilic Microorganisms in Fungal Hosts

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.

Simultaneous expression of the bacterial Dictyoglomus thermophilum xynB gene under three different Trichoderma reesei promoters

Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.

Activity-based identification of secreted serine proteases of the filamentous fungus, Ophiostoma

A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease.

Use of the Biolistic Particle Delivery System to Transform Fungal Genomes

Biolistic delivery of transforming DNA into fungal genomes, especially when performed on uninucleate haploid conidia, has proven successful in bypassing the time-consuming repetitive purification of protoplasts used for the widely applied polyethylene glycol-mediated method. Biolistic transformation is also relatively quick compared to other available methods and provides a high percentage of stable transformants.

Comparative Genomic Analysis of Glycoylation Pathways in Yeast, Plants and Higher eukaryotes

N-linked glycosylation is an essential modification of secretory and membrane proteins in all eukaryotic cells. Here, we review the current metabolic pathways of N-linked oligosaccharide biosynthesis in the endoplasmic reticulum and in the Golgi apparatus for yeasts: Saccharomyces cerevisiae and Schizosaccharomyces pombe, and higher eukaryotes: plants and human. The evolutionarily conserved proteins, processed in the cytosolic and the luminal side of the ER membrane, and the unique genes and their specific functions, occurring in the Golgi complex, for each selected organism, will be collated and discussed. This precise knowledge of the glycosylation pathway contributes to better understanding of the N-linked glycoprotein biosynthesis among different species, resulting in the recently successfully engineered strains for heterologous gene expression systems for industrial and therapeutic protein production.