1)study of the kinetics of secretion of heterologous proteins and interaction of transcription, translation and secretion rates.
2)molecular characterisation of the secretion pathway and analysis of protein folding and post-translational protein-modification processes.
3)improvement of protein-fusion strategies.
During the report period the following achievements have been obtained within the different tasks of the project.
Task 1: Study of the kinetics of secretion of heterologous proteins and interaction of transcription, translation and secretion rates
The group of Penttilä. To study the rate-limiting steps in protein production, we have developed a metabolic protein labelling technique for T. reesei. The protein production kinetics has been studied in a chemostat cultivation system at different growth rates. We have been able to follow the time course of protein synthesis, glycosylation and transport into the culture medium, as well as to obtain information on the effect of growth rate on the protein synthesis and secretion capacity. The results obtained with the major cellulase (CBHI) produced by T. reesei indicate that especially at low growth rates the protein production is limited by the overall capacity of the secretory machinery of the fungus. A system to analyse heterologous protein production (production of antibody Fab fragments) and the effect of CBHI fusion on the production kinetics of recombinant molecules has been set up.
Task 2a: Isolation and functional analysis of genes involved in heterologous protein secretion
The group of Van den Hondel. The functionality of cloned SAR1-like genes of A. niger and T. reesei was demonstrated by complementation of S. cerevisae sar1 and sec12 mutants. Furthermore, replacement experiments of the wt sarA gene of A.niger with putative ts mutants alleles did not result in the isolation of the desired ts mutants but in mutants with a cold sensitive phenotype. Further analysis indicated that the putitative ts alleles were ts dominant negative alleles. More useful derivatives from one of the available 'dominant-negative' mutants were obtained. By using a two step gene replacement strategy isolation of a new sarA mutant was initiated. Furthermore, by using a specific PCR approach several small GTPase encoding gene fragments were obtained from two available A.niger cDNA libraries. At least 3 classes of clones were identified carrying the A.niger sagA/B/C genes, homologous to S.cerevisiae YTP1,SEC4, YPT6.
The group of Archer. Two genes have been isolated from A.niger that encode endoplasmic reticulum (ER)-resident proteins within the family of protein disulphide isomerases; pdiA and tigA. Both genes have been completely sequenced and shown to encode secretion signal sequences which direct the proteins to the lumen of the ER. The proteins are retained within the ER by C-terminal signals, KDEL (tigA) and HDEL (pdiA). The transcription of both genes is up-regulated 2-3 fold by tunicamycin, an inhibitor of N-linked glycosylation, indicating their likely involvement in the secretion and post-translational modification of secretory proteins.
Task 2b: Analysis of post-translational protein-modification processes
The group of Peberdy/Hemming. A. niger 402 secretes two proteins with invertase activity when grown in minimal medium containing sucrose as the sole carbon source. These secreted proteins have been shown to be heavily glycosylated and have been purified. The two invertases have shown to differ in their molecular weight (as shown by SDS-page), their isoelectric points, their glycosylation and in their substrate specificity. A system to allow the use of protoplasts to study protein secretion and glycosylation has been developed. Two forms of invertase have been shown to be secreted by protoplasts. An immunodetection system has been developed and introduced for Aspergillus invertase.
The group of Contreras. A rough screening of a collection of different yeasts and filamentous fungi have been carried out to search for oligosaccharides that are acceptor substrate for glycosyl-transferases from higher eukaryotes. The major fraction of the screened species has mannose as the terminal sugar, but other species seem to contain N-Acetylglucosamine or alfa- and beta-linked galactose. The latter sugars also appear in the complex type carbohydrates found in mammalian cells. Some species were also found to be acceptors for N-acetylglucosamine.Subsequently N-linked, mannose containing carbohydrates (present on cellobiohydrolase I from Trichoderma reesei and alpha-amylase from Aspergillus niger) have been elongated with N-acetylglucosamine, galactose and sialic acid in vitro. In this way we have synthesized a glycosyl structure which is very similar, or identical to a mammalian hybrid carbohydrate. In parallel experiments, an alpha-mannosidase from T. reesei was cloned from which we expect that it is involved in the trimming of the alpha-1,2 mannoses which have to be removed before the N-acetylglucnactransferase I can transfer a Glc-NAc-sugar. Furthermore, the precise structure of the predominant N-linked carbohydrate structures present on CBH1 from T. reesei was determined by NMR, ANTS-gel analysis, mannosidase degradation, and HPAE-PAD analysis.
The group of Kubicek. The involvement of a KEX2-like endoprotease in the secretory pathway of Trichoderma reesei was demonstrated by the in vivo and in vitro specific inhibitor for KEX2-type proteases (pAPMSF) on the secretion of various cellulases and xylanases. To investigate the target specificity of the inhibitor in vivo, T. reesei was transformed with 7 vectors containing chimeric signal peptides fused to the S. hindustanus bleomycin resistance gene as a marker. A targeted integration system, yet not available for T.reesei, has been constructed and is currently analysed. We have cloned a pool of DNA-fragments flanked by the regions around the active site residues His and Ser, which may represent a partial serin-like protease-library of T.reesei. Screening this pool several fragments of T.reesei exhibiting high similarity to the subtilisin-like serine protease-family were cloned. A mov34-homologue of T.reesei was cloned by accident, which may be important for heterologous protein formation in T. reesei.
The group of Archer. The tools necessary to study the effects of up-regulating the expression of the PDI-family genes isolated from A.niger (Task 2a) are being constructed. The gene pdiA and the Saccharomyces cerevisiae PDI-encoding gene have been cloned into expression vectors.
Task 3: Improvement of gene-fusion systems with the goal of obtaining more efficient secretion of biologically active, heterologous proteins.
The group of Parriche. Eleven secretion vectors have been constructed using various carrier proteins, linker peptides and signal sequences. They have been tested in three T. reesei host strains presenting different properties with respect to protein secretion. Active human lysozyme production, never exceeding 1mg/l with standard expression vectors, was raised up to 250 mg/l by using a carrier protein (shake flask culture).The best carrier in all strains was found to be a chimeric construct made with the phleomycin-resistance protein (Sh-ble) fused, at the C-terminus, to A. Niger glucoamylase O-glycosylated region(GAM hinge). While the chimeric construct retained the phleomycin-resistance capabilities of the She-ble protein, the addition of the o-glycosylated region was a significant enhancement over the production capability of vectors with the She-ble alone. A. Niger glucoamylase performed as well in one other strain (11D5). Reduction of the GAM to its catalytic moiety lead to the loss of the carrier ability. Various designs of linker peptide (notably one with an N-glycosylation site) have failed to improve secretion. Detection of lysozyme with anti-HLZ antibodies in intra and extra-cellular extracts showed that cleavage of the fusion protein occurred outside of the fungal cells. These finding have been applied to the production of T. Reesei of the commercially valuable human anti-RhD Fab fragment antibodies; production levels equivalent to the best-producing hybridoma have been achieved (that is 100 times more than production in E. Coli).
MAJOR SCIENTIFIC BREAKTHROUGHS AND/OR INDUSTRIAL APPLICATIONS:
- Analysis of the protein synthesis and secretion by metabolic labelling, and characterization of the capacity of protein synthesis and secretion in different physiological states of the fungus.
- Defined sarA mutations have been constructed, which show to be termosensitive, dominant negative.
- Three smGTPase encoding genes from A.niger, involved in the major transport steps in the secretion pathway have been isolated.
- Two genes, pdiA and tigA, have been isolated from A.niger that encode PDI-family proteins. Both genes have been sequenced and their expression shown to be up-regulated by tunicamycin.
- Purification and partial characterisation of two invertases (glucoproteins) secreted by A. niger.
- Glycoproteins of T. reesei were converted to a mammalian type. The presence of terminal sialic acid is supposed to prevent the protein from binding to mannose or galactose receptors which initiates fast clearing from the blood (patent applied with the company Primalco, Helsinki, Finland).
- Heterologous protein production by gene fusion has been improved by 50% with a new secretion vector consisting of Sh-ble::GAM-hinge
- Anti-RhD Fab fragment antibody production by T. reesei was highly successful with the use of the new secretion vectors developed.
- Several gene fragments of T.reesei exhibiting high similarity to KEX-2 were cloned
- A putative mov34-homologue of T.reesei has been cloned
Funding SchemeCSC - Cost-sharing contracts
NR4 7UA Norwich
NG7 2RD Nottingham