Jaana Uusitalo

The Effects of Drugs Inhibiting Protein Secretion in the Filamentous Fungus Trichoderma reesei EVIDENCE FOR DOWN-REGULATION OF GENES THAT ENCODE SECRETED PROTEINS IN THE STRESSED CELLS

To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca2+-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.

ENZYME PRODUCTION BY RECOMBINANT TRICHODERMA REESEI STRAINS

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.

Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei: a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30

ABSTRACT We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) of the industrially important fungus Trichoderma reesei. This subunit is the catalytic part of the glucosidase II heterodimeric enzyme involved in the structural modification within the endoplasmic reticulum (ER) of N-linked oligosaccharides present on glycoproteins. The gene encoding GIIα (gls2α) in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product. Based on the peculiar monoglucosylated N-glycan pattern on proteins produced by the strain, we concluded that the truncated protein can still hydrolyze the first α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc2Man9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα protein. Since glucosidase II is also involved in the ER quality control of nascent polypeptide chains, its transcriptional regulation was studied in a strain producing recombinant tissue plasminogen activator (tPA) and in cultures treated with the stress agents dithiothreitol (DTT) and brefeldin A (BFA), which are known to block protein transport and to induce the unfolded protein response. While the mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement was observed after DTT addition.

In vivo synthesis of complex N-glycans by expression of human N-acetylglucosaminyltransferase I in the filamentous fungus Trichoderma reesei

The human N‐acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT‐D‐80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N‐glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.

Monitoring the kinetics of glycoprotein synthesis and secretion in the filamentous fungus Trichoderma reesei: cellobiohydrolase I (CBHI) as a model protein

The authors have developed methodology to study the kinetics of protein synthesis and secretion in filamentous fungi. Production of cellobiohydrolase I (CBHI) by Trichoderma reesei was studied by metabolic labelling of the proteins in vivo with [35S]methionine or [14C]mannose, and subsequent analysis of the labelled proteins using two-dimensional gel electrophoresis. Analysis of the different pl forms of the nascent proteins allowed monitoring of the maturation of CBHI during the transport along the biosynthetic pathway. The maturation of the pi pattern of CBHI as well as secretion into culture medium was prevented by treatment with the reducing agent DTT. The pl forms of CBHI detectable in the presence of DTT corresponded to the early endoplasmic reticulum forms of the protein. Removal of N-glycans by enzymic treatment (endoglycosidase H or peptide-N-glycosidase F), or chemical removal of both N- and O-glycans, changed the pl pattern of CBHI, showing that glycan structures are involved in formation of the different pl forms of the protein. By quantifying the labelled proteins during a time course, parameters describing protein synthesis and secretion were deduced. The mean synthesis time for CBHI under the conditions used was 4 min and the minimum secretion time was 11 min. The methodology developed in this study provides tools to reveal the rate-limiting factors in protein production and to obtain information on the intracellular events involved in the secretion process.

Hot water extraction and steam explosion as pretreatments for ethanol production from spruce bark

Spruce bark is a source of interesting polyphenolic compounds and also a potential but little studied feedstock for sugar route biorefinery processes. Enzymatic hydrolysis and fermentation of spruce bark sugars to ethanol were studied after three different pretreatments: steam explosion (SE), hot water extraction (HWE) at 80 °C, and sequential hot water extraction and steam explosion (HWE+SE), and the recovery of different components was determined during the pretreatments. The best steam explosion conditions were 5 min at 190 °C without acid catalyst based on the efficiency of enzymatic hydrolysis of the material. However, when pectinase was included in the enzyme mixture, the hydrolysis rate and yield of HWE bark was as good as that of SE and HWE+SE barks. Ethanol was produced efficiently with the yeast Saccharomyces cerevisiae from the pretreated and hydrolysed materials suggesting the suitability of spruce bark to various lignocellulosic ethanol process concepts.

Reed canary grass as a feedstock for 2nd generation bioethanol production.

The enzymatic hydrolysis and fermentation of reed canary grass, harvested in the spring or autumn, and barley straw were studied. Steam pretreated materials were efficiently hydrolysed by commercial enzymes with a dosage of 10-20FPU/g d.m. Reed canary grass harvested in the spring was hydrolysed more efficiently than the autumn-harvested reed canary grass. Additional β-glucosidase improved the release of glucose and xylose during the hydrolysis reaction. The hydrolysis rate and level of reed canary grass with a commercial Trichoderma reesei cellulase could be improved by supplementation of purified enzymes. The addition of CBH II improved the hydrolysis level by 10% in 48hours hydrolysis. Efficient mixing was shown to be important for hydrolysis already at 10% dry matter consistency. The highest ethanol concentration (20g/l) and yield (82%) was obtained with reed canary grass at 10% d.m. consistency.

Expression and characterisation of chymosin pH optima mutants produced in Trichoderma reesei

The production of chymosin mutants designed to have altered pH optima using the cellulolytic filamentous fungus Trichoderma reesei is described. The strong promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has been used for the expression and secretion of active calf chymosin. Structural analysis of the hydrogen bonding network around the two active site aspartates 32 and 215 in chymosin have suggested that residues Thr 218 and Asp 303 may influence the rate and pH optima for catalysis. The chymosin mutants Thr218Ala and the double mutant Thr218Ala/Asp303Ala have been made by site-directed mutagenesis and expressed in T. reesei. Enzyme kinetics of the active enzyme T218A indicate a pH optimum of 4.2 compared to 3.8 for native chymosin B using a synthetic octa-peptide substrate, confirming the previous analysis undertaken in E. coli. The double mutant T218A/D303A exhibits a similar optimum of 4.4 to that reported for the D303A, indicating that the combination of these changes is not additive. The application of protein engineering in the rational design of specific modifications to tailor the properties of enzymes offers a new approach to the development of industrial processes.