The role of CRE1 in nucleosome positioning within the cbh1 promoter and coding regions of Trichoderma reesei.

Nucleosome positioning within the promoter and coding regions of the cellobiohydrolase-encoding cbh1 gene of Trichoderma reesei was investigated. T. reesei is a filamentous fungus that is able to degrade dead plant biomass by secreting enzymes such as cellulases, a feature which is exploited in industrial applications. In the presence of different carbon sources, regulation of one of these cellulase-encoding genes, cbh1, is mediated by various transcription factors including CRE1. Deletion or mutation of cre1 caused an increase in cbh1 transcript levels under repressing conditions. CRE1 was shown to bind to several consensus recognition sequences in the cbh1 promoter region in vitro. Under repressing conditions (glucose), the cbh1 promoter and coding regions are occupied by several positioned nucleosomes. Transcription of cbh1 in the presence of the inducer sophorose resulted in a loss of nucleosomes from the coding region and in the re-positioning of the promoter nucleosomes which prevents CRE1 from binding to its recognition sites within the promoter region. Strains expressing a non-functional CRE1 (in strains with mutated CRE1 or cre1-deletion) exhibited a loss of positioned nucleosomes within the cbh1 coding region under repressing conditions only. This indicates that CRE1 is important for correct nucleosome positioning within the cbh1 coding region under repressing conditions.

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.

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.

Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin.

Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 angstroms resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in Km with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of kappa-casein, the natural substrate, revealed an increase in the kcat compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.

Isolation of Trichoderma reesei genes highly expressed on glucose-containing media: characterization of the tef1 gene encoding translation elongation factor 1 alpha.

Genes that are highly expressed on glucose-containing media were isolated from the filamentous fungus, Trichoderma reesei. A cDNA bank was prepared from glucose-grown fungus, the bank was screened with the same cDNA as a probe, and clones giving the strongest signal were isolated. This resulted in the isolation of previously uncharacterized genes. Five of the genes, representing the most abundant transcripts, corresponded to 1-3% of the total mRNA population and were clearly more highly expressed than the phosphoglycerate kinase-encoding gene (pgk1) of T. reesei. Based on sequence homology, one of the genes was identified as tef1, encoding translation elongation factor 1 alpha (TEF). The T. reesei TEF is most related to the Mucor racemosus TEF3, showing an overall amino acid similarity of 85%. Interestingly, an exon of only 2 bp seems to be present in T. reesei tef1, comprising the first 2 bp of the Gly15 codon.

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

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.

Hydrolytic properties of two cellulases of Trichoderma reesei expressed in yeast.

Two cellulases of the filamentous fungus Trichoderma reesei, cellobiohydrolase II (CBHII, EC 3.2.1.91) and endoglucanase I (EGI, EC 3.2.1.4), produced in recombinant strains of the yeast Saccharomyces cerevisiae, were tested in the hydrolysis of cellulose, xylan and other polymeric substrates. Both enzymes were active against unsubstituted, insoluble cellulose. CBHII had greater activity than EGI against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The EGI was also active against soluble substituted cellulose derivatives, whereas the activity of CBHII against these substrates was insignificant. Both enzymes were active against barley (1-->3,1-->4)-beta-glucan, but were inactive against (1-->3,1-->6)-beta-glucan (laminarin). An apparent low mannan-degrading activity of EGI against locust-bean (Ceratonia siliqua) gum galactomannan was not confirmed when homopolymeric mannan was used as substrate in a prolonged hydrolysis test. EGI exhibited considerably greater activity against insoluble, unsubstituted hardwood xylan than against amorphous cellulose. Soluble 4-O-methyl-glucuronoxylan was also attacked by EGI, although to a somewhat lesser extent than the unsubstituted xylan. By comparison with two purified xylanases of T. reesei, EGI produced xylo-oligosaccharides with a longer mean chain length when acting on both substituted and unsubstituted xylan substrates. CBHII was inactive against xylan.

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

An electrophoretic karyotype of Trichoderma longibrachiatum (reesei) was obtained using contour-clamped 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-celluloytic 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).

Pilot scale production of a Trichoderma reesei endo-beta-glucanase by brewer's yeast.

Endo-beta-glucanase I (EGI) of Trichoderma reesei was produced in laboratory and pilot scale using recombinant strains of "bottom-fermenting" Saccharomyces cerevisiae. The gene eg/1 was integrated in the chromosome or an expression cassette was inserted on a multicopy plasmid. Expression levels were compared in a laboratory scale bioreactor. The best EGI-producing strain was cultivated in pilot scale. Adsorbent treatment was used to remove endogenous yeast proteins and other impurities from the culture filtrate during concentration. Effective pilot scale one-step purification of the EGI protein was obtained using DEAE-Sepharose, on which EGI was weakly bound. The purified enzyme reacted with antibodies prepared against T. reesei EGI and catalyzed the hydrolysis of both insoluble and soluble substrates.

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.

Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae.

Cellobiohydrolase II of Trichoderma reesei was produced in laboratory and pilot scale using a transformant strain of Saccharomyces cerevisiae harbouring a multicopy expression plasmid. Different strategies were compared for concentration and partial purification of the enzyme produced in a 200 1 pilot cultivation. After efficient separation of biomass and sub-cellular particulate matter, a combination of ultrafiltration and adsorbent treatment for removal of protein impurities was used to provide a concentrate for chromatographic purification. Effective purification of the CBH II protein was obtained by passing the concentrate through a column of DEAE Sepharose, on which almost all the yeast proteins were adsorbed. The purified enzyme reacted with antibodies prepared against T. reesei CBH II and catalyzed partial solubilization of crystalline cellulose to soluble sugars.

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.

Expression of two Trichoderma reesei endoglucanases in the yeast Saccharomyces cerevisiae.

The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.