Gérard Tiraby

Genetic improvement of Trichoderma reesei for large scale cellulase production

A strategy based on plate screening tests was designed for the selection of mutant strains of the fungus Trichoderma reesei suitable for cellulase (EC 3.2.1.4) production on an industrial scale. Six mutant generations were successively isolated, each of them fulfilling all of the three criteria: (1) improved productivity compared to the previous one, (2) high stability, (3) ability to be further improved. The mutant ultimately selected, CL 847, exhibited a four-fold increase in cellulase productivity in cellulose media as compared to the starting strain QM 9414, resistance to catabolite repression, increased β-d-glucosidase (EC 3.2.1.21) specific activity and it was partially constitutive. High cellulase concentrations were obtained in simple media with lactose or glucose as sole carbon source. The strain is currently used in an industrial fermentation plant.

Cassettes of the Streptoalloteichus hindustanus ble gene for transformation of lower and higher eukaryotes to phleomycin resistance

Phleomycins are glycopeptide antibiotics related to bleomycins nucleotide sequence of the Sh ble gene and its polylinker and tallysomycins which are active at low concentrations on both fragments. prokaryotic and eukaryotic cells (1). We have cloned a phleomycin-resistance gene (Sh ble) from the genomic DNA of REFERENCES Streptoalloteichus hindustanus (ATCC 31158), a tallysomycin 1 Berdy,J. (1980) Handbook of.Antibiotic Compounds. Vol.4, pp. 459-491, producer. The Sh ble gene encodes a small acidic protein (MW CRC Press, Boca Raton, FL, USA 13665) which has been crystallised for X-ray structural studies 2. Rondeau,J.M., Cagnon,C., Moras,D. and Masson,J.M. (1989) J. MoI. Biol. (2). The Sh protein is a binding protein with a strong affinity 207, 645-646. for the phleomycin family of antibiotics (3). When these 3. Gatignol,A., Durand,H. and Tiraby,G. (1988) FEBS Lent. 230, 171-175. antibiotics are bound by the Sh protein, then phleomycins can 4. Glumoff,V., Kappeli,O., Fiechter,A. and Reiser,J. (1989) Gene 84, nolonger areactivated by tneffous ions and oxyen teobreakdcan 311-318. no longer be activated by ferrous ions and oxygen to break down 5. Mattern,J.E. and Punt,P.J. (1988) Fungal Genet. Newslett. 35, 25. DNA (3). The Sh ble gene has been used in a number of 6. Perez,P., Tiraby,G., Kallerhoff,J. and Perret,J. (1989) Plant Mol. Biol. 13, laboratories as a selectable marker for lower (4, 5) and higher 365-373. eukaryotes (6, 7, 8). In order to facilitate vector construction 7. Mulsant,P., Gatignol,A., Dalens,M. and Tiraby,G. (1988) Somat. Cell. Mol. exeukryoetes(, 8).theinordgoers wto fac tipltevctoricositruction Genet. 14, 243-252. periment , synthetic oligom with multiple cloning sites have 8. Cosset,F.L., Legras,C., Chebloune,Y., Savater,P., Thoraval,P., been added at both ends of the Sh ble gene to give the two pUC Thomas,J.L., Samarut,J., Nigon,U.M. and Verdier,G. (1990) J. Virol. 64, based derivatives, pUT56 and pUT58. We report here the 1070-1078.

Bleomycin resistance conferred by a drug-binding protein

The protein coded by a bleomycin‐resistance gene (ble) cloned from producing actinomycetes was purified from a culture of a recombinant E.coli strain and its action on bleomycin was determined by in vitro assays. The protein binds reversibly in a one to one ratio to bleomycin which can no longer cleave DNA. The bleomycin resistance of cells harboring a ble gene could be accounted for by a sequestering effect of the bleomycin‐binding protein.

Comparative study of cellulases and hemicellulases from four fungi: mesophiles Trichoderma reesei and Penicillium sp. and thermophiles Thielavia terrestris and Sporotrichum cellulophilum

Abstract The enzymes produced by two thermophilic fungi claimed to produce heat-stable cellulases [see 1,4-(1,3;1,4)-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4] have been compared to those of two mesophilic fungi on the basis of the following criteria: polysaccharolytic spectrum, heat and pH effects on stability and on activity of the different enzymes, and the ability to hydrolyse raw natural substrates. The cellulases produced by one of the thermophiles, Sporotrichum cellulophilum, appeared to be as heat-labile as those from the mesophile Trichoderma reesei ; moreover, the former enzyme preparation is the least efficient of the four tested. Thielavia terrestris enzymes are the most thermostable; on the basis of the other properties tested, T. terrestris enzymes are comparable to, or in some cases better than, those from mesophilic strains. However, the differences are not so great as to compensate for the much lower productivity of T. terrestris compared to the improved T. reesei and Penicillium sp. strains.

Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae

SummaryPhleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.

Proteolytic events in the processing of secreted proteins in fungi.

Secreted heterologous proteins have been found to be produced much less efficiently by fungi than secreted homologous ones. This could be due, at least in part, to proteolytic cleavage by site-specific endoproteases of the secretory pathway, similar to the yeast KEX2 protease and the mammalian dibasic endoproteinases found in secretory pathways. Mature secreted fungal proteins may be protected from such cleavage due to the absence of cleavable sites in exposed regions. A comparison of the dipeptide distributions of 33 secreted and 34 cytoplasmic proteins from fungal producers of extracellular enzymes indicated a significant bias for some doublets, including the basic dipeptides Lys-Arg, Arg-Arg and Arg-Lys which have also been demonstrated to be KEX2 substrates. Other combinations were also found to be rare in secreted proteins, which could indicate either a broader specificity of the considered endopeptidase, or the presence either in the secretory organelles or among the secreted proteins of additional proteases with different specificities. Experimental evidence that the Lys-Arg site is processed in Tolypocladium geodes was provided by cloning a synthetic prosequence upstream of a phleomycin resistance (Sh ble) gene and analyzing the N-terminus of the corresponding protein purified from the culture supernatant. This system also provides a tool for further studies of specific proteases of fungi.

Cloning of Saccharomyces cerevisiae promoters using a probe vector based on phleomycin resistance

Vectors that confer high levels of phleomycin (Ph) resistance to Saccharomyces cerevisiae have been constructed with the TEF1 and ENO1 promoters, the Tn5 ble gene and the CYC1 terminator. They are able to transform yeast cells grown on rich glucose medium containing a moderate level of Ph (10 micrograms/ml, corresponding to 100-fold the minimal inhibitory concentration). Frequencies of transformation are identical to those obtained with the URA3 marker on a defined medium. A promoter probe vector, based on the same ble marker, enabled us to isolate sequences from chromosomal yeast DNA that had promoter activities. These DNA fragments have been sequenced and those which promote the highest levels of Ph resistance have been found to be either A + T-rich or have a potentially new and more efficient translation start site.

Phleomycin resistance as a dominant selectable marker in CHO cells.

The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.

Dectin-1 Is Essential for Reverse Transcytosis of Glycosylated SIgA-Antigen Complexes by Intestinal M Cells

This work reports the long-awaited identification of Dectin-1 and Siglec-5 as the M cell co-receptors that mediate the reverse transcytosis of secretory IgA molecules to mount a gut immune response.

First-in-man Phase 1 Clinical Trial of Gene Therapy for Advanced Pancreatic Cancer: Safety, Biodistribution, and Preliminary Clinical Findings

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.