Patent Application
20080014620
The anaerobic fungus Piromyces sp. E2 (ATCC 76762), isolated from faeces of an Indian elephant, was grown anaerobically under N2/CO2 (80%/20%) at 39° C. in medium M2 supplemented with various carbon sources (24). Carbon sources used were Avicel (microcrystaline cellulose type PH 105, Serva, Germany), fructose or xylose (all 0.5%, w/v). After growth ceased, as judged by hydrogen production, the cells were harvested by centrifugation (15,000×g, 4° C., 15 min;) or by filtration over nylon gauze (30 μm pore size).
The fungal cells were washed with deionized water to remove medium components. Cell-free extracts were prepared by freezing the cells in liquid nitrogen and subsequent grinding with glass beads (0.10-0.11 mm diameter) in a mortar. Tris/HCl buffer (100 mM, pH 7.0) was added to the powder (1:1, w/v) and after thawing for 15 min the suspension was centrifuged (18,000×g, 4° C., 15 min). The clear supernatant was used as a source of intracellular enzymes.
Xylose isomerase activity was assayed at 37° C. in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 10 mM xylose, 10 mM MgCl2 and a suitable amount of cell-free extract. The amount of xylulose formed was determined by the cysteine-carbazole method (9). Xylulose kinase and xylose reductase activities were assayed as described by Witteveen et al. (28). One unit of activity is defined as the amount of enzyme producing 1 nmol of xylulose per min under the assay conditions. Xylulose formed was determined by the method of Dische and Borenfreund (Dische and Borenfreund, 1951, J. Biol. Chem. 192: 583-587) or by HPLC using a Biorad HPX-87N Column operated at 80° C. and eluated at 0.6 ml/min using 0.01 M Na2BPO4 as the eluens. Xylose and xylulose were detected by a Refractive Index detector at an internal temperature of 60° C.
Specific activity is expressed as units per mg protein. Protein was determined with the Bio-Rad protein reagent (Bio-Rad Laboratories, Richmond, Calif., USA) with bovine γ-globulin as a standard.
Random Sequencing of a Piromyces sp. E2 cDNA Library
The cDNA library constructed in the vector lambda ZAPII as described previously (2) was used. An aliquot of this library was converted to pBluescript SK-clones by mass excission with the ExAssist helper phage (Stratagene, La Jolla, Calif., USA). Randomly selected clones were sequenced with the M13 reverse primer to obtain 5′ part sequences. Uncomplete cDNAs were used to synthesize probes which were used to rescreen the library. To obtain fall length sequences subclones were generated in pUC18. Sequencing was performed with the ABI prism 310 automated sequencer with the dRhodamine terminator cycle sequencing ready reaction DNA sequencing kit (Perkin-Elmer Applied Biosystems).
Randomly selected clones from a cDNA library of the anaerobic fungus Piromyces sp. E2 were sequenced and this resulted in two clones (pH97 and pAK44) which sequences showed high homology to xylose isomerase and D-xylulokinase genes, respectively. The clones were analysed in detail.
Clone pH97 did not contain a complete ORF and therefore the cDNA library was rescreened with a probe designed on the basis of sequence data from clone pH97. This resulted in a clone pR3 with an insert of 1669 bp. An ORF encoding a protein of 437 amino acids with high similarity to xylose isomerases could be identified. Although the 5′ untranslated region comprises only 4 bp, the presumed starting methionine residue fitted well into an alignment of known xylose isomerase sequences. The 3′ untranslated region was 351 bp long and had a high AT content, which is typical for anaerobic fungi. The ORF contained the amino acids shown to be important for interaction with the substrate (catalytic triad His 102, Asp 105, Asp 340 and Lys 235) and binding of magnesium (Glu 232) (14, 26). Further, the two signature patterns (residues 185-194 and 230-237) developed for xylose isomerases (20) were present. The Piromyces sp. E2 xylose isomerase (XylA) shows the highest homology to the enzymes of Haemophilus influenza (52% identity, 68% similarity) and Hordeum vulgare (49% identity, 67% similarity). The polypeptide deduced from the cDNA sequence corresponds to a molecular mass of 49,395 Da and has a calculated pI of 5.2.
The second clone, pAK44, had an insert of 2041 bp and contained a complete ORF encoding a protein of 494 amino acids with a molecular weight of 53,158 Da and a pI of 5.0. The first methionine is preceeded by a 111 bp 5′ untranslated region, while the 3′ untranslated region comprised 445 bp. Both regions are AT-rich. BLAST and FASTA searches revealed high similarity to xylulokinases. The two phosphate consensus regions defined by Rodriguez-Peña et al. (22) were found at positions 6-23 and 254-270 as shown in a partial alignment. Moreover the signatures for this family of carbohydrate kinase as described in the Prosite database were identified (131-145 and 351-372). The Piromyces sp. E2 xylulokinase (XylB) showed highest homology with the XylB protein of Haemophilus influenza (46% identity, 64% similarity).
cDNA from Piromyces sp. E2 was used in a PCR reaction with pfu polymerase (Stratagene). The primers were designed using the sequences from the 5′ and 3′ ends of the xylose isomerase gene and also contained a Sfi I and a XbaI restriction site. The PCR product was cloned in the pPICZα vector (Invitrogen, Carlsbad, Calif., USA). To obtain the xylose isomerase gene, the pPICZα vector was digested with EcoRI and XbaI. The digestion product was ligated-into the pYes2 vector (Invitrogen). The pYes2 plasmid with the xylose isomerase gene was transformed into Saccharomyces cerevisiae (stam BJ1991, gift from Beth Jones, UvA). The genotype of this strain is: matα, leu2, trp1, ura 3-251, prb1-1122 and pep4-3.
Transformants were plated on SC plates (0.67% YNB medium+0.05% L-Leu+0.05% L-Trp+2% glucose+2% agarose). Untransformed cells can not grow on these plates.
Transformed Saccharomyces cerevisiae cells were grown on glucose medium at 25° C. for 72 h (raffinose can be used as an alternative for glucose). Cells were harvested and resuspended in SC medium with galactose instead of glucose. After 8 h of induction cells were harvested and lysed using glass beads (0.10-0.11 mm diameter) and “breaking buffer” (50 mM phosphate buffer+5% glycerol+protease inhibitor). After lysis the mixture was centrifuged (18,000×g, 4° C., 15 min). The clear supernatant was used to determine xylose isomerase activity using the method described above (Example 1). An activity of 10 U per mg protein was measured at 37° C.
Saccharomyces cerevisiae strains were grown on SC-medium with the following composition: 0.67% (w/v) yeast nitrogen base; 0.01% (w/v) L-tryptophan; 0.01% (w/v) L-leucine and either glucose, galactose or xylose, or a combination of these substrates (see below). For agar plates the medium was supplemented with 2% (w/v) bacteriological agar.
Saccharomyces cerevisiae strain BJ1991 (genotype: matα, leu2, trp1, ura 3-251, prb1-1122, pep4-3) transformed with pYes2 without insertion and three selected transformants (16.2.1; 16.2.2 and 14.3) containing pYes2 with the Piromyces sp. E2 xylose isomerase gene were grown on SC-agar plates with 10 mM glucose as carbon source. When colonies were visible, single colonies were used to inoculate liquid SVC-medium with 100 mM xylose and 25 mM galactose as carbon sources. Growth was monitored by measuring the increase in optical density at 600 nm on a LKB Ultrospec K spectrophotometer.
The results of the growth experiments are compiled in
The pPICZα vector, containing the Piromyces sp. E2 gene coding for xylose isomerase, was used as a template for PCR with VentR DNA polymerase (New England Biolabs). The primers were designed using the 5′ and 3′ sequences of the gene coding for xylose isomerase and included an EcoRI and an SpeI site. Additionally the primers were designed to remove the XbaI site found in the pPICZα construct, replacing it with a stopcodon (TAA). The final product was designed to restore the original open reading frame, without the added aminoacids (his and c-Myc tags) found in the pPICZα construct. The PCR product was cut with EcoRI and SpeI. The final product was cloned into a vector derived from pYES2 (Invitrogen). In this vector the GAL1 promoter found in pYES2 was replaced by the TPI1 promoter in order to ensure constitutive expression of the xylose isomerase, thereby eliminating the need for galactose in the medium. The TPI1 promoter was cloned from a modified form of plasmid pYX012 (R&D systems). The promoter was cut out as a NheI-EcoRI fragment. Both the TPI1 promoter and the PCR product of the gene coding for the xylose isomerase were ligated into pYES2 cut with SpeI and XbaI. This plasmid was used to transform Saccharomnyces cerevisiae strain CEN.PK113-5D (gift from Peter Kõtter, Frankfurt). The genotype of the strain is: MatA ura3-52. Transformants were selected on mineral medium plates (Verduyn et al.: Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. (1992) Yeast 8(7):501-17) with 2% glucose as the carbon source. Untransformed cells cannot grow on these plates.
Transformants were grown on glucose/xylose mixtures in carbon-limited chemostat cultures. Transformants grown under these conditions exhibit high xylose isomerase activities (800 units per mg at 30° C.) according to a specific enzyme assay as developed by Kersters-Hildersson et al. (Kinetic characterization of D-xylose isomerases by enzymatic assays using D-sorbitol dehydrogenase. Enz. Microb. Technol. 9 (1987) 145-148). The in vitro activity of xylose isomerase in the cell-free extracts of the transformed S. cerevisiae strain was dependent on bivalent cations (Mg2+ or Co2+) and a relatively low Km value for xylose of approximately 20 mM was measured.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02075266.3 | Jan 2002 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NL03/00049 | 1/23/2003 | WO | 00 | 12/6/2004 |
