Masahiro Nogawa

l-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei

Abstractl-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner, different from that of sophorose in Trichoderma reesei. Through Northern blot analysis however, l-sorbose was found to regulate coordinately six cellulase genes (including egl3, whose behavior has not been studied so far) at transcriptional level, as is the case with sophorose in T. reesei strains PC-3-7 and QM9414. Dot blot analysis showed that the proportions of each cellulase mRNA to cbh1 mRNA, the largest amount of mRNA transcribed in T. reesei, did not change when l-sorbose or sophorose was used as an inducer in the PC-3-7 and QM9414 strains. cbh2 and egl1 mRNAs were about 45–60% and 20–30% of the cbh1 transcript, whereas small amounts of mRNA, 1–2% of cbh1, were observed on other endoglucanase genes. Furthermore, the PC-3-7 strain showed an enhanced level of cellulase gene transcription, about two- and four- to six-fold higher than that of the QM9414 strain with sophorose and l-sorbose, respectively.

A third xylanase from Trichoderma reesei PC-3-7

Abstract A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III.

Chitosanase from the Plant Pathogenic Fungus, Fusarium solani f. sp. Phaseoli—Purification and Some Properties

Among 162 strains of the genus Fusarium tested, 22 strains, mainly from F. solani and F. splendens, formed halos on chitosan-containing agar medium. Chitosanase secreted in the culture by the most effective producer, F. solani f. sp. phaseoli SUF386, was further investigated. N-Acetylglucosamine (GlcNAc) used as a carbon source was most effective for production of chitosanase. Chitosan, a substrate for chitosanase, inhibited cell growth completely and abolished production of chitosanase when used as a carbon source in the liquid medium. Chitosanase purified from the culture filtrate had a molecular mass of 36 kDa, and showed a maximum activity at pH 5.6 and 40°C. The enzyme catalyzed the hydrolysis of chitopentaose, chitosan (70% and 100% deacetylation), and glycolchitosan, but showed little activity toward chitobiose, chitotriose, chitotetraose, glycol chitin, and carboxymethyl cellulose. A rapid reduction in the viscosity of chitosan solutions suggested that the enzyme catalyzed an endo-type cleavage reaction.

Regulation of xyn3 gene expression in Trichoderma reesei PC-3-7.

Abstract The characteristics of regulation of the gene encoding the third xylanase (Xyn III) of a filamentous fungus, Trichoderma reesei PC-3–7, were studied by Northern blot analysis. A partial DNA sequence (185 bp) of the xyn3 gene was obtained by PCR amplification of genomic DNA of T. reesei PC-3–7 and sequenced. This xyn3 gene fragment was used as a probe for Southern and Northern blot analysis. The expression of the xyn3 gene was regulated at the transcriptional level. The xyn3 mRNA was expressed in mycelia of T. reesei PC-3–7 induced by Avicel, l-sorbose and sophorose, but not by xylose, xylooligosaccharides and birchwood xylan. Furthermore, it was observed that xyn3 was synchronously expressed with egl1 but not with xyn1 and xyn2 by l-sorbose, indicating that the xyn3 gene may be coordinately expressed with cellulase genes. By Southern blot analysis, the xyn3 gene was confirmed to exist as a single copy in both strains of T. reesei PC-3–7 and QM9414. However, no xyn3 mRNA appeared in the mycelia induced by any kind of inducers in T. reesei QM9414 even when total RNA was used in large excess, suggesting that the xyn3 gene in T. reesei QM9414 is in the dormant state and cannot be expressed. Therefore, T. reesei PC-3–7 may be a very useful strain for elucidating the induction mechanism of xylanase biosynthesis by cellulosic and xylanosic substrates, and also the regulatory correlation between cellulase and xylanase induction.

Cloning and Characterization of a Chitosanase Gene from the Plant Pathogenic Fungus Fusarium solani

The plant pathogenic fungus Fusarium solani f. sp. phaseoli SUF386 secretes a chitosanase in the absence of exogenous chitosan. Based on partial amino acid sequences of the purified chitosanase, two degenerate oligonucleotides were synthesized and used as reverse transcription-mediated PCR (RT-PCR) primers to amplify a 500-bp fragment of corresponding cDNA. The PCR product was used as a probe to isolate the genomic copy of the gene (csn). F. solani csn has an open reading frame encoding a polypeptide of 304 amino acid residues with a calculated molecular mass of 31,876 Da and containing a putative 19-amino acid residue signal sequence. Comparison between the genomic and cDNA sequences revealed that three introns are present in the coding region. Southern blot analysis results indicated that csn is present as a single copy in the genome of F. solani SUF386. The cDNA fragment corresponding to the mature enzyme was introduced into E. coli using an expression vector driven by the T7 promoter. The resulting E. coli transformant overproduced proteins with chitosanolytic activity.

In Planta Transformation of Kenaf Plants (Hibiscus cannabinus var. aokawa No. 3) by Agrobacterium tumefaciens

Kenaf was transformed by inoculation of Agrobacterium tumefaciens onto the meristems of young plants in pots. The transformation was demonstrated by three lines of evidence: a phenotypic inheritance from T(0) to T(1) plants, detection of the transgene in both T(0) and T(1) plants, and rescue of plasmids composed of T-DNA of the binary vector and flanking plant genomic DNA from T(1) plants.

Identification of active site carboxylic residues in Trichoderma reesei endoglucanase Cel12A by site-directed mutagenesis

Abstract cDNA of Cel12A (formerly EG III), one of five endoglucanases of Trichoderma reesei , was expressed in Escherichia coli by using the tac promoter of E. coli . Transformants of E. coli harboring a plasmid pAGmegl3 containing mature form Cel12A cDNA produced Cel12A protein largely as insoluble inclusion bodies in the cytoplasm of the cells. The insoluble fraction was solubilized with urea from which Cel12A was purified by cation chromatography to electrophoretic homogeneity. The purified enzyme was immunologically and enzymologically identical to that of Cel12A purified from T. reesei . E116 and E200 of Cel12A of T. reesei are completely conserved in family-12 cellulases. In order to investigate the role of these two glutamate residues in the enzymic function of Cel12A, two mutant enzymes were produced at each position, namely E116D/Q and E200D/Q. The specific activity of these mutant enzymes is reduced by more than 98%, revealing the importance of these two residues to the catalytic function of Cel12A. The data demonstrated that E116 and E200 are the nucleophilic and acid-base residues, respectively.

The bacterium Burkholderia gladioli strain CHB101 produces two different kinds of chitinases belonging to families 18 and 19 of the glycosyl hydrolases

Two genes (chiA and chiB) coding for chitanases A and B (ChiA and ChiB) were isolated from the chitinolytic bacterium, Burkholderia gladioli strain CHB101. chiA contains an open reading frame that encodes a protein of 343 amino acids, whereas chiB encodes a protein of 307 amino acids. The deduced amino acid sequence of ChiA showed a high similarity to those of microbial chitinases belonging to family 18 of the glycosyl hydrolases, while ChiB showed significant sequence similarity to plant chitinases and Streptomyces spp. chitinases belonging to family 19.

Xylanase induction by L-sorbose in a fungus, Trichoderma reesei PC-3-7

Xylanase induction by L-sorbose was studied in a resting cell system of a filamentous fungus, Trichoderma reesei PC-3-7, a hypercellulolytic mutant, and compared with that by other inducers. L-Sorbose induced xylanase activity as well as cellulase. It induced a higher level of xylanase activity than sophorose and xylose did. Three main xylanases, xylanase I (Xyn I), xylanase II (Xyn II), and a non-specific endoglucanase I (EG I), were separated using cation-exchange chromatography, and their activity were measured. Xyn II was induced in about the same proportion (60-80% of the total xylanase activity) by all inducers used. On the other hand, Xyn I was apparently induced by L-sorbose, xylose, and xylooligosaccharides, but only a little by sophorose. Northern blot analysis showed that L-sorbose induced Xyn I and Xyn II at the transcriptional level, and more xyn1 mRNA was transcribed after L-sorbose addition than after sophorose. These results suggested that the expressions of both Xyn I and Xyn II are regulated, at least in part, in a different manner. Furthermore, the Xyn I induction by L-sorbose indicated that an unknown common regulatory mechanism may exist between Xyn I and cellulase inductions.

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments.