To construct strains of the filamentous fungus Trichoderma reesei with low cellobiohydrolases while high endoglucanase activity, the Pcbh1-eg3-Tcbh1 cassette was constructed and the coding sequence of the cellobiohydrolase I (CBHI) gene was replaced with the coding sequence of the eg3 gene by homologous recombination. Disruption of the cbh1 gene was confirmed by PCR, Southern dot blot and Western hybridization analysis in two transformants denoted as L13 and L29. The filter paper-hydrolyzing activity of strain L29 was 60% of the parent strain Rut C30, and the CMCase activity was increased by 33%. This relatively modest increase suggested that the eg3 cDNA under the control of the cbh1 promoter was not efficiently transcribed as the wild type cbhl gene. However our results confirmed that homologous recombination could be used to construct strains of the filamentous fungus Trichoderma reesei with novel cellulase profile. Such strains are of interest from the basic science perspective and also have potential industrial applications.

A mitochondrial gene cluster, encoding proteins homologous to NADH dehydrogenase subunits II and III (ND2 and ND3) and seven tRNAs, from Trichoderma reesei QM9414 was cloned and sequenced. These genes are clustered tandemly on the mitochondrial genome of QM9414. Phylogenetic analysis showed that ND2 and ND3 were most closely related to the mitochondrialND subunits II (71% identity) and III (70% identity) from Podospora anserine. Northern dot blot analysis showed that the nd-and nd3 genes are actively transcribed in the T. reesei mitochondria.

The filamentous fungi from the Huanghai sea sludge were screened according to their ability to produce cold-ac-tive α-amylase. The strain with the highest amylase activity was identified as Penicillium species. The α-amylase purified by ammonium sulphate precipitation and column chromatography on DEAE2sepharose and sephadex G2100 shows a molecular weight of about 55000 and a p I of 4. 38. The enzyme is stable in a pH range of 5. 5-8. 0 and has a maximum activity at pH 6. 0. Compared with the α-amylase from mesophiles and thermophiles, the cold-ac-tive enzyme shows a high enzyme activity at lower temperatures and a high sensitivity at temperatures higher than 50℃. The optimal temperature is 40℃and the activity decreases dramatically at temperatures above 50℃. Ca2+shows a significant effect on maintaining the structure and the activity of the enzyme. EDTA and Cu2 + are its in-hibitors. The products from the hydrolysis of soluble starch with the cold-active enzyme are maltose and other oligosaccharides.

To construct a yeast one-hybrid system and isolate transcriptional activators. Methods and Results: A 1

To inhibit xylitol dehydrogenase (XDH) in Trichoderma reesei by antisense inhibition strategy and construct novel strains capable of accumulating xylitol. Methods and Results: The xdh1 antisense expression plasmid pGTA-xdh was constructed by inserting xdh1 DNA fragment inversely between the gpdA promoter and the trpC terminator from Aspergillus nidulans into a pUC19 plasmid backbone. Trichoderma reesei protoplasts were co-transformated with pGTA-xdh and hygromycin B resistance plasmid pAN7-1. Of 20 transformants screened from the selective medium, one transformant with the highest xylitol accumulation, designated ZY15, showed a distinct reduction (c. 52%) in XDH activity compared with the original strain Rut-C30. The results of Southern hybridization and PCR assay showed that the antisense expression cassette of xdh1 was integrated into the genome of T. reesei. The RT-PCR analysis proved that antisense RNA effectively inhibited XDH expression (c. 65%). Xylitol accumulation 2