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Fungal laccases have been widely used in industry. The expression of laccase often is repressible by the primary carbon source glucose in many fungi. The underlying basis is largely unclear. We demonstrate here that a gene, TSP2 - 1 , was required for laccase repression by glucose in the basidiomycete Cryptococcus neoformans . TSP2 - 1 encodes a Tsp2-type tetraspanin. The disruption of TSP2 - 1 resulted in constant melanin formation and the expression of the laccase gene LAC1 . This derepression phenotype was restorable by 10 mM exogenous cyclic AMP (cAMP). A capsule defect in the mutant tsp2 - 1 Δ also was restored by cAMP. The results indicate an interaction of Tsp2-1 with the cAMP-dependent protein kinase A (PKA) pathway that has been shown to modulate laccase repression and capsule biosynthesis in this fungus. Other roles of TSP2 - 1 , e.g., in maintaining cell membrane integrity and stress resistance, also were defined. This work reveals a Tsp2-1-dependent glucose repression in C. neoformans . The function of Tsp2-type tetraspanin Tsp2-1 is described for the first time.

We previously reported a taxol-producing fungus Pestalotiopsis malicola . There, we described the transformation of the fungus mediated by Agrobacterium tumefaciens. T-DNA carrying the selection marker was transferred into the fungus and randomly integrated into the genome as shown by Southern blotting. Approximately 66% of the transformants obtained a single copy of T-DNA. Conditions of transformation were optimized. The method can be used for taxol-related genes searching and manipulation in P. malicola . This is the first report of T-DNA-mediated transformation in taxol-producing fungi. Key words: T-DNA, random insertional mutagenesis, taxol, Pestalotiopsis .

Use of renewable plant biomass is an active area in current biotechnology research. This report explores the cellulose-degradation system and the factors affecting cellulase gene expression in Chaetomium globosum NK102.In the sequenced genome, we identified 10 cellulase genes by sequence homology alignment. We used a high-throughput sequencing technology (RNA-Seq) to monitor the differential expression of the genes under different culture conditions.We observed that cellulase activity increased with time in the fungal cultures. Accordingly, transcription of the genes encoding cellobiohydrolase, cellobiose dehydrogenase and endoglucanase (cbh1, cdh and eglI) was higher than the others. Expression of the transcriptional repressors, ACE I and CreA, decreased with the time, whereas expression of Hap2/3/5 complex was upregulated. In different carbon sources, cellulase activity and their gene transcription were repressed by glucose and were activated by cellobiose. Sorbitol had no significant effect. Interestingly, light affected positively the expression of these cellulase genes.Differential RNA-seq analysis can make preliminary exploration on the expression regulation of cellulase genes. Expression of the genes in C. globosum was determined by different culture conditions. This study has shown a molecular system of cellulose-degradation in C. globosum and provides information for interpretation of carbohydrate metabolism.

We developed the transformation methods of the strain Chaetomium globosum NK-102.We constructed plasmid pUCATPH-Pgap and compared the transformation efficiency with pUCATPH and pCM768. We established the PEG mediated protoplast transformation and Agrobacterium tumefaciens EHA105 mediated transformation methods.In protoplast approach, approximately 3 -5 transformants/microg DNA could be obtained. The highest efficiency of transformation was obtained by employing pUCATPH-Pgap. A. tumefaciens EHA105 successfully mediated T-DNA insertion into the genome of C. globosum NK-102 and the transformation rate was 3.2 x 10(2) transformants/10(7) spores. The transformants retained stable after generations. Southern blot analyses confirmed that the DNA had integrated into the chromosomal DNA of C. globosum NK-102.The transformation systems were good basis for selection of C. globosum mutant strains that effectively utilizing cellulose.