Use of laccase as a novel, versatile reporter system in filamentous fungi.

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

A genome-wide transcription analysis of a fungal riboflavin overproducer.

The production of many fine chemicals such as vitamins and amino acids is carried out in bioreactors using microorganisms. Usually, these strains are developed from wild-type organisms by classical mutation and selection. After several generations of strain improvement, no further enhancement can be achieved. Therefore, metabolic engineering (ME) is a rational approach to optimise such producer organisms beyond this point, or for starting all over from the beginning. Metabolic Engineering involves detailed analysis of the organism's metabolic and genetic properties, leading to the identification of new target genes. The fungal riboflavin overproducer Ashbya gossypii converts vegetable oil to vitamin B2 in a "one-step reaction". The productivity and selectivity of this microorganism have been optimised significantly over the years, first following a classical approach and now a rational one. The improvement is based on our understanding of vitamin B2 metabolism. We have been able to selectively enhance the pathways that are necessary for the formation of riboflavin and to inhibit those leading to unwanted side products. New targets for further improvements of this process have been found using a genome-wide transcript expression analysis; namely massive parallel signature sequencing (MPSS). With this analysis even completely unknown genes can be used for strain improvement.

THTA, a thermotolerance gene of Aspergillus fumigatus.

Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.

Molecular analysis of CPRalpha, a MATalpha-specific pheromone receptor gene of Cryptococcus neoformans.

The putative Cryptococcus neoformans pheromone receptor gene CPRalpha was isolated and studied for its role in mating and filamentation. CPRalpha is MATalpha specific and located adjacent to STE12alpha at the MATalpha locus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-protein-coupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of the CPRalpha gene exhibited drastic reductions in mating efficiency but were not completely sterile. Delta cpr alpha cells displayed wild-type mating efficiency when reconstituted with the wild-type CPRalpha gene. Hyphal production on filament agar was not affected in the delta cpr alpha strain, indicating no significant role for CPRalpha in sensing environmental cues during haploid fruiting. The wild-type MATalpha CPRalpha strain produced abundant hyphae in response to synthetic MATa pheromone; however, the hyphal response to pheromone by delta cpr alpha cells was significantly reduced. Exposure of wild-type cells to synthetic MATa pheromone for 2 h induced MFalpha pheromone expression, whereas unexposed cells showed only basal levels of the MFalpha transcript. The delta cpr alpha cells, however, exhibited only basal levels of MFalpha message with or without pheromone exposure, suggesting that CPRalpha and MFalpha are components of the same signaling pathway.