Development of the recombinant aequorin method and its evaluation for calcium measurement in filamentous fungi

The aim of this work was to express the Ca2+ sensitive photoprotein, apoaequorin, in filamentous fungi to a high level in order to allow routine and simple measurement of changes in cytosolic Ca2+ ([Ca2+]c) concentration in living hyphae. Analysis of the codon bias of the available apoaequorin genes showed a large discrepancy with the codon usage of the target fungi, Neurospora crassa, Aspergillus niger and A. awamori. To overcome this discrepancy, a synthetic apoaequorin gene with optimal codon usage for Neurospora and Aspergillus was designed and synthesised. An expression vector was generated for the production of apoaequorin from the synthetic gene using a constitutive Aspergillus promoter and suitable selectable markers for transformation. An expression vector was also produced for use in N. crassa to allow comparison of the aequorin levels obtained between the synthetic gene and the native gene. Strains of A. niger and A. awamori were transformed with the Aspergillus apoaequorin expression vector, and transformants expressing large amounts of active aequorin were selected and purified. Transformants of N. crassa were also produced using the Neurospora expression vector, and the highest expression transformants isolated and purified. Comparisons of the aequorin expression levels obtained in these transformants showed a 200-fold higher level of expression in A. awamori compared with that obtained in N. crassa. Also, the comparison between native and synthetic aequorin production showed a 280-fold higher level in the synthetic aequorin transformant. One high expression transformant from each Aspergillus species was chosen for investigation of [Ca2+]c responses to a variety of external stimuli. A successful method of growing cultures in small volume, still liquid culture in 96-well microtitre plates was developed which allowed efficient analysis of [Ca2+]c in growing cultures. Using such cultures, [Ca2+]c was monitored during germination, growth and conidiation. A suitable age of culture (18-36 h) was determined for investigation of changes in [Ca2+]c in response to external stimuli. The first stimulus investigated was the application of high extracellular concentration (50 mM) of Ca2+, which elicited a large [Ca2+]c increase.