Characterisation of the Mucor circinelloides regulated promoter gpd1P.

The promoter of the Mucor circinelloides gpd1 gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd1P) was recently cloned and used for the production of recombinant proteins, such as the Aspergillus niger glucose oxidase 1 (GOX). This represents the first example of the application of a strong and regulated promoter from this fungus for recombinant protein production. The original 741-bp gpd1P promoter fragment conferred hexose-dependent expression of GOX in M. circinelloides. To understand the regulatory mechanisms involved in gpd1P-driven expression and to develop improved promoter fragments, deletion derivatives of gpd1P were constructed. These derivatives were fused to the A. niger gox1 gene and used to construct strains containing a single copy of the expression cassette. GOX activity was detected in strains containing the full-length gpd1P and also in strains containing a 713-bp or a 361-bp derivative. Expression levels for the 361-bp derivative were high and comparable, regardless of the carbon source used. This promoter represents a useful derivative for constitutive heterologous gene expression in M. circinelloides.

Cloning of glyceraldehyde-3-phosphate dehydrogenase-encoding genes in Mucor circinelloides (Syn. racemosus) and use of the gpd1 promoter for recombinant protein production.

Three genes (gpd1, gpd2, and gpd3) encoding glyceraldehyde-3-phosphate dehydrogenase were isolated from the dimorphic zygomycete Mucor circinelloides by PCR using degenerated primers. Transcription of gpd1 could be detected during vegetative growth under both aerobic and anaerobic conditions, whereas neither gpd2 nor gpd3 transcription was detected, indicating that gpd1 is the major transcribed gpd gene. The transcription of gpd1 was regulated by carbon source. The gpd1 promoter was successfully used for recombinant expression of genes of both homologous (crgA encoding a regulator of carotene biosynthesis) and heterologous (gox1 from Aspergillus niger encoding glucose oxidase; GOX) nature. Growth of a gox1 transformant strain resulted in the secretion of enzymatically active GOX. The potential advantages of using a dimorphic fungus for heterologous protein production are discussed.

Identification and analysis of genes involved in the control of dimorphism in Mucor circinelloides (syn. racemosus).

Mucor circinelloides (syn. racemosus) is a non-pathogenic dimorphic fungus belonging to the class of zygomycetes. We are developing a novel system for heterologous protein production exploiting the dimorphic growth characteristics of M. circinelloides. In order to identify potential genetic regulators of morphology we have initiated a characterisation of key genes involved in signal transduction in Mucor. We have cloned and characterised pkaR and pkaC encoding the regulatory subunit (PKAR) and the catalytic subunit (PKAC), respectively, of the cAMP-dependent protein kinase A (PKA) of M. circinelloides. In anaerobically grown yeast cells, the levels of expression of both pkaR and pkaC were significantly higher than the levels of expression in aerobically grown mycelium. However, during the dimorphic shift, i.e. during the transition from anaerobic yeast growth to aerobic filamentous growth, the expression of pkaR was found to increase approximately two-fold. These results indicate that regulation of PKA activity is conferred at different levels according to growth and environmental conditions. Overexpression of pkaR resulted in a multi-branched colony phenotype on solid medium indicating that PKAR plays a role in filamentation and branching. Fragments of genes encoding factors of the mitogen-activated protein (MAP) kinase (MAPK) pathway have also been cloned: mpk1 (mitogen-activated protein kinase 1) encoding a MAPK homologue and ste12 encoding a transcription factor.