| Summary: | Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2006. === Filamentous fungi have been employed for production of heterologous proteins such as
enzymes, antibiotics and vaccines due to their good secretion capacities and effective posttranslational
modifications of these proteins. With an improvent in recombinant DNA
technologies it has become possible to express many useful proteins in species such as the
Aspergilli. However the submerged cultivation of filamentous fungi is complicated by the
difficulties in mixing and oxygen and nutrient transfer in the highly viscous culture fluids
that result.
The purpose of the project was to investigate the potential of simultaneous control of
morphology and production of enzymes in the dimorphic fungus, Mucor circinelloides, in
order to overcome problems associated with the submerged cultivation of filamentous
fungi. Dimorphic M. circinelloides, a zygomycete in the order Mucorales, occurs in a
filamentous form or a yeast-like morphology in response to environmental conditions.
Recently, advances were made in transformation of Mucor, and it has become possible to
transform M. circinelloides to express heterologous proteins. The first example of a
strong, regulated promoter from M. circinelloides being used for recombinant protein
production was the expression of the glucose oxidase gene (from Aspergillus niger) under
the control of the glyceradehyde-3-phosphate dehydrogenase (gpd1) promoter. Glucose
oxidase (GOX) is an enzyme used to prevent oxidation of foods to extend shelf-life, to
produce low-kilojoule beverages and to measure glucose levels in medical diagnostic
applications.
The scope of this project was to establish the conditions for yeast and filamentous growth
of M. circinelloides in order to allow control of morphology, and to evaluate enzyme
production under these conditions. Enzyme production of the GOX producing mutant
strain, that was recently constructed, was compared to that of a wild type M.circinelloides
strain. M. circinelloides was cultured in two-stage batch fermentations, firstly a yeast stage and
then a filamentous stage. The yeast morphology was induced by anaerobic conditions
while the filamentous morphology was achieved by exposure to air. The enzyme, biomass
and metabolite production of the glucose-oxidase producing mutant strain and the wild
type were monitored during the two-stage fermentations. GOX from the mutant and native
amylase activity levels from the wild type were compared to each other and to other
production systems for these enzymes.
The morphology could be maintained in a yeast form under N2 with addition of ergosterol
and Tween 80. The GOX activity levels in the culture fluid were comparable to some of
the unoptimized GOX production systems in literature, but much lower than the optimized,
recombinant GOX production systems that employ certain yeasts, or Aspergilli or
Penicillium. The intracellular GOX levels were almost 6-fold higher than the extracellular
levels which was unexpected as GOX is usually well-secreted. The morphological control
improved the morphology for the initial yeast-stage of the fermentation but did not
improve the morphology during the filamentous, enzyme-producing stage and it decreased
the biomass yield and enzyme production by 50%.
The constraint of Mucor to its yeast-like form did not improve the broth homogeneity or
enzyme production and increased the time required for enzyme production. In this study
M. circinelloides did not perform that well against other species already used to produce
these enzymes. However, M. circinelloides could be used to produce enzymes from
zygomycetes that systems such as A. niger do not produce well.
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