Evaluation of the effect of morphological control of dimorphic Mucor circinelloides on heterologous enzyme production
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.