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

[摘要] Filamentous fungi have been employed for production of heterologous proteins such asenzymes, antibiotics and vaccines due to their good secretion capacities and effective posttranslationalmodifications of these proteins. With an improvent in recombinant DNAtechnologies it has become possible to express many useful proteins in species such as theAspergilli. However the submerged cultivation of filamentous fungi is complicated by thedifficulties in mixing and oxygen and nutrient transfer in the highly viscous culture fluidsthat result.The purpose of the project was to investigate the potential of simultaneous control ofmorphology and production of enzymes in the dimorphic fungus, Mucor circinelloides, inorder to overcome problems associated with the submerged cultivation of filamentousfungi. Dimorphic M. circinelloides, a zygomycete in the order Mucorales, occurs in afilamentous form or a yeast-like morphology in response to environmental conditions.Recently, advances were made in transformation of Mucor, and it has become possible totransform M. circinelloides to express heterologous proteins. The first example of astrong, regulated promoter from M. circinelloides being used for recombinant proteinproduction was the expression of the glucose oxidase gene (from Aspergillus niger) underthe control of the glyceradehyde-3-phosphate dehydrogenase (gpd1) promoter. Glucoseoxidase (GOX) is an enzyme used to prevent oxidation of foods to extend shelf-life, toproduce low-kilojoule beverages and to measure glucose levels in medical diagnosticapplications.The scope of this project was to establish the conditions for yeast and filamentous growthof M. circinelloides in order to allow control of morphology, and to evaluate enzymeproduction under these conditions. Enzyme production of the GOX producing mutantstrain, that was recently constructed, was compared to that of a wild type M.circinelloidesstrain. M. circinelloides was cultured in two-stage batch fermentations, firstly a yeast stage andthen a filamentous stage. The yeast morphology was induced by anaerobic conditionswhile the filamentous morphology was achieved by exposure to air. The enzyme, biomassand metabolite production of the glucose-oxidase producing mutant strain and the wildtype were monitored during the two-stage fermentations. GOX from the mutant and nativeamylase activity levels from the wild type were compared to each other and to otherproduction systems for these enzymes.The morphology could be maintained in a yeast form under N2 with addition of ergosteroland Tween 80. The GOX activity levels in the culture fluid were comparable to some ofthe unoptimized GOX production systems in literature, but much lower than the optimized,recombinant GOX production systems that employ certain yeasts, or Aspergilli orPenicillium. The intracellular GOX levels were almost 6-fold higher than the extracellularlevels which was unexpected as GOX is usually well-secreted. The morphological controlimproved the morphology for the initial yeast-stage of the fermentation but did notimprove the morphology during the filamentous, enzyme-producing stage and it decreasedthe biomass yield and enzyme production by 50%.The constraint of Mucor to its yeast-like form did not improve the broth homogeneity orenzyme production and increased the time required for enzyme production. In this studyM. circinelloides did not perform that well against other species already used to producethese enzymes. However, M. circinelloides could be used to produce enzymes fromzygomycetes that systems such as A. niger do not produce well.

[发布机构] Stellenbosch University