Rodrigo Mora-Lugo

• Solid-state fermentation (SSF) processes with the filamentous fungus Aspergillus sojae ATCC 20235 (A. sojae) are known to yield good levels of pectinolytic enzymes which are valuable biocatalysts in the food industry. However, in fungal-based SSF systems there is a poor availability of oxygen in inner regions of the fermented mass, leading to limited cell growth and protein production. Previous studies showed that the heterologous expression of the bacterial Vitreoscilla hemoglobin (VHb) –commonly referred to as the “vgb/VHb technology”- can favor respiratory metabolism and cell growth of prokaryotes and eukaryotes under oxygen-limited conditions. The main purpose of this thesis was to genetically engineer A. sojae with VHb aiming to enhance its cell growth and protein production under conditions of SSF. An Agrobacterium tumefaciens-mediated transformation procedure was established to successively integrate the VHb gene (vgb) into the genome of A. sojae. The transformed A. sojae strain harboring vgb (A. sojae vgb+) yielded more biomass and enzymatic units of exo-pectinases and protease in comparison to its parental strain (A. sojae wt) in solid-state cultures in Erlenmeyer flasks. Fermentations were up-scaled (100 times) in a drum-type reactor and the recombinant fungus produced up to 38% more pectinases and 33% more proteases in comparison to its wild-type. Furthermore, enzymatic extracts of A. sojae vgb+ clarified apple juice and blood orange juice with up to 37% and 50% more efficiency, respectively in comparison to the enzymatic extracts of A. sojae wt. Overall, the results of this dissertation show that the vgb/VHb technology can improve cell growth and protein production of A. sojae in SSF and that the protein complex of the recombinant A. sojae vgb+ was substantially more efficient in the clarification of fruit-based beverages compared to A. sojae wt.