Filamentous co-cultivation for the conversion of cellulose into pigmented antibiotics

  • Filamentöse Ko-Kultivierung für die Umwandlung von Zellulose in Pigmentierte Antibiotika

Finger, Maurice; Büchs, Jochen (Thesis advisor); Agler-Rosenbaum, Miriam (Thesis advisor)

Aachen : RWTH Aachen University (2023)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2023

Abstract

The demand for novel natural products such as antibiotics is greater than ever. Driven by emerging antibiotic resistances, new production processes are of particular interest. Especially filamentous co-cultivations offer vast potential for the discovery of novel natural products or process intensification. However, these benefits go hand in hand with an inherently increased complexity compared to axenic cultivations, impeding utilization due to the lack of process understanding. This thesis focused on establishing a model filamentous co-cultivation process with the cellulolytic fungus Trichoderma reesei RUT-C30 and the soil bacterium Streptomyces coelicolor A3(2) for the production of natural products. By providing cellulose as the primary carbon source, a dependency was created between the co-cultivation partners. The cellulose was hydrolysed by cellulases, releasing soluble sugars. Cellulases were only formed and secreted by T. reesei. The complexity of this system was reduced by the utilization of high-throughput online monitoring systems. Biomass formation of the co-cultivation partners was individually tracked by monitoring the fluorescence protein mNeonGreen in S. coelicolor and mCherry in T. reesei with an in-house built 48 well microtiter plate-based system. In combination with the information on the oxygen transfer rates, the influence of the inoculation ratio, the osmolality and the specific power input on the population composition were successfully derived. The co-cultivation was transferred from the microtiter plate into a stirred tank reactor. In the stirred tank reactor, a more than 2-fold increased titer for the pigmented antibiotic actinorhodin was achieved compared to glucose-limited axenic S. coelicolor cultivations. As substrate affinity is a critical parameter in carbon source limited co-cultivations, a novel respiratory method was developed for the fast initial estimation. The method was utilized to estimate the glucose affinity of Corynebacterium glutamicum and T. reesei as well as the fructose affinity of a genetically modified Gluconobacter oxydans. The potential of respiration monitoring was further demonstrated for agar cultivations. Instead of a visual examination, the respiration represents an objective parameter for evaluating cultivations on solid media. The method was successfully demonstrated for several agar-based applications. The presented work provides valuable insights and tools for the development of defined filamentous co-cultivations, which assist in the production of novel natural product.

Institutions

  • Chair of Biochemical Engineering [416510]

Identifier

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