Process development for heterotrophic terpene production in Cupriavidus necator
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The global demand for the sesquiterpene α-humulene is steadily increasing due to its wide range of applications in the food, fragrance, cosmetics, and pharmaceutical industries. The process parameters for microbial α-humulene production using Cupriavidus necator still offer optimization potential to fully exploit the significant advantages of biotechnological production over traditional production methods. The primary objective of this work was, therefore, the optimization of heterotrophic α-humulene production using C. necator, with a focus on in-depth investigation and optimization of various process parameters.In initial studies, the used statistical experimental design demonstrated that a temperature range of 25 °C to 28 °C resulted in increased plasmid-based α-humulene production, along with improved product/substrate and product/biomass yield coefficients compared to the established standard temperature of 30 °C. Subsequent optimization of individual process parameters led to enhanced biomass formation and α-humulene production. The fructose concentration was increased from 4 g/L to 8 g/L, the iron (II) sulfate heptahydrate concentration from 0.75 mg/L to 3.75 mg/L, and the L-rhamnose inducer concentration from 0.2 % to 2 % (w/v). In addition, the cultivation temperature was divided into two stages at 30 °C and 25 °C, whereas previously it was constant at 30 °C. The combination of these optimized parameters resulted in a 241 % increase in α-humulene production compared to the non-optimized standard process.
A robustness assessment of these optimized parameters indicated a highly stable production process using C. necator pKR-hum. For α-humulene production under varying process conditions, a robustness value of -0.155 ± 0.143 was observed, while biomass formation demonstrated even greater robustness with a value of -0.002 ± 0.002, approaching the ideal robustness value of 0. Even under the influence of simulated process disturbances, the process maintained 79 % of the maximum α-humulene level compared to the undisturbed process run, with a robustness value of -0.045 ± 0.001, highlighting the robust properties of the process. Furthermore, for the first time, a dose-dependent anti-inflammatory effect of α-humulene on lipopolysaccharide-induced human THP-1 cells was observed, with a maximum reduction in interleukin-6 levels of 60 % following the administration of 100 μM α-humulene. These findings are expected to significantly contribute to the optimization of microbial-based terpenoid production processes. Additionally, initial insights were obtained that confirm α-humulene’s potential as an alternative, nature-based, and promising therapeutic approach for the reduction of elevated interleukin-6 levels and chronic inflammation.
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Diese Dissertation wurde im Rahmen der Promotion über das Promotionszentrum für Ingenieurwissenschaften (PZI) am Forschungscampus Mittelhessen erstellt.