Process development for heterotrophic terpene production in Cupriavidus necator

Abstract

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 <i>Cupriavidus necator</i> 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 <i>C. necator</i>, with a focus on in-depth investigation and optimization of various process parameters.<br>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.<br>A robustness assessment of these optimized parameters indicated a highly stable production process using <i>C. necator</i> 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.