Production of tailor-made P(HB-co-HHx) by engineered Ralstonia eutropha at laboratory and pilot scale

A transition from petroleum-based plastics to sustainable bioplastics is critical for addressing the environmental challenges posed by plastic pollution. Polyhydroxyalkanoates (PHAs), particularly the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], are promising candidates due to their biobased and biodegradable nature, coupled with their versatile material properties. To realize their commercialization at a competitive price, highly efficient bioprocesses with improved production hosts need to be developed. Ralstonia eutropha is the model organism for PHA production, yet the regulatory mechanisms governing PHA synthesis, storage and degradation are poorly understood, limiting the development of engineered production strains. During this thesis promoter pull-down assays followed by LC-MS/MS analysis enabled the identification of three novel transcriptional regulators involved in the expressional control of phasin phaP1 and depolymerases phaZ3 and phaZ5. Functional characterization revealed first insights into an intricate regulatory network: the novel transcriptional regulator XRE-family-like protein H16_B1672 specifically binds the phaP1 promoter in vitro with a KD of 175 nM and represses gene expression from this promoter in vivo. Additionally, H16_B1672 was found to interact with both depolymerase promoters in vivo and in vitro, suggesting a broader role in the regulation of PHA metabolism. Moreover, in vivo assays revealed that the putative transcription factors H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB strongly repress gene expression from the phaP1 and phaZ3 promoters, respectively. Apart from strain engineering, process development for optimized PHA production can be driven by designing novel cultivation strategies that deliver high product yields while enabling to target various applications. This thesis provides a framework for the high-yield production of tailor-made P(HB-co-HHx) at laboratory and pilot scale. Precise modulation of the HHx monomer content between 2 – 17 mol% was achieved by developing a robust feeding strategy using mixtures of fructose and canola oil. Comprehensive material characterization showed a significant extension of the processing window with increasing HHx contents (melting temperature: 165 °C to 126 °C and glass transition temperature: 4 °C to -5.9 °C) accompanied by a striking increase in flexibility up to 700 % while maintaining a fairly constant tensile strength, around 20 MPa, within the examined range. By this, the capability of the cultivation strategy to fine-tune the material properties of the biopolymer to meet diverse application requirements was demonstrated while also achieving differential degradability rates. The cultivation strategy was optimized to high-cell-densities above 100 g L−1 containing 70 – 80 wt% of tailored-PHA reaching remarkable productivities of up to 1.5 g L−1 h−1. Scalability could be demonstrated from the mL-scale in deep-well-plates to 150-L pilot-scale being the first study to demonstrate control over P(HB-co-HHx) compositions at an industrially relevant scale while showing the highest ever reported pilot-scale productivity. Ultimately, a solution for the production of tailored PHA in the kg-range was provided, addressing the need for large polymer quantities for testing and adapting manufacturing processes to target diverse applications. The work of this thesis offers a contribution to the intensification of PHA commercialization by i) advancing the engineering of R. eutropha through the implementation of regulatory models and ii) developing scalable, high-yield cultivation strategies that enable the production of PHA bioplastics with tailored properties for various industrial applications.