A step-by-step roadmap involving physiological characterization, genomics, fluxomics, and tailored fermentation to empower Paracoccus pantotrophus DSM 2944 as a metabolically versatile SynBio chassis

Introduction:

Paracoccus pantotrophus DSM 2944, a Gram-negative bacterium, was selected for its metabolic robustness and potential as a SynBio (synthetic biology) chassis due to its diverse substrate utilization including cheap and renewable feedstocks, CO2, and other C1 and C2 compounds coupled with the production of biopolymer polyhydroxyalkanoates (PHAs).

Goals:

The study aims to transform P. pantotrophus DSM 2944 into a proficient SynBio chassis through in-depth physiological, metabolic, computational, and phylogenetic studies coupled with industrially related bioreactor scale-up.

Results:

To investigate the suitability as a new chassis organism, extensive studies exploring substrate utilization and tolerance, optimal growth temperature, pH values, and phylogenetic studies were performed in P. pantotrophus DSM 2944. Moreover, to elucidate the strain"s metabolic flux in vivo, an in-depth analysis of flux distributions was also using 13C labeled glucose. The flux map showed that P. pantotrophus DSM 2944 prefers the pentose phosphate and Entner-Doudoroff pathways over glycolysis offering a surplus co-factor regeneration coupled with energy generation. Furthermore, to facilitate in-silico simulation of an organism"s growth and metabolism, the genome-scale metabolic model was constructed and validated with experimental data. A genetic toolbox was also designed comprising promoters with tunable strengths, gene integration and deletion strategies, and compatible origins of replication. Highlights include improved growth on the non-native carbon-source terephthalic acid along with native carbon-source ethylene glycol, the two monomers from polyethylene terephthalate (PET). Finally, tailor-made DO-based fermentation strategies were established, showcasing the applicability of the strain in bioreactors regarding PHA production.

Conclusions:

Through the implementation of the chassis roadmap and comprehensive research, P. pantotrophus DSM 2944 emerges as a promising SynBio chassis. Its metabolic versatility and engineered capabilities highlight its potential in advancing sustainable bioeconomy initiatives, paving the way for a greener future.