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bioRxiv · 10.1101/2024.03.28.587248

Isolation and Characterization of a Halomonas Species for Non-Axenic Growth-Associated Production of Bio-Polyesters from Sustainable Feedstocks

Woo, S.-G.·Averesch, N. J.·Berliner, A. J.·Deutzmann, J. S.·Payne, V. E.·Chatterjee, S.·Criddle, C. S.
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Abstract

Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01". Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strains exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon-feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further also glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to [~]60% of its biomass [dry w/w] in poly(3-hydroxbutyrate), while reaching a doubling time of 1.7 h at 30{whitebullet}C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but can also facilitate the release of intracellular products through osmolysis. Development of a minimal medium also allowed the estimation of maximum PHB production rates, which were projected to exceed 5 gPHB/h. Finally, also the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid-vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCEThe urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing. Especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

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Woo, S.-G., Averesch, N. J., Berliner, A. J., Deutzmann, J. S., Payne, V. E., Chatterjee, S., Criddle, C. S.. 2024-04-02. Isolation and Characterization of a Halomonas Species for Non-Axenic Growth-Associated Production of Bio-Polyesters from Sustainable Feedstocks. https://doi.org/10.1101/2024.03.28.587248

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