PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003

Among the different tools which can be studied and managed to tailor-make polyhydroxyalkanoates (PHAs) and enhance their production, bacterial strain and carbon substrates are essential. The assimilation of carbon sources is dependent on bacterial strain’s metabolism and consequently cannot be dissociated. Both must wisely be studied and well selected to ensure the highest production yield of PHAs. Halomonas sp. SF2003 is a marine bacterium already identified as a PHA-producing strain and especially of poly-3-hydroxybutyrate (P-3HB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P-3HB-co-3HV). Previous studies have identified different genes potentially involved in PHA production by Halomonas sp. SF2003, including two phaC genes with atypical characteristics, phaC1 and phaC2. At the same time, an interesting adaptability of the strain in front of various growth conditions was highlighted, making it a good candidate for biotechnological applications. To continue the characterization of Halomonas sp. SF2003, the screening of carbon substrates exploitable for PHA production was performed as well as production tests. Additionally, the functionality of both PHA synthases PhaC1 and PhaC2 was investigated, with an in silico study and the production of transformant strains, in order to confirm and to understand the role of each one on PHA production. The results of this study confirm the adaptability of the strain and its ability to exploit various carbon substrates, in pure or mixed form, for PHA production. Individual expression of PhaC1 and PhaC2 synthases in a non-PHA-producing strain, Cupriavidus necator H16 PHB¯4 (DSM 541), allows obtaining PHA production, demonstrating at the same time, functionality and differences between both PHA synthases. All the results of this study confirm the biotechnological interest in Halomonas sp. SF2003.

Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate–glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.