Enhanced production of polyhydroxybutyrate in recombinant Escherichia coli for sustainable material

The increasing production and consumption of petroleum-derived plastics have led to environmental problems because of its non-biodegradability. This concern has driven the development of biopolymer alternative plastic, like polyhydroxybutyrate (PHB). In this paper, we reported a high production of PHB in recombinant Escherichia coli BL21(DE3) containing two-plasmid or one-plasmid systems carrying the three genes involved in PHB biosynthesis. PCR approach was used to isolate these three genes from Halomonas elongata BK-AG25 local strain, namely phbA, phbB, and phbC . Among the three genes, phbC is the largest, encoded crucial enzyme for polymerization, and located far away from the other two genes in the chromosome. Therefore, phbC was cloned separately into pET-30a(+), while phbA and phbB were inserted together into pET-21b(+), hence creating a two-plasmid system (pET- phbC/ pET- phbAB ). On the other hand, a one-plasmid system (pET- phbABC ) was also created for expression comparison. Astonishingly, the PHB production observed higher in the two-plasmid system compared to the one-plasmid system. The PHB produced by these two recombinant clones were analyzed using FT-IR and NMR to verify its functional groups and purity. SEM analysis indicated that both PHBs have sheet-like structure morphologically. Thermal analyses including TGA, DTG, and DTA revealed that the PHB produced by two-plasmid system exhibited higher thermal stability. These results indicate that the two-plasmid system enhances yield, purity, and thermal stability of PHB, hence significantly contribute for effective and efficient production of PHB as the next-generation sustainable material, as well as addressing the growing demand for eco-friendly alternatives in various industries. • PHB biosynthetic genes have been successfully inserted into the two-plasmid system. • PHB production was increased by employing a two-plasmid with the same origin. • PHB from two-plasmid system has higher yield, purity, and thermal stability. • The two-plasmid approach proved more efficient for large-scale PHB production.