Increased Production and Molecular Weight of Artificial Polyhydroxyalkanoate Poly(2-hydroxybutyrate) Above the Glass Transition Temperature Threshold

Abstract

Poly(2-hydroxybutyrate) [P(2HB)] is an artificial polyhydroxyalkanoate (PHA) synthesized using engineered 2-hydroxyalkanoate-polymerizing PHA synthase. In the present study, the effect of temperature on P(2HB) synthesis was investigated. Recombinant Escherichia coli harboring PHA synthetic genes were cultivated with 2HB and 3-hydroxybutyrate (3HB) supplementation at varied temperatures ranging from 24 to 36°C for the synthesis of P(2HB) and natural PHA P(3HB), respectively. P(2HB) production and its molecular weight increased considerably at a threshold temperature of 32–34°C. The trend was not observed during the synthesis of P(3HB). Notably, the threshold temperature was close to the glass transition temperature ( T g ) of P(2HB) (30°C), while the T g of P(3HB) (4°C) was much lower than the cultivation temperature. The results suggest that thermal motion of the polymer chains influenced the production and molecular weight of the obtained polymer. According to the results, the production and molecular weight of PHA drastically changes at the threshold temperature, which is linked to the T g of the polymer. The hypothesis should be applicable to PHAs in general, and potentially explains the inability to biosynthesize high-molecular-weight polylactate homopolymer with a T g of 60°C.

Biosynthesis of Random-Homo Block Copolymer Poly[Glycolate-ran-3-Hydroxybutyrate (3HB)]-b-Poly(3HB) Using Sequence-Regulating Chimeric Polyhydroxyalkanoate Synthase in Escherichia coli

“…The T g of the polymer product also influences polymer synthesis. Our recent study revealed that P(2HB) biosynthesis is efficient when the cultivation temperature is higher than the T g of the polymer ( Matsumoto and Kageyama, 2019 ). Given the fact that the random copolymer possesses a lower T g than that of polyglycolate (35–40°C), P(GL- ran -3HB) chains are more readily synthesized than the homopolymer.…”

Microbial Polyhydroxyalkanoates Granules: An Approach Targeting Biopolymer for Medical Applications and Developing Bone Scaffolds

“…The polymerization and molecular weight are affected by C:N ratio [39] and are governed by the specificity of the substrate of PHA synthase [36]. The reaction temperature [36] is another vital component contributing to its polymerization and high molecular weight. Nutrient limiting media one of the essential factors contributing to PHA granule formation.…”

“…The polymerization of the monomeric components of the PHA nano-sized granule depends on the activity of PHA synthase [ 36 ] This helps to achieve the desired high molecular weight of the resultant PHA homopolymer and copolymers. The gel permeation chromatography results showed that in marine bacteria, the number average molecular weight of the PHAs after purification ranged from 3 × 10 3 –9.94 × 10 5 g/mol [ 37 ].…”

“…The PHA isolated from the recombinant gram-negative bacteria, E. coli has a molecular weight of nearly 2 × 10 6 g/mol. The polymerization and molecular weight are affected by C:N ratio [ 39 ] and are governed by the specificity of the substrate of PHA synthase [ 36 ]. The reaction temperature [ 36 ] is another vital component contributing to its polymerization and high molecular weight.…”

“…In recombinant E. coli , a feasible incubation temperature of 24–36 °C was optimum whereby the threshold temperature was close to the glass transition temperature. The thermal motion also greatly influenced the molecular weight of the polymer chain [ 36 ]. It is usually desirable to choose bacterial species yielding PHA polymer with a low polydispersity index [ 38 ] and another with high polydispersity index [ 44 ] or ultra-high molecular weight using mixed cultures so that the resultant polymer can be applicable for different industrial applications [ 44 ].…”