Biosynthesis of P(3HB-co-3HHx) Copolymers by a Newly Engineered Strain of Cupriavidus necator PHB−4/pBBR_CnPro-phaCRp for Skin Tissue Engineering Application

Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable polymers synthesized by certain bacteria and archaea with functions comparable to conventional plastics. Previously, our research group reported a newly PHA-producing bacterial strain, Rhodococcus pyridinivorans BSRT1-1, from the soil in Thailand. However, this strain’s PHA synthase (phaCRp) gene has not yet been characterized. Thus, this study aims to synthesize PHA using a newly engineered bacterial strain, Cupriavidus necator PHB−4/pBBR_CnPro-phaCRp, which harbors the phaCRp from strain BSRT1-1, and characterize the properties of PHA for skin tissue engineering application. To the best of our knowledge, this is the first study on the characterization of the PhaC from R. pyridinivorans species. The results demonstrated that the expression of the phaCRp in C. necator PHB−4 had developed in PHA production up to 3.1 ± 0.3 g/L when using 10 g/L of crude palm kernel oil (CPKO) as a sole carbon source. Interestingly, the engineered strain produced a 3-hydroxybutyrate (3HB) with 2 mol% of 3-hydroxyhexanoate (3HHx) monomer without adding precursor substrates. In addition, the 70 L stirrer bioreactor improved P(3HB-co-2 mol% 3HHx) yield 1.4-fold over the flask scale without altering monomer composition. Furthermore, the characterization of copolymer properties showed that this copolymer is promising for skin tissue engineering applications.

Statistical optimization of P(3HB-co-3HHx) copolymers production by Cupriavidus necator PHB−4/pBBR_CnPro-phaCRp and its properties characterization

“…The 1 H NMR was performed to verify the presence of 3HHx monomer in the copolymer synthesized by the strain C. necator PHB − 4/pBBR_CnPro- phaC Rp . Figure 3 illustrates the 1 H NMR band of H4, corresponding to the C4 methylene groups, and the 1 H NMR band of H6, corresponding to the C6 methyl group, indicating the formation of the P(3HB- co -3HHx) copolymer 15 , 24 , 29 . The monomer fractions of the copolymer were calculated according to the 1 H spectrum intensity ratio of the methyl components 30 .…”

“…Previously, Han et al reported that the wild-type strains, Aeromonas spp., can produce P(3HB- co -3HHx) from vegetable oils and fatty acids via PHA synthase, which has a uniquely broad substrate specificity to (R)-3-hydroxyacyl-CoAs of C4-C6 39 . In this study, the P(3HB- co -3HHx) copolymer was synthesized by C. necator PHB − 4/pBBR_CnPro- phaC Rp 23 , 24 , a newly engineered bacterial strain, and the P(3HB- co -3HHx) production was improved using RSM. The results showed that the highest 3.6 ± 0.4 g/L of P(3HB- co -4 mol% 3HHx) was obtained using the RSM-optimized conditions in the flask scale.…”

“…The recombinant strain C. necator PHB − 4/pBBR_CnPro- phaC Rp was cultivated as described by Trakunjae et al 24 . Briefly, the bacterial strain was cultured on nutrient-rich (NR) agar supplemented with 50 µg/mL kanamycin at 30 °C for 24 h. Then, three full loops of a bacterial colony were transferred in NR medium supplemented with 50 µg/mL kanamycin to prepare the bacterial inoculum.…”

“…The recombinant PHA-producing strains with genes encoding PHA synthesis enzymes from various bacteria have been developed to generate short chain length-medium chain length (SCL-MCL) PHAs more effectively 22 . Our research group recently engineered a new P(3HB- co -3HHx)-producing bacterial strain, C. necator PHB − 4/pBBR_CnPro- phaC Rp 23 , 24 . This strain produced 3.1 ± 0.3 g/L of P(3HB- co -3HHx) copolymer containing 2 mol% of 3HHx monomer composition when using 10 g/L of crude palm kernel oil (CPKO) as a sole carbon source 24 .…”

“…Our research group recently engineered a new P(3HB- co -3HHx)-producing bacterial strain, C. necator PHB − 4/pBBR_CnPro- phaC Rp 23 , 24 . This strain produced 3.1 ± 0.3 g/L of P(3HB- co -3HHx) copolymer containing 2 mol% of 3HHx monomer composition when using 10 g/L of crude palm kernel oil (CPKO) as a sole carbon source 24 . However, an optimal culture medium and conditions for PHA production by this strain need to be improved to maximize the yield of P(3HB- co -3HHx).…”