Dataset Information

Biosynthesis of P(3HB-co-3HHx) Copolymers by a Newly Engineered Strain of Cupriavidus necator PHB^-4/pBBR_CnPro-phaC_Rp 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 (phaC_Rp) 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-phaC_Rp, which harbors the phaC_Rp 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 phaC_Rp 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.

SUBMITTER: Trakunjae C

PROVIDER: S-EPMC9570888 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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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.

Trakunjae Chanaporn C Sudesh Kumar K Neoh Soon Zher SZ Boondaeng Antika A Apiwatanapiwat Waraporn W Janchai Phornphimon P Vaithanomsat Pilanee P

Polymers 20220928 19

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, Cupri ...[more]

PMID: 36236022

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Project description:BackgroundExisting plasmid systems offer a fundamental foundation for gene expression in Cupriavidus necator; however, their applicability is constrained by the limitations of conjugation. Low segregational stabilities and plasmid copy numbers, particularly in the absence of selection pressure, pose challenges. Phytases, recognized for their widespread application as supplements in animal feed to enhance phosphate availability, present an intriguing prospect for heterologous production in C. necator. The establishment of stable, high-copy number plasmid that can be electroporated would support the utilization of C. necator for the production of single-cell protein from CO2.ResultsIn this study, we introduce a novel class of expression plasmids specifically designed for electroporation. These plasmids contain partitioning systems to boost segregation stability, eliminating the need for selection pressure. As a proof of concept, we successfully produced Escherichia coli derived AppA phytase in C. necator H16 PHB- 4 using these improved plasmids. Expression was directed by seven distinct promoters, encompassing the constitutive j5 promoter, hydrogenase promoters, and those governing the Calvin-Benson-Bassham cycle. The phytase activities observed in recombinant C. necator H16 strains ranged from 2 to 50 U/mg of total protein, contingent upon the choice of promoter and the mode of cell cultivation - heterotrophic or autotrophic. Further, an upscaling experiment conducted in a 1 l fed-batch gas fermentation system resulted in the attainment of the theoretical biomass. Phytase activity reached levels of up to 22 U/ml.ConclusionThe new expression system presented in this study offers a highly efficient platform for protein production and a wide array of synthetic biology applications. It incorporates robust promoters that exhibit either constitutive activity or can be selectively activated when cells transition from heterotrophic to autotrophic growth. This versatility makes it a powerful tool for tailored gene expression. Moreover, the potential to generate active phytases within C. necator H16 holds promising implications for the valorization of CO2 in the feed industry.

Dataset Information

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

Publications

Biosynthesis of P(3HB-<i>co</i>-3HHx) Copolymers by a Newly Engineered Strain of <i>Cupriavidus necator</i> PHB<sup>-</sup>4/pBBR_CnPro-<i>phaC<sub>Rp</sub></i> for Skin Tissue Engineering Application.

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