Production of P(3HB-co-4HB) Copolymer with High 4HB Molar Fraction by Burkholderia contaminans Kad1 PHA Synthase

In this study, the PHA synthase gene of B. contaminans Kad1 (phaCBcon) was successfully cloned and characterized. Heterologous expression of phaCBcon conferred PHA synthesis to the mutant Cupriavidus necator PHB¯4, confirming that phaCBcon encoded a functional active protein. The transformant C. necator PHB¯4 (Cn-Kad1) harboring phaCBcon was able to synthesize PHA when grown on sodium-4-hydroxybutyrate (sodium-4-HB) as the sole carbon source in a two-stage cultivation. NMR results confirmed the presence of poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB). Cn-Kad1 has the ability to produce a broad range of copolymer with a molecular weight (Mw) in the range of 8.0 × 105 to 10 × 105 Dalton. However, the number average molecular weight (Mn) decreased from 5.0 × 105 to 4.7 × 105 Da, when the sodium-4-HB concentration increased from 0.1 to 0.4 M. Higher concentration of sodium-4-HB, resulted in higher 4HB compositions in the polymer and consequently gave lower values for both the melting temperature (Tm) and the glass transition temperature (Tg). Interestingly, Cn-Kad1 accumulated P(3HB-co-4HB) with 4HB monomer up to 51 mol% from sodium-4-HB, while, the wild type B. contaminans Kad1 has limited ability to accumulate PHA containing 4HB monomer.Graphical abstract
Source: Biochemical Engineering Journal - Category: Biochemistry Source Type: research