Production of P(3HB-co-4HB) copolymer with high 4HB molar fraction by Burkholderia contaminans Kad1 PHA synthase

Abstract

Burkholderia sp. has been reported to synthesize polyhydroxyalkanoates (PHAs) with different monomers such as 3-hydroxybutyrate and 3-hydroxyvalerate. 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.