Metabolic Engineering Of Cupriavidus Necator For The Production Of Poly (3-Hydroxybutyrate-Co-3-Hydroxyhexanoate
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Date
2017-05
Authors
Ab Ghani, Balqis
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Publisher
Universiti Sains Malaysia
Abstract
Polyhydroxyalkanoate (PHA) is a family of polyesters produced by bacteria that possesses physical properties for various industrial applications. In this study, PHA synthase (PhaC) gene isolated from Chromobacterium sp. USM2 (phaCCs USM2) was explored for its potential to synthesize polymer in Cupriavidus necator. A broad-host range plasmid pBBR1MCS-2 was modified to construct an expression plasmid pYEB-100, which was designed to facilitate cloning of PHA synthases in order to assess the performance of various PhaCs and the composition of PHA accumulated in a standard environment. To prove this concept, phaCCs USM2 was expressed in C. necator PHB-4, which produced 68 wt% of P(3HB-co-3HHx) copolymer using crude palm kernel oil as the sole carbon source. The effect of other key enzymes; β-ketothiolase (PhaA) and NADPH-dependent acetoacetyl-CoA reductase (PhaBI) on growth and PHA production in C. necator were also investigated. Deletion of phaA and phaB1 in C. necator (both H16 and PHB-4 strains) were generated by homologous recombination using the suicidal plasmid pDM4. The deletion mutants were observed to produce lower cell dry weights but higher 3HHx fraction in the P(3HB-co-3HHx) copolymer produced. The (R)-specific enoyl-CoA hydratase gene (phaJ) from Aeromonas sp. AE6, which was co-expressed with phaCCs USM2 increased P(3HB-co-3HHx) copolymer but the 3HHx fraction remained constant at 2 mol%. Transmission electron microscopy analysis revealed that PHA granules of P/CCsJAs were larger and elongated compared to P/CCs granules. In deletion mutants, HΔAB1/CCsJAs granules were smaller in size, but greater in number compared to HΔAB1.
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Keywords
Metabolic engineering of Cupriavidus necator , the production of 3-hydroxybutyrate