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Cloning and characterization of genes involved in PHA biosynthesis

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TEM micrographs of Chromobacterium sp. after
negative staining (left and upper right) and dark
purple colonies of this bacterium on nutrient rich agar
plate (lower right)

 

Chromobacterium sp. is a bacterium capable of producing PHA. The Chromobacterium sp. PHA synthase (PhaC) is able to polymerize short-chain-length PHA monomers, such as 3-hydroxybutyrate and 3-hydroxyvalerate. This bacterium has been long recognized for its production of violacein, a violet non-diffusible pigment making the colonies appear dark purple to black. Recently, this unique bacterium was isolated from the famous ‘7 Wells’ in Langkawi, Malaysia. 16S rRNA and biochemical analyses confirmed the identity of the isolate.

The PHA synthase (phaC) gene of Chromobacterium sp. was successfully cloned by using PCR amplification techniques. Preliminary studies suggested that the PhaC of this bacterium has an unusual specificity towards 3-hydroxyvaleryl-CoA. This enzyme specificity was further supported by the ability of this isolate to synthesize poly(3-hydroxyvalerate) of 100 mol% purity from suitable substrates.

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Heterologous expression of Chromobacterium sp.
synthase gene inCupriavidus necator PHB-4
complemented the mutant’s PHA producing ability