Heterologous expression of lipase gene (lip) and PHA synthase gene (PHAC1) from pseudomonas spp. in escherichia coli for the biosynthesis of polyhydroxyalkanoates (PHA) / Aung Shuh Wen

Polyhydroxyalkanoates (PHA) are natural biodegradable and biocompatible plastic materials which potentially have a wide range of applications. The potential commercial value of these polyesters prompts a demand for intensive studies to maximise their production and applications. Generally, PHAs are produced and accumulated intracellular by numerous bacteria as energy storage. The accumulation of medium-chain-length (mcl) PHA in bacteria is dependent on the type of carbon source available to the bacteria. It has been reported that palm oil, palm kernel oil and their derivatives were suitable carbon source for the production of mcl-PHA. However, there are few bacteria strains that are able to synthesis PHA by utilising oil because they can produce lipase enzyme. This study describes the construction of a recombinant strain of Escherichia coli that is able to digest palm oil for the biosynthesis of PHA. A lipase gene (lip) from Pseudomonas fluorescens and a PHA synthase gene (phaC1) from Pseudomonas putida were cloned, separately and together, into fab B- E. coli using the pBAD-TOPO vector. The constructed fab B¯ E. coli strain LS_pT-phaC1 which harboured the phaC1 gene only, and fab B¯ E. coli strain LS_M3 which harboured both the lip and phaC1 genes were tested for the accumulation of PHA. The results revealed that up to 7.8% cell dry weight of PHA was detected in fab B¯ E. coli strain LS_M3 when it was cultivated in medium containing palm kernel oil (PKO) as the sole carbon source. No PHA was detected in fab B¯ E.coli strain LS_pT-phaC1 grown with PKO as the sole carbon source. This showed that both the lip and phaC1 genes were successfully cloned and expressed in the fab B- E. coli strain LS_M3