Eicosapentaenoic acid (EPA) and arachidonic acid (ARA) are long-chain polyunsaturated fatty acids (PUFAs) that play a significant role in human growth and development, which deficiency can trigger several metabolic-related diseases. Since the availability of PUFA sources is limited, there arises a need to explore alternative sources. Therefore, the present study aimed to investigate whether an Escherichia coli which are engineered with Delta 5Des-Iso gene isolated from Isochrysis sp. could be utilized to synthesize PUFAs. Full-length gene Delta 5Des-Iso (1149 bp) was isolated from Isochrysis sp. that encodes 382 amino acids and identified as Delta 5-desatruase gene using different bioinformatic analysis. Heterologous gene expression was carried out in E. coli having Delta 5Des-Iso with precursor fatty acids. The Delta 5Des-Iso produced novel fatty acids of EPA (omega-3) and ARA (omega-6) as respective products were identified by GC-MS. Gene expression and PUFA synthesis in E. coli were optimized by temperature, time, and concentrations of precursor fatty acid substrates. Delta 5Des-Iso RNA transcript level was inversely proportional to the time and fatty acid synthesis. And, the significant production of EPA (4.1 mg/g) and ARA (8.3 mg/g) in total fatty acids was observed in E. coli grown at 37 degrees C for 24 h with 25 mu M of external fatty acid substrate as an optimum growth conditions. E. coli could be used as alternative organism to synthesis PUFAs and widely applicable in many nutraceuticals and pharmaceuticals industry for human use.