Heterologous expression of actinomycetes terpene cyclases and cytochrome P450 in escherichia coli for terpene production

Secondary metabolites are natural products that have been proven to be valuable sources of bioactive agents for use in the pharmaceutical and agrochemical industries. Actinomycetes are high-guanine and cytosine Gram-positive, rod-shaped bacteria and are well known as producers of many high value bioactive metabolites such as terpenes. Characterization of terpene cyclase from several actinomycetes species demonstrated that these enzymes are not membrane-bound and can be overproduced in heterologous hosts such as Escherichia coli. Whereas, cytochrome P450 (CYP450) is an enzyme in bacteria that has the ability to catalyse diverse range of reactions, making it an attractive biocatalyst for numerous biotechnology applications. Few researches were carried out to understand interaction of both enzymes. Thus, this research aims to study the terpenes production by heterologous co-expression of terpene cyclase and CYP450 in E. coli. Synthetic genes from Streptomyces clavuligerus and Streptomyces griseus which encode for terpene cyclase and synthetic genes from S. griseus, S. griseus NBRC13350 and Streptomyces coelicolor which encode for CYP450 genes were synthesized. The genes of interest, promoters and terminators were amplified by PCR followed by in vivo vector construction via homologous recombination in yeast, resulting in five expression vectors, pET/2U-T1, pET/2U-T2, pET/2U-T1Sc, pET/2U-T2Sc and pET/2U-T2Sg. The expression vectors were individually transformed into E. coli. Terpenes production by recombinant E. coli were screened and analysed using gas chromatography-mass spectrometry (GC-MS). pET/2U-T1Sc is the only recombinant that produced two compounds of monoterpene which are terpinen-4-ol and α–terpineol.