Cloning and Expression of a Gene Encoding Phosphomevalonate Kinase from an Endophytic Talaromyces species

Cloning of PMK gene

Abstract

Terpinoids, a diverse class of important bioactive compounds with therapeutic value, are produced through Mevalonate pathway (MVA) in fungi, plants, animals and some bacteria. Phosphomevalonate kinase (PMK) is a key enzyme and catalyses one the rate limiting steps of this pathway. This enzyme is generally expressed in very low levels and strategies for metabolic engineering of this enzyme may pave way for increased production of this important pathway metabolites.  In an earlier study, an endophytic Talaromyces sp. strain YS-1 isolated from a medicinal plant Syzygium samarangense was subjected to whole genome sequencing.  Genome mining and bioinformatics analysis with AntiSMASH (Antibiotics and Secondary Metabolite Analysis Shell) annotation identified 9 terpenoid gene clusters including the PMK gene which may be involved in biosynthesis of important bioactive terpenoids. In the present study this PMK gene of 1455 bp length ORF encoding 484 amino acids was cloned into pRSF expression vector and expressed in Escherichia coli as a His-tag fusion protein.  The recombinant PMK was purified to near homogeneity using Nickel-CL agarose column.  Its molecular weight was found to by 50 kDa when separated on SDS-PAGE. The expressed protein can be further characterized for its biological activities and can be metabolically engineered for production of value-added products