Identification and expression analysis of the gene associated with geosmin production in Lyngbya kuetzingii UTEX 1547 (cyanobacteria)

Cyanobacteria are the major producers of geosmin in natural waters. To identify a gene involved in geosmin biosynthesis in cyanobacteria, the polymerase chain reaction (PCR) was used to amplify a 2298-bp open reading frame (ORF) from the geosmin-producing cyanobacterium Lyngbya kuetzingii UTEX 1547. This ORF encoded a protein of 765 amino acids. Alignment of the deduced amino acid sequence demonstrated that geoL had high similarity to the corresponding genes of Oscillatoria sp. PCC 6506 (100% identity), Calothrix sp. PCC 7507 (89%), Anabaena ucrainica CHAB 1432 (88%), A. ucrainica CHAB 2155 (87%), Nostoc punctiforme PCC 73102 (87%), Phormidium sp. P2r (84%) and Cylindrospermum stagnale PCC 7417 (83%), and modest similarity to myxobacteria (61-73%). It also indicated geoL with low similarity to the corresponding genes of actinomycetes (<60%). The encoded protein GEOL was estimated to have two geosmin synthase domains, and each contained two strictly conserved Mg2+-binding motifs (aspartate-rich motif and NSE triad). The geoL gene was shown to be responsible for geosmin biosynthesis in L. kuetzingii UTEX 1547. Then, geoL had been cloned into pET21a(+) vector and expressed in Escherichia coli BL21(DE3) with the isopropyl-β-d-thiogalactoside (IPTG) induction. The recombinant GEOL protein was purified and exhibited a single band (MW ∼ 90 kDa) on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was consistent with the predicted molecular weight (MW) of 87,046 Da. In conclusion, this study has confirmed that geosmin synthase gene and its expression product can be identified and characterized from cyanobacteria, which will help understand the fundamental biological mechanism of geosmin biosynthesis in cyanobacteria.