Recent years have seen a remarkable increase in the global interest for mining isoprenoid based fine-chemicals and nutraceuticals from GRAS (Generally regarded as Safe) status microbes capable of metabolizing complex carbon sources. In the present study, soil bacterium Bacillus subtilis was explored for its ability to produce hemiterpene-based alcohols, isopentenol (isoprenol and prenol) from simple to complex carbon sources. Isopentenol production in B. subtilis was enhanced by exploiting the over-expression of an endogenous 1-deoxy-D-xylulose-5-phosphate synthase (dxs) gene along with another native gene ADP-ribose pyrophosphatase (nudF). Genetic modulation enhanced isopentenol production in the recombinant strain (MTCC121-DxNu) by two-folds over the wild type strain. Further, the modulation of physico-chemical factors such as temperature (25 degrees C), pH regulator (K2HPO4) and carbon source (sorbitol) improved protein expression and isopentenol production in the recombinant strain. The maximum production of isopentenol achieved was similar to 2100 mu g L-1 in shake flask containing Luria Broth (LB) with 0.1 M Sorbitol, 0.2 M K2HPO4 at 25 degrees C, whereas, the non-detoxified rice-straw hydrolysate which is a low-cost substrate yielded 640 mu g L(-1 )of isoprenol. This proof-of-concept study can be scaled up for the economical production of other commercially important isoprenoid-based flavors and pharmaceuticals from industrially competent microbial strains.