Removal of sulfur from petroleum improves the performance of refineries and reduces air pollution from fuel combustion. Biodesulfurization can selectively remove sulfur from petroleum, but improved biocatalysts are needed. This study illustrates a new approach to obtain improved desulfurization biocatalysts. A synthetic gene (Sulpeptide, S1) encoding high proportions of the sulfur containing amino acids methionine and cysteine was designed, constructed and cloned, as part of the Rhodococcus dszABC (desulfurization) operon, in vector pRESX, under control of the Rhodococcus kstD promoter. pRESXdszABC and pRESXdszAS1BC were electroporated into desulfurization-negative Rhodococcus opacus and the strains were transferred through 40 passages in medium with dibenzothiophene (DBT) as the sole sulfur source. Modest increases in desulfurization activity were achieved after 10 passages selecting for rapid growth with DBT. After selection for 40 passages, both the dszABC and dszAS1BC expressing R. opacus strains showed a greater than 20-fold increase in specific desulfurization ability, and exceeded the specific activity of the control, desulfurization positive strain Rhodococcus erythropolis IGTS8. This study serves as a proof of concept that engineering with Sulpeptide genes, combined with selective pressure for rapid growth with DBT, can drive evolution of improved desulfurization biocatalysts. (C) 2013 Elsevier Ltd. All rights reserved.