AbstractThe dibenzothiophene biodesulfurization pathway has shown significant potential for improving the processing of sulfur‐containing fossil fuels. However, the rate of desulfurization is limited by the last enzyme in the pathway, DszB. Genetic constructs designed to produce increased DszB activity were not functional due to low production of DszB, even when using a consensus ribosome binding site. To increase DszB production, the untranslated region 5′ of dszB was mutated using degenerate oligonucleotides and translational fusions with gfp to detect increased translation of dszB. After screening only 96 mutants, several showed increased green fluorescence and two showed increased DszB activity. When cotransformed with the full dszABC operon, the mutant dszB increased the rate of desulfurization ninefold relative to that using the native dszB. © 2004 Wiley Periodicals, Inc.