One model system for reducing the sulfur content of petroleum in an environmentally safe manner is the biological desulfurization (biodesulfurization) of dibenzothiophene (DBT) via the 4S pathway. Although the 4S pathway’s efficiency is still too low for a commercial oil biodesulfurization process despite a lot of work, it may soon be utilized for commercial processes that produce antibiotics, polythioesters, surfactants, and other chemicals as well as for the detoxification of some chemical warfare agents. Although disulfide-rich proteins are difficult to produce in microbial hosts, proteins with disulfide links are resistant to temperature, pH, and solvents. The 4S pathway can be studied to learn how to produce as many disulfide-rich proteins as possible. By raising the dietary need for sulfur, engineering the operon encoding the 4S pathway to contain higher levels of methionine and cysteine may be able to connect using DBT as the only source of sulfur to increased 4S pathway activity. The development of biocatalysts appropriate for an oil biodesulfurization process may come from this approach, but research on the 4S pathway can also improve our knowledge of microbial physiology to maximize the activity of a multi-step co-factor-requiring pathway and produce highly stable, industrially relevant enzymes for a variety of uses.