Growth assays reveal that Rhodococcus rhodochrous IGTSS uses a wide range of organosulphur compounds as the sole source of sulphur, yet none of the compounds serve as carbon sources. Compounds that are utilized include thiophenes, sulphides, disulphides, mercaptans, sulphoxides, and sulphones. A convenient spectrophotometric assay (Gibbs assay), based on the chromogenic reaction of 2,6-dichloroquinone-4-chloroimide with aromatic hydroxyl groups, was developed and used in conjunction with GC/MS analyses to examine the kinetics of dibenzothiophene metabolism by axenic and mixed cell cultures of Rhodococcus rhodochrous IGTSS. The desulphurization trait is expressed at increasing levels during the exponential phase of growth and then declines in stationary-phase cells. Mixtures of streptomycin-resistant Rhodococcus rhodochrous IGTSS and Enterobacter cloacae (an organism incapable of cleaving carbon-sulphur bonds in relevant test compounds) were prepared in ratios that varied over six orders of magnitude. Growth studies revealed that E. cloacae was able to gain access to sulphur liberated from organosulphur compounds by IGTSS ; however, cell-to-cell contact appears to be required. These experiments also indicate that the desulphurization activity, on a per cell basis, is higher in mixed cultures than in axenic cultures.

The Chinese hamster ovary cell (CHO) is the major host cell factory for recombinant production of biological therapeutics primarily because of its "human-like" glycosylation features. CHO is used for production of several O-glycoprotein therapeutics including erythropoietin, coagulation factors, and chimeric receptor IgG1-Fc-fusion proteins, however, some O-glycoproteins are not produced efficiently in CHO. We have previously shown that the capacity for O-glycosylation of proteins can be one limiting parameter for production of active proteins in CHO.

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An organism, identified as Mycobacterium phlei GTIS10, was isolated based on its ability to use dibenzothiophene (DBT) as a sole source of sulfur for growth at 30-52 degrees C. Similar to other biodesulfurization-competent organisms, M. phlei GTIS10 converts DBT to 2-hydroxybiphenyl (2-HBP), as detected by HPLC. The specific desulfurization activity of the 50 degrees C M. phlei GTIS10 culture was determined to be 1.1+/-0.07 micromol 2-HBP min(-1) (g dry cell)(-1). M. phlei GTIS10 can also utilize benzothiophene and thiophene as sulfur sources for growth. The dszABC operon of M. phlei GTIS10 was cloned and sequenced and was found to be identical to that of Rhodococcus erythropolis IGTS8. The presence of the R. erythropolis IGTS8 120-kb plasmid pSOX, which encodes the dszABC operon, has been demonstrated in M. phlei GTIS10. Even though identical dsz genes are contained in both cultures, the temperature at which resting cells of R. erythropolisIGTS8 reach the highest rate of DBT metabolism is near 30 degrees C whereas the temperature that shows the highest activity in resting cell cultures of M. phlei GTIS10 is near 50 degrees C, and activity is detectable at temperatures as high as 57 degrees C. In M. phlei GTIS10, the rate-limiting step in vivo appears to be the conversion of DBT to dibenzothiophene sulfone catalyzed by the product of the dszC gene, DBT monooxygenase. The thermostability of individual desulfurization enzymes was determined and 2-hydroxybiphenyl-2-sulfinate sulfinolyase, encoded by dszB, was found to be the most thermolabile. These results demonstrate that the thermostability of individual enzymes determined in vitro is not necessarily a good predictor of the functional temperature range of enzymes in vivo.

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