We illuminate the ecological, physiological and genetic characteristics of the yeast Debaryomyces hansenii in the view of our belief that this metabolically versatile, non-pathogenic, osmotolerant and oleaginous microorganism represents an attractive target for fundamental and applied biotechnological research. To this end, we give a broad overview of extant biotechnological procedures using D. hansenii, e.g. in the manufacture of various foods, and propose research into the heterologous synthesis of a range of fine chemicals.

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated ␤-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

Reliable compound-specific isotope enrichment factors are needed for a quantitative assessment of in situ biodegradation in contaminated groundwater. To obtain information on the variability on carbon and hydrogen enrichment factors (epsilonC, epsilonH) the isotope fractionation of methyl tertiary (tert-) butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) upon aerobic degradation was studied with different bacterial isolates. Methylibium sp. R8 showed a carbon and hydrogen isotope enrichment upon MTBE degradation of -2.4 +/- 0.1 and -42 +/- 4 per thousand, respectively, which is in the range of previous studies with pure cultures (Methylibium petroleiphilum PM1) as well as mixed consortia. In contrast, epsilonC of the beta/-proteobacterium L108 (-0.48 +/- 0.05 per thousand) and Rhodococcus ruber IFP 2001 (-0.28 +/- 0.06 per thousand) was much lower and hydrogen isotope fractionation was negligible (epsilonH < or = -0.2 per thousand). The varying isotope fractionation pattern indicates that MTBE is degraded by different mechanisms by the strains R8 and PM1 compared to L108 and IFP 2001. The carbon and hydrogen isotope fractionation of ETBE by L108 (epsilonC = -0.68 +/- 0.06 per thousand and epsilonH = -14 +/- 2 per thousand) and IFP 2001 (epsilonC = -0.8 +/- 0.1 per thousand and epsilonH = -11 +/- 4 per thousand) was very similar and seemed slightly higher than the fractionation observed upon MTBE degradation by the same strains. The low carbon and hydrogen enrichment factors observed during MTBE and ETBE degradation by L108 and IFP 2001 suggest a hydrolysis-like reaction type of the ether bond cleavage compared to oxidation of the alkyl group as suggested for the strains PM1 and R8. The variability of carbon and hydrogen enrichment factors should be taken into account when interpreting isotope pattern of fuel oxygenates with respect to biodegradation in contamination plumes.

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The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter ؊1 ectoine with a space-time yield of 40 mg liter ؊1 h ؊1 , with the vast majority of the ectoine being excreted.Halophilic microorganisms live in highly saline environments. There are two major strategies of adaptation to these hostile conditions. Extreme halophiles such as Halobacterium salinarum accumulate salt in the cytosol to maintain the osmotic balance (the salt-in strategy). Other halophiles synthesize and accumulate small organic molecules as osmotic counterweights (the organic-osmolyte strategy). Unlike intracellular salt, the small organic compounds do not affect the metabolism of the organism and are thus called compatible solutes. The enzymes of organic-osmolyte strategists do not need to be haloadapted, allowing these organisms to cope with strong salinity fluctuations. Non-salt-tolerant bacteria like Escherichia coli are usually unable to synthesize large amounts of compatible solutes but may resist halostress to a certain extent by taking up and accumulating compatible solutes (8,9,12,27).Different classes of chemical compounds, including polyols, sugars, methylamines, and linear and cyclic amino acids and betaines, have been found to act as compatible solutes. Besides functioning as osmotic counterweights, compatible solutes were shown to protect biomolecules and whole cells against denaturation caused by heating, freezing, desiccation, or chemical agents (15,19,22). This property has attracted commercial attention. Compatible solutes can be used as chemical chaperones for protein folding, enhancers of PCR performance, cryoprotectants of microorganisms, cosmeceuticals, and pharmaceuticals (20, 32). A potentially promising future application could be the enhancement of drought tolerance or salt tolerance of transgenic plants (1, 37).The best-investigated compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), is biotechnologically produced with the halophilic bacterium Halomonas elongata (34). The physiology and genetics of ectoine biosynthesis in this bacterium have been studied in detail (7,23,24,25,26,29). The technical bioprocess for the synthesis of ectoine exploits the salt adaptation strategy of H. elongata and is called "bacterial milking" (34). When the medium has a high salt concentration, the bacterium s...