Constitutive expression of the cytochrome P450 EthABCD monooxygenase system enables degradation of synthetic dialkyl ethers in Aquincola tertiaricarbonis L108.

In Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005, and Gordonia sp. strain IFP 2009, the cytochrome P450 monooxygenase EthABCD catalyzes hydroxylation of methoxy and ethoxy residues in the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME). The expression of the IS3-type transposase-flanked eth genes is ETBE dependent and controlled by the regulator EthR (C. Malandain et al., FEMS Microbiol. Ecol. 72:289-296, 2010). In contrast, we demonstrated by reverse transcription-quantitative PCR (RT-qPCR) that the betaproteobacterium Aquincola tertiaricarbonis L108, which possesses the ethABCD genes but lacks ethR, constitutively expresses the P450 system at high levels even when growing on nonether substrates, such as glucose. The mutant strain A. tertiaricarbonis L10, which is unable to degrade dialkyl ethers, resulted from a transposition event mediated by a rolling-circle IS91-type element flanking the eth gene cluster in the wild-type strain L108. The constitutive expression of Eth monooxygenase is likely initiated by the housekeeping sigma factor σ(70), as indicated by the presence in strain L108 of characteristic -10 and -35 binding sites upstream of ethA which are lacking in strain IFP 2001. This enables efficient degradation of diethyl ether, diisopropyl ether, MTBE, ETBE, TAME, and tert-amyl ethyl ether (TAEE) without any lag phase in strain L108. However, ethers with larger residues, n-hexyl methyl ether, tetrahydrofuran, and alkyl aryl ethers, were not attacked by the Eth system at significant rates in resting-cell experiments, indicating that the residue in the ether molecule which is not hydroxylated also contributes to the determination of substrate specificity.

Production of the chiral compound (R)-3-hydroxybutyrate by a genetically engineered methylotrophic bacterium.

In this study, a methylotrophic bacterium, Methylobacterium rhodesianum MB 126, was used for the production of the chiral compound (R)-3-hydroxybutyrate (R-3HB) from methanol. R-3HB is formed during intracellular degradation of the storage polymer (R)-3-polyhydroxybutyrate (PHB). Since the monomer R-3HB does not accumulate under natural conditions, M. rhodesianum was genetically modified. The gene (hbd) encoding the R-3HB-degrading enzyme, R-3HB dehydrogenase, was inactivated in M. rhodesianum. The resulting hbd mutant still exhibited low growth rates on R-3HB as the sole source of carbon and energy, indicating the presence of alternative pathways for R-3HB utilization. Therefore, transposon mutagenesis was carried out with the hbd mutant, and a double mutant unable to grow on R-3HB was obtained. This mutant was shown to be defective in lipoic acid synthase (LipA), resulting in an incomplete citric acid cycle. Using the hbd lipA mutant, we produced 3.2 to 3.5 mM R-3HB in batch and 27 mM (2,800 mg liter(-1)) in fed-batch cultures. This was achieved by sequences of cultivation conditions initially favoring growth, then PHB accumulation, and finally PHB degradation.

Optimization of preservation conditions of As (III) bioreporter bacteria.

Long-term preservation of bioreporter bacteria is essential for the functioning of cell-based detection devices, particularly when field application, e.g., in developing countries, is intended. We varied the culture conditions (i.e., the NaCl content of the medium), storage protection media, and preservation methods (vacuum drying vs. encapsulation gels remaining hydrated) in order to achieve optimal preservation of the activity of As (III) bioreporter bacteria during up to 12 weeks of storage at 4 degrees C. The presence of 2% sodium chloride during the cultivation improved the response intensity of some bioreporters upon reconstitution, particularly of those that had been dried and stored in the presence of sucrose or trehalose and 10% gelatin. The most satisfying, stable response to arsenite after 12 weeks storage was obtained with cells that had been dried in the presence of 34% trehalose and 1.5% polyvinylpyrrolidone. Amendments of peptone, meat extract, sodium ascorbate, and sodium glutamate preserved the bioreporter activity only for the first 2 weeks, but not during long-term storage. Only short-term stability was also achieved when bioreporter bacteria were encapsulated in gels remaining hydrated during storage.

Internal arsenite bioassay calibration using multiple bioreporter cell lines.

Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error-prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli-based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or ß-galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for ß-galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10 µg l(-1)).

Artificial neural network study of whole-cell bacterial bioreporter response determined using fluorescence flow cytometry.

Genetically engineered bioreporters are an excellent complement to traditional methods of chemical analysis. The application of fluorescence flow cytometry to detection of bioreporter response enables rapid and efficient characterization of bacterial bioreporter population response on a single-cell basis. In the present study, intrapopulation response variability was used to obtain higher analytical sensitivity and precision. We have analyzed flow cytometric data for an arsenic-sensitive bacterial bioreporter using an artificial neural network-based adaptive clustering approach (a single-layer perceptron model). Results for this approach are far superior to other methods that we have applied to this fluorescent bioreporter (e.g., the arsenic detection limit is 0.01 microM, substantially lower than for other detection methods/algorithms). The approach is highly efficient computationally and can be implemented on a real-time basis, thus having potential for future development of high-throughput screening applications.

Calorimetric bioprocess monitoring by small modifications to a standard bench-scale bioreactor.

Heat released during whole cell bioconversions provides information about the stoichiometry and kinetics of growth and product formation. It is considered as a potent control variable because it reflects immediately changes in metabolism. Furthermore, the sensitivity of calorimetric measurements is improving with increasing ratios of heat producing volume to heat exchanging surface and therefore with any scaling-up. However, the development and testing of calorimetry-based control strategies at the bench scale is difficult. Common microcalorimeters are unsuited due to their high price, the reactor volume (1-100mL) and the reactor design. Reaction calorimeters are even more expensive, less sensitive than microcalorimeters and often not adapted to bioprocesses. This paper describes a way-out by integrating calorimetric measurement principles into bench-scale standard bioreactors. The proposed solution benefits from all the measurement and control features of bench-scale bioreactors for, e.g. aeration, stirring, pH-control and substrate dosage. The additional calorimetric option can be easily constructed and integrated into an existing bioreactor at relatively low cost. The achieved sensitivity of 50 mW L(-1), stability of 0.2 mW L(-1)h(-1) and response time of a 1-2 min is in the same magnitude or even better than those of established reaction calorimeters. The calorimetrically monitored growth of the halophilic strain Halomonas elongata DMSZ 2581(T) demonstrates the applicability of the proposed solution for bioprocess analysis and control.

Variations in 13C/12C and D/H enrichment factors of aerobic bacterial fuel oxygenate degradation.

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.

Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium.

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.

The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway.

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 beta-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.

Debaryomyces hansenii--an extremophilic yeast with biotechnological potential.

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.

A wide-range integrative yeast expression vector system based on Arxula adeninivorans-derived elements.

An Arxula adeninivorans integration vector was applied to a range of alternative yeast species including Saccharomyces cerevisiae, Debaryomyces hansenii, Debaryomyces polymorphus, Hansenula polymorpha and Pichia pastoris. The vector harbours a conserved A. adeninivorans-derived 25S rDNA sequence for targeting, the A. adeninivorans-derived TEF1 promoter for expression control of the reporter sequence, and the Escherichia coli-derived hph gene conferring resistance against hygromycin B for selection of recombinants. Heterologous gene expression was assessed using a green fluorescent protein (GFP) reporter gene. The plasmid was found to be integrated into the genome of the various hosts tested; recombinant strains of all species exhibited heterologous gene expressions of a similar high level.

Flow calorimetry and dielectric spectroscopy to control the bacterial conversion of toxic substrates into polyhydroxyalcanoates.

The microbial conversion of toxic substrates into valuable products in continuous culture requires the equivalent of a tight rope walk between formation of the desired product and intoxication of the microbial catalyst. The condition of the latter is reflected immediately by changes in heat flow rate and beta-dispersion in an electrical RF field. Therefore, these were applied to the example of the continuous growth-associated synthesis of polyhydroxyalcanoates (PHA) from phenol by the bacterial strain Variovorax paradoxus DSM 4065. By controlling the supply of phenol to the chemostat, the rates of degradation, biomass formation, and synthesis of target product, respectively, were increasingly elevated until the onset of poisoning the organisms. The boundary between the maximum rates and the initiation of intoxication coincided with a sudden change in the heat flux. Using this occurrence, it was possible to develop a control strategy and test it successfully for a time period of 80 h. After 40 h the process stabilized at mean values, i.e., at rates of 92% phenol degradation, 100% biomass formation, and 70 - 75% of PHA formation compared with the situation shortly before poisoning the organisms. Using a moving-average technique to filter the raw dielectric spectroscope data, changes were followed in biomass concentration of approximately 100 mg/L. However, this technique was not sensitive or rapid enough to control the process.