Escherichia coli kduD encodes an oxidoreductase that converts both sugar and steroid substrates.

A previously unidentified oxidoreductase from Escherichia coli catalyzes the regioselective reduction of eukaryotic steroid hormone 11-deoxycorticosterone (11-DOC) to the valuable bioactive product 4-pregnen-20,21-diol-3-one. In nature, a reduction of C-20 carbonyl of C21 steroids is catalyzed by diverse NAD(P)H-dependent oxidoreductases. Enzymes that possess 20-ketosteroid reductase activity, however, have never before been described in E. coli. Our present study aimed to identify and characterize the E. coli enzyme which possesses 20-ketosteroid reductase activity against eukaryotic steroid hormone 11-DOC. We partially purified the enzyme from E. coli DH5α using protein chromatography techniques. Mass spectrometry revealed the presence of three NADH-specific oxidoreductases in the sample. The genes encoding these oxidoreductases were cloned and overexpressed in E. coli UT5600 (DE3). Only the overexpression of 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase (KduD) encoded by kduD gene enabled the whole-cell biotransformation of 11-DOC. A 6xHis-tagged version of KduD was purified to homogeneity and found to reduce several eukaryotic steroid hormones and catalyze the conversion of novel sugar substrates. KduD from E. coli is therefore a promiscuous enzyme that has a predicted role in sugar conversion in vivo but can be used for the production of valuable bioactive 20-hydroxysteroids.

Autodisplay of nitrilase from Klebsiella pneumoniae and whole-cell degradation of oxynil herbicides and related compounds.

Using the Autodisplay system, a recombinant Escherichia coli strain displaying the dimeric nitrilase from Klebsiella pneumoniae subsp. ozaenae (NitKp) on the cell surface was constructed. Localization of the nitrilase in the cell envelope of E. coli was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis and surface exposure was verified by its accessibility to externally added protease. The whole-cell biocatalyst obtained converted the substrates analyzed in the following order: chloroxynil > bromoxynil > ioxynil > 3-bromo-4-hydroxybenzonitrile (1.67, 0.89, 0.13, and 0.09 mM product formation within 72 h, respectively), indicating the same substrate specificity for the displayed enzyme as for the free enzyme. The whole-cell biocatalyst was also able to convert 3-fluoro-4-hydroxybenzonitrile and 3,5-dimethyl-4-hydroxybenzonitrile to the corresponding carboxylic acids. In contrast, it was not possible to detect any enzyme activity when 4-methoxybenzonitrile was used as substrate. The temperature optimum determined was 45 °C for the surface-displayed enzyme instead of 35 °C for the purified enzyme. In addition, the optimum activity of the displayed nitrilase was shifted to more acidic pH in comparison to the free enzyme.

Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.