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.

Experimental determination and data-driven prediction of homotypic transmembrane domain interfaces.

Interactions between their transmembrane domains (TMDs) frequently support the assembly of single-pass membrane proteins to non-covalent complexes. Yet, the TMD-TMD interactome remains largely uncharted. With a view to predicting homotypic TMD-TMD interfaces from primary structure, we performed a systematic analysis of their physical and evolutionary properties. To this end, we generated a dataset of 50 self-interacting TMDs. This dataset contains interfaces of nine TMDs from bitopic human proteins (Ire1, Armcx6, Tie1, ATP1B1, PTPRO, PTPRU, PTPRG, DDR1, and Siglec7) that were experimentally identified here and combined with literature data. We show that interfacial residues of these homotypic TMD-TMD interfaces tend to be more conserved, coevolved and polar than non-interfacial residues. Further, we suggest for the first time that interface positions are deficient in ß-branched residues, and likely to be located deep in the hydrophobic core of the membrane. Overrepresentation of the GxxxG motif at interfaces is strong, but that of (small)xxx(small) motifs is weak. The multiplicity of these features and the individual character of TMD-TMD interfaces, as uncovered here, prompted us to train a machine learning algorithm. The resulting prediction method, THOIPA (www.thoipa.org), excels in the prediction of key interface residues from evolutionary sequence data.

Autotransporter mediated esterase display on Zymomonas mobilis and Zymobacter palmae.

Autodisplay, i.e. surface expression of recombinant proteins by virtue of the autotransporter secretion pathway, has been used predominantly with Escherichia coli as host organism, which often limits the applicability of this technique to laboratory purposes and scales. The aim of this study was to investigate if the fermentative bacteria Zymomonas mobilis and Zymobacter palmae, representing attractive candidates for industrial applications, can serve as host organisms for autodisplay. We therefore used the carboxylesterase EstA from Burkholderia gladioli as an autotransporter passenger to display it on the surfaces of Z. palmae and Z. mobilis. Expression and outer membrane localization of the EstA-autotransporter fusion protein were verified by SDS-PAGE, and surface display of the enzyme was demonstrated by ELISA and flow cytometer analysis. Whole-cell activity assays revealed that EstA retained its activity on the cell surface. Recombinant Z. palmae cells exhibited significant higher esterase activity (294mU/mL/OD 1) in comparison to Z. mobilis (88mU/mL/OD 1) and the control E. coli (113mU/mL/OD 1). This appears even more noteworthy, as about 30% of EstA was released from the cell surface of Z. palmae. Nevertheless, our results indicate that both species are suitable autodisplay hosts, in particular Z. palmae for displaying esterase, opening up new horizons for biocatalytic applications.

Autodisplay of enzymes--molecular basis and perspectives.

To display an enzyme on the surface of a living cell is an important step forward towards a broader use of biocatalysts. Enzymes immobilized on surfaces appeared to be more stable compared to free molecules. It is possible by standard techniques to let the bacterial cell (e.g. Escherichia coli) decorate its surface with the enzyme and produce it on high amounts with a minimum of costs and equipment. Moreover, these cells can be recovered and reused in several subsequent process cycles. Among other systems, autodisplay has some extra features that could overcome limitations in the industrial applications of enzymes. One major advantage of autodisplay is the motility of the anchoring domain. Enzyme subunits exposed at the cell surface having affinity to each other will spontaneously form dimers or multimers. Using autodisplay enzymes with prosthetic groups can be displayed, expanding the application of surface display to the industrial important P450 enzymes. Finally, up to 105-106 enzyme molecules can be displayed on a single cell. In the present review, we summarize recent achievements in the autodisplay of enzymes with particular attention to industrial needs and process development. Applications that will provide sustainable solutions towards a bio-based industry are discussed.

Autodisplay of functional CYP106A2 in Escherichia coli.

Cytochrome P450 enzymes catalyse a wide variety of reactions, including the hydroxylation and epoxidation of CC bonds, and dealkylation reactions. There is high interest in these reactions for biotechnology and pharmaceutical processes. Many P450s require membrane surroundings and have substrates that do not cross biological membranes. To circumvent these obstacles, CYP106A2 from Bacillus megaterium was expressed on the outer membrane of Escherichia coli cells by Autodisplay. Exposure on the surface was confirmed by a protease accessibility test and flow cytometry after immunolabelling. HPLC assays showed that 0.5 ml of cells displaying the enzyme (OD578 = 6) converted 9.13 µmol of deoxycorticosterone to 15ß-OH-deoxycorticosterone within 1h. Imipramine and abietic acid were also accepted as substrates. The number of active enzyme molecules per cell was calculated to be 20,000. Surprisingly, surface-exposed CYP106A2 was active in E. coli BL21 without the external addition of the heme group. However, when CYP106A2 was expressed on the surface of an E. coli strain lacking the TolC channel protein (JW5503), enzymatic activity was almost completely abolished. The activity of CYP106A2 on the surface of E. coli JW5503 could be restored by the external addition of the heme group. This suggests, as has been reported before, that E. coli uses a TolC-dependent mechanism to export heme into the growth media, where it can be scavenged by a surface-displayed apoenzyme. Our results indicate that Autodisplay enables the functional surface display of P450 enzymes and provides a new platform to access their synthetic potential.