| Abstract [eng] |
A novel enzyme, indole-3-carboxylic acid monooxygenase (Icm), which was able to convert indole-3-carboxylic acid into indigo, was identified from metagenome and characterized. Due to this activity, Icm enzyme was used as an auxiliary enzyme for the construction of enzyme-based screening system, where an enzyme of interest converts a substrate to indole-3-carboxylic acid, which is then oxidized to indigo by Icm, visually indicating the presence of the desired enzyme. The potential of this system was showcased by using indole-3-carboxaldehyde as a prochromogenic substrate for screening of the metagenomic libraries for aldehyde dehydrogenases. In addition to Escherichia coli, this system was also found to be applicable in a grampositive host – Rhodococcus erythropolis. Also, Icm tolerated certain modifications in the indole ring of indole-3-carboxylic acid that could allow for the selection of enzymes with different specificity. Given these points, Icm-based selection system represents a feasible method for obtaining metagenome-encoded enzymes with desired activities. Hind8 oxygenase, active towards indole, was selected as a promising tool for the conversion of a wide range of indole derivatives. By using E. coli cells with the recombinant Hind8 oxygenase as a bioconversion platform, a series 10 of novel indigoid compounds were produced for the first time. Among those, indigo and indirubin dicarboxylic acids showed increased solubility in water (from micromolar to millimolar range). Hence, the oxygenase Hind8 represents a novel tool with wide substrate specificity for the biosynthesis of indigoid compounds. On top of that, a novel approach can be suggested for increasing the water solubility of indigoids by the introduction of carboxyl groups. Novel biological functions of indigo-producing and related enzymes are shown in this work. Although indole detoxification function has been suggested for the enzyme IifC, no functions for other Iif proteins have been assigned. By identifying that Iif proteins catalyze the initial steps of indole biodegradation, this study elucidates the long-searched genes and proteins responsible for indole catabolism, suggesting that indole can be detoxified by certain bacteria through Iif-mediated assimilation. Following the elucidation of bacterial indole biodegradation, several important aspects of bacterial degradation of indole-3-acetic acid (IAA, a plant growth hormone) have been addressed. It has been known that Iac proteins are responsible for the initial steps of IAA biodegradation. This study clarifies that IacB protein does not participate in IAA biodegradation. Also, experimental evidence presented here suggest an H2O-dependent formation of DOAA, an intermediate compound of IAA biodegradation. In addition, IacA and IacE enzymes were found to be capable of converting indole-3-propionic acid and indole-3-butyric acid into corresponding DOAA derivatives. As no biodegradation data has been available so far regarding these biologically active derivatives of indole, IacA and IacE enzymes could be a potential constituent of such hypothetical pathway. |
