|dc.description.abstracteng||A novel screening approach for identification of biocatalysts by reverse omics techniques was developed. Basic principle is the differential analysis of metatranscriptomes obtained from cultures treated either with the substrate or the product of the enzymatic reaction of interest. All genes upregulated in the substrate-containing culture respond to the added compound and could be involved in its degradation or belong to respective transporters or resistance mechanisms.
Nitrilases, enzymes degrading often toxic nitriles to the corresponding carboxylic acids and ammonia, were chosen as biocatalysts of interest. As effect of nitriles on microbial life is barely known, their influence had to be determined before establishment of the screening approach. For that purpose, agar plates containing different concentrations of nine tested nitriles (phenylacetonitrile, succinonitrile, acetonitrile, crotononitrile, 4-hydroxybenzonitrile, acetone cyanohydrin, cyclohexanecarbonitrile, 2-phenylpropionitrile, and pyruvonitrile) were prepared. Growth of Agrobacterium tumefaciens, Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli was monitored on these plates and revealed first insights into nitrile toxicity. Subsequently, highest non-toxic concentrations were used to treat liquid cultures containing a microbial community derived from compost. Growth was monitored regularly and cultures were transferred to fresh medium every second day to avoid nutrient depletion. Acetone cyanohydrin, 2-phenylpropionitrile, and pyruvonitrile exhibited a lethal effect on the microbial community. In contrast, cultures containing succinonitrile, acetonitrile, and crotononitrile showed higher optical densities than the control, indicating a growth-supporting effect. Furthermore, community composition was determined by 16S rRNA gene analysis and metagenome sequencing, revealing specific community-shaping effects for every compound, i.e. Pseudomonas was detected in most cultures whereas Paenibacillus was highly abundant in cultures containing growth-suppressing nitriles. In general, Gram-positive bacteria showed higher nitrile tolerance than Gram-negative bacteria.
Growth-supporting effect of acetonitrile during nitrile toxicity analysis indicated metabolization of this compound and therefore presence of nitrile-degrading enzymes. To increase knowledge on nitrile-degrading organisms and obtain strains for establishment of the screening approach, isolation of respective organisms was performed. Finally, eight different isolates belonging to Flavobacterium, Pseudomonas, Rhodococcus, and Variovorax were identified. Nitrile-degradation by the latter three is common, but only weak degradation by Flavobacterium has been reported before. Genome sequencing of the isolates revealed various nitrile-degrading enzymes for all strains except Flavobacterium, indicating a novel mechanism for nitrile degradation in this genus.
Analysis of metagenomes obtained during nitrile toxicity test revealed 70 putative nitrilases in nitrile-treated cultures. Due to their origin, they were promising candidates for identification of novel nitrile-degrading biocatalysts. For fast and simple screening of this number of enzymes, a novel high-throughput assay was developed. The new method combines real-time measurement of enzymatic activity and high sensitivity without dependency on purified proteins. Six putative enzymes exhibited nitrilase activity and subsequently, the most interesting biocatalysts was further characterized. The novel nitrilase exhibits a broad pH optimum and unusually high long-term stability. Furthermore, it is highly specific for phenylacetonitrile and belongs to the class of arylacetonitrilases, which are of industrial importance but rarely studied.
Finally, the proposed screening approach for identification of biocatalysts by reverse omics techniques was developed. For initial tests, the previously isolated acetonitrile-degrading Rhodococcus was used. Growth condition, cell harvesting, and isolation of DNA and RNA were optimized with this strain. Subsequently, a protocol for normalization and differential analysis of transcriptomes was established. Only one operon consisting of three genes and a nearby amidase were significantly upregulated in the nitrile-treated cultures. The operon did not encode for known nitrile-degrading proteins, but proximity to the amidase supports the respective activity. Subsequently, the established parameters were applied to a complex microbial community. Metatranscriptomes were normalized based on the abundance of single species to allow statistically valid analyses. More than 500 up- and 280 downregulated genes could be identified under nitrile-treatment, indicating complex interactions between members of the community. For one species, a highly upregulated nitrilase and amidase were found, whereas no significant upregulation of respective genes was recorded for other species. Therefore, unknown nitrile-degrading enzymes may hide behind upregulated putative proteins, demonstrating the potential of this novel metatranscriptomic screening approach.||de