A new chemoenzymatic route from carboxylic acids to nitriles
Biocatalysis has become an important part of organic synthesis but some desirable routes are still missing in its toolbox, among them the conversion of carboxylic acids into nitriles. Therefore, we propose to develop a mild and eco-friendly chemoenzymatic cascade combining two enzymes, carboxylate reductase (CAR) and aldoxime dehydratase (AOxD) with a chemical conversion of the products of CARs (aldehydes) into the substrates of AOxDs (aldoximes). The target process (acid to nitrile) is formally a reversed nitrilase reaction, which was never achieved using the nitrilase enzymes. To this end, E. coli strains will be furnished with genes coding for i) a CAR and its helper protein and ii) an AOxD, while stable CARs and AOxDs with broad substrate specificities will be selected. The optimum solution will reside in co-expression of these genes and establishing an acid-to-nitrile transformation in vivo. The environmental impact of the new route mainly resides in avoiding the use of cyanide and enabling the use of renewable materials, e.g., lignin, as sources of the carboxylic acids.
Stress responses in bacterial degrader of toxic pollutants Rhodococcus erythropolis
Rhodococcus erythropolis CCM2595, a versatile degrader of toxic aromatic pollutants, whose complete genome sequence was determined, is the object of this study. The main aim is to reveal the mechanisms of stress response in cells grown on phenol as a model toxic pollutant. Expression of the genes involved in phenol stress response will be studied both at the genome level using RNA sequencing and at the single gene level using in vivo and in vitro techniques. Role of the alternative sigma factors of RNA polymerase in stress response will be analyzed. Promoters of stress genes will be defined and their regulatory links to stress response and functions of sigma factors will be elucidated. A complex regulatory network of sigma factors and the respective stress regulons will be designed. Expression of the chosen genes encoding stress proteins, whose involvement in phenol stress response will be proved, will be modified with the aim to increase resistance of R. erythropolis strains to stress factors and improve their
efficiency of degrading toxic compounds.
Construction of a model of regulatory network controlled by sigma factors of RNA polymerase in Corynebacterium glutamicum
The project is focused on elucidating the roles of the alternative sigma factors of RNA polymerase (RNAP) in expression of Corynebacterium glutamicum genes and on integrating all available data into a model of sigma factor regulatory network. The particular methodical aims are the improvement of the unique in vitro transcription system for C. glutamicum, use of the reconstituted holo-RNAP for genome-wide in vitro transcription combined with sequencing of the in vitro-generated transcripts, primer extension analysis using in vitro-generated templates and construction of a two-plasmid system for in vivo assignment of sigma factors to particular promoters. All available techniques will be used for studies of sigma-specific regulons and the respective promoters of different classes in C. glutamicum. We will strive to define the regulons and their overlaps, assign the sigma factors to the localized promoters, discover the promoters recognized by two or more sigma factors, specify the consensus sequences of promoters of different classes and reveal some regulatory mechanism of expression of the sig genes. A model of a regulatory network controlled by sigma factors of RNAP in C. glutamicum is the final aim. The project will benefit from combining the in vivo/in vitro methods and genome-wide/single-gene analyses. Moreover, in silico modeling of recognition of the key promoter nucleotides by RNAP+sigma proteins will contribute to a complex view on the relations of individual holo-RNAPs to different classes of promoters. In future, the accumulated data can be used to the development of an expression system based on deletion, overexpression or modification of sigma factor genes in C. glutamicum.