Applied Biochemistry and Microbiology

The review summarizes the problems, achievements, and prospects of various approaches related to the delivery of components of DNA editing systems to plant cells, the regeneration of whole plants with an edited genome, and the development of transgene-free or transgene-clean crops. Here, an attempt was made to systematize the results of various studies describing the successful production of genome-edited (GE) plants with various genome modifications/mutations via the application of nuclease DNA–editing systems (ZFN, TALEN, and CRISPR/Cas). We discuss the main directions for the development of nuclease-based genome-editing methods to obtain GE plants free of foreign sequences from genome-editing tools.

It is shown that, unlike eukaryotic ubiquitin-specific proteinases (deubiquitinating enzymes, DUBs), the E. coli deubiquitinase ElaD can process sites containing not only GlyGly but also GlyAla with equal efficiency. At the same time, the bacterial enzyme, like eukaryotic DUBs, fails to process sites containing AlaGly, AlaAla, or GlyPro dipeptides. In light of the functional mission of the E. coli deubiquitinating agent, its decreased specificity can be considered a valuable evolutional acquisition that allows expansion of the list of molecular targets attacked during pathogenesis.

A Rhodococcus rhodochrous strain, M33-2nit, has been constructed with two copies of the nitrilase gene from A. denitrificans В-9582 under the control of nitrile hydratase promoter from R. rhodochrous M8. The optimized cultivation of this strain made it possible to obtain the enzyme in a concentration of up to 17 g of dry cells/L with a specific activity of up to 7 U/mg cdw with a two-substrate culturing scheme in a fed-batch reactor with the sequential addition of glucose and acetate. The capacities of A. denitriificans В-9582 and R. rhodochrous M33-2nit cells to synthesize ammonium acrylate from acrylonitrile under conditions imitating industrial synthesis are compared. It is shown that R rhodochrous M33-2nit cells can synthesize ammonium acrylate under higher rates of acrylonitrile feeding than A. denitriificans В-9582 cells. The potential to obtain a highly concentrated solution of ammonium acrylate (450 g/L) with R. rhodochrous M33-2nit cells as a biocatalyst was demonstrated. The conversion of acrylonitrile to ammonium acrylate reached 99.5%.

Efficient expression of the man1 gene for Aspergillus aculeatus β-1,4-mannanase was demonstrated in Pichia pastoris yeast for the first time, and industrially important characteristics of the synthesized enzyme were confirmed. β-Mannanases are industrial enzymes that can be used, in particular, in the feed industry. The most important requirements for feed enzymes are a broad pH range, thermal stability, and high specific activity. The obtained data indicate that the man1 gene from A. aculeatus is potentially useful for the construction of industrial mannanase producers based on Pichia pastoris yeast.

Marker-free transgenic Camelina sativa (L.) plants carrying a synthetic gene for cecropin P1, an antimicrobial peptide, under the control of the cauliflower mosaic virus 35S RNA promoter have been obtained and analyzed. The plants were transformed with an agrobacterial binary vector free of selective genes of antibiotic and herbicide resistance. The marker-free transformants were screened via measurement of the antibacterial activity of cecropin P1 and enzyme immunoassay. The obtained plants exhibited an increased resistance to infection with the bacteria Erwinia carotovora, the fungi Fusarium graminearum, and oxidative stress during infection. Analysis of the fatty acid composition of seed oil showed an increased amount of α-linolenic acid in the transgenic Camelina lines as compared to unmodified plants. The results indicate that the cecropin P1 gene can be included in an integral antistress plant-protective system.