PURPOSE
Regulatory assessment and analysis of the compliance of the characteristics of the genetically modified microorganism (GMM) Escherichia coli K-12 VKPM B-13285 producer of succinic acid (UC), containing the heterologous pyruvate carboxylase gene — pycA from the Bacillus subtilis strain, with the requirements of Russian and international legislation applicable to industrial GMM producers of biologically active substances. Comparative analysis of a number of characteristics of the initial E. coli K-12 MG1655 and GMM under the influence of environmental factors.
MATERIALS AND METHODS
Methods for studying the growth characteristics of strains, methods for assessing the influence of such parameters as temperature, UV radiation, incubation in water, soil, and wastewater on the viability of bacteria are presented. The method of identification of GMM among other representatives of enterobacteria by PCR analysis using pairs of locus-specific primers in the areas that distinguish GMM from the original E. coli K-12 strain, as well as the method of identification of enterobacteria on diagnostic media, is presented.
RESULTS
The article analyzes the regulatory documents of the Russian and international legislation regarding the requirements for microorganisms obtained using the methodology of genetic engineering. The characteristics of E. coli K-12 VKPM B-13285, a plasmid-free, non-antibiotic-resistant UC producer, meet the requirements of legislative documents. It is shown that GMM differs from E. coli K-12 MG1655 in terms of growth parameters, the level of biomass accumulation, is characterized by reduced viability under UV irradiation, temperature increase, cultivation in soil and wastewater. The possibility of identifying GMM in various conditions was established and the sensitivity of methods using PCR analysis and diagnostic media was determined.
CONCLUSION
Based on a comparative analysis of regulatory documents, characteristics and behavior of E. coli K-12 MG1655 and GMM under the influence of environmental factors, it is possible to state the validity of using precision genomic editing using chromosomal recombination engineering to design microorganisms that meet international safety requirements in industrial biotechnology.
