Regulatory Assessment of Characteristics of Genetically Modified E. coli K-12 VKPM B-13285 Strain, Succinic Acid Producer. Analysis of Strain Behavior under the Influence of Environmental Factors

Abstract—Regulatory assessment and analysis were carried out of the compliance of the characteristics of a genetically modified microorganism (GMM) Escherichia coli K-12 VKPM B-13285, a producer of succinic acid (SA) containing a heterologous pyruvate carboxylase gene (pycA from the Bacillus subtilis strain) in the chromosome, with the requirements of Russian and international legislation applied to industrial GMM producers of biologically active substances. Comparative analysis was performed of a number of characteristics of the initial E. coli K-12 MG1655 and GMM under the influence of environmental factors. Methods for studying the growth characteristics of the strains, methods for estimating the effect of such parameters as temperature, UV radiation, incubation in water, soil, wastewater on the viability of bacteria are presented. A method of the identification of GMM among other representatives of enterobacteria by PCR analysis using pairs of locus-specific primers in the regions that distinguish a GMM from the initial E. coli K-12 strain, as well as a method of identification of enterobacteria on diagnostic media, are presented. The regulatory documents of Russian and international legislation with regard to the requirements for microorganisms obtained using the methods of genetic engineering were analyzed. The compliance of characteristics of E. coli K-12 VKPM B-13285 (a plasmid-free SA producer containing no antibiotic resistance genes) with the requirements of legislative documents was established. It was demonstrated that GMM differs from E. coli K-12 MG1655 in terms of growth parameters and the level of biomass accumulation and is characterized by reduced viability under UV irradiation, an increase in temperature, and cultivation in soil and wastewater. The possibility of identifying GMM under different conditions was established and the sensitivity of the methods using PCR analysis and diagnostic media was determined. Based on a comparative analysis of regulatory documents and of the characteristics and behavior of E. coli K-12 MG1655 and GMM under the influence of environmental factors, it is possible to state that the application of precision genomic editing using chromosomal recombination engineering is valid for the construction of microorganisms that meet international safety requirements in industrial biotechnology.