Abstract
An E. coli strain in which all known pathways of threonine catabolism were inactivated (Δtdh, ΔltaE, ΔilvA, ΔtdcB, ΔyiaY) has been constructed. It was shown that the expression of heterologous citramalate synthase from Leptospira interrogans in an E. coli strain carrying the ΔilvA deletion can serve as an alternative pathway for isoleucine synthesis. It was observed that cimA overexpression has a negative effect on threonine production. We developed a system for regulated gene expression based on the inducible promoter PLtetO and TetR, a repressor of the tetracycline operon. The threonine-producing strain B-1201, in which the cimA gene is expressed under the control of the regulated promoter, was constructed. When the B-1201 strain was cultivated in a fermenter, a correlation was established between the threonine productivity and the expression level of the cimA gene, and the optimal inductor content for the maximum threonine accumulation was also determined.
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ACKNOWLEDGMENTS
The work was carried out on the equipment of the Multipurpose Scientific Installation of the Russian State Collection of Industrial Microorganisms, Kurchatov Institute National Resource Center (NRC GOSNIIGENETIKA).
The authors are grateful to Dr. I.V. Manukhov for the provision of the MG1655Z1 strain and pZE21-lux plasmid and for assistance with the development of techniques to measure bioluminescence.
Funding
The work was supported by the Ministry of Education and Science of the Russian Federation (Project Unique Identifier no. RFMEFI61017X0011).
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Translated by I. Gordon
Abbreviations: ATc—anhydrotetracycline; FB—fermentation broth; HPLC—high-performance liquid chromatography; LB medium—lysogeny broth medium; NAD—nicotinamide adenine dinucleotide; OD660—optical density at a wavelength of 660 nm; SD sequence—Shine—Dalgarno sequence; 5-UTR—5'-untranslated region; VKPM—Russian State Collection of Industrial Microorganisms.
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Vybornaya, T.V., Yuzbashev, T.V., Fedorov, A.S. et al. Use of an Alternative Pathway for Isoleucine Synthesis in Threonine-Producing Strains of Escherichia coli . Appl Biochem Microbiol 56, 759–769 (2020). https://doi.org/10.1134/S0003683820070066
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DOI: https://doi.org/10.1134/S0003683820070066