Use of an Alternative Pathway for Isoleucine Synthesis in Threonine-Producing Strains of Escherichia coli
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UCERS, BIOLOGY, SELECTION, AND GENE ENGINEERING
Use of an Alternative Pathway for Isoleucine Synthesis in Threonine-Producing Strains of Escherichia coli T. V. Vybornayaa, *, T. V. Yuzbasheva, A. S. Fedorova, D. M. Bubnova, S. S. Filippovaa, F. V. Bondarenkoa, and S. P. Sineokya a
State Research Institute for Genetics and Selection of Industrial Microorganisms, Kurchatov Institute National Research Center (Kurchatov Institute NRC, GOSNIIGENETIKA), Moscow, 117545 Russia *e-mail: [email protected] Received June 6, 2019; revised July 5, 2019; accepted July 22, 2019
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. Keywords: Escherichia coli, strain, threonine, citramalate synthase DOI: 10.1134/S0003683820070066
INTRODUCTION Inactivation of the degradation pathways of the target product is the most important task in the construction of producer strains. At present, there are three known threonine degradation pathways in E. coli cells. Amino-acid catabolism is carried out by three enzyme groups: threonine dehydrogenases, threonine aldolases, and threonine deaminases. The enzymes of the first group, which are encoded by the tdh gene, play the key role in threonine degradation. It was shown that inactivation of this gene has a positive effect on threonine production [1, 2]. It is believed that there is another gene, yiaY, that encodes NAD-dependent threonine dehydrogenase [3]. The literature contains conflicting data on the phenotype of strains carrying a deletion in the yiaY gene [3, 4]. Inactivation of the ltaE gene, which encodes threonine aldolase, also causes an increase in threonine production [5]. Serine hydroxymethyltransferase, which is encoded by the glyA gene, has treonine aldolase activAbbreviations: 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.
ity [6]. However, inactivation of the glyA gen
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