On the role of isoleucyl-tRNA synthetase in multivalent repression

An isoleucine auxotroph of Salmonella typhimurium was derived from a merodiploid strain (containing the F-14 episome from Escherichia coli) that contained two copies of the structural genes concerned with isoleucine and valine biosynthesis. A haploid derivative, strain TU6001, having the same growth properties as the original merodiploid mutant was found to have normal biosynthetic enzymes and an altered isoleucyl-tRNA synthetase. The K _m for isoleucine was increased by about 200-fold over that for the wild-type enzyme. All five enzymes in the isoleucine and valine biosynthetic pathway were derepressed relative to wild-type enzyme levels. A partial revertant of strain TU6001 was isolated which had properties that were intermediate between those of the mutant and the wild type (i.e., intermediate growth dependence on exogenous isoleucine, intermediate activity of isoleucyl-tRNA synthetase, and intermediate derepression of biosynthetic enzymes). The properties of strain TU6001 were demonstrated to be simultaneously transferable by transduction (using P_LT22 H₄ bacteriophage) of a single genetic locus, linked to pyr A, which has been designated ilv S. It is concluded that some function of the isoleucyl-tRNA synthetase is important in repression of the isoleucine and valine biosynthetic enzymes.Supported by grant GM 12522 from the National Institute of General Medical Sciences, U.S. Public Health Service. J. M. B. received a U.S. Public Health Service Postdoctoral Fellowship 1-F02-GM-30, 650-02.     

Regulation of isoleucine and valine biosynthesis in Salmonella typhimurium: the effect of hisU on repression control.

The control of isoleucine and valine biosynthesis was examined in a hisU mutant of Salmonella typhimurium. It was found that the levels of expression of the ilvEDA operon and the ilvC gene were significantly reduced relative to an isogenic normal strain when grown in unsupplemented medium. In contrast, this hisU mutant exhibited only a slight reduction in total acetohydroxy acid synthase activity relative to that of the wild type. The hisU and hisU⁺ strains were examined to determine their derepressibility upon either leucine, valine or isoleucine limitation. Only during leucine limitation did the hisU strain exhibit impaired derepressibility relative to the hisU⁺ strain. In addition, repression control of threonine deaminase (the ilvA product of the ilvEDA operon) in this hisU mutant was refractory to exogenous supplementation with either leucine or valine. This response is in distinct contrast to that of the normal strain, in which the single addition of leucine or valine results in a significant reduction in the level of threonine deaminase.     

Multivalent repression and genetic depression of isoleucine-valine biosynthetic enzymes in Serratia marcescens

The regulation of the formation of isoleucine-valine biosynthetic enzymes was examined to elucidate the mechanism of isoleucine-valine accumulation by alpha-aminobutyric acid-resistant (abu-r) mutants of Serratia marcescens. In the isoleucine-valine auxotroph, l-threonine dehydratase, acetohydroxy acid synthetase, and transaminase B were repressed when isoleucine, valine, and leucine were simultaneously added to minimal medium. These enzymes were derepressed at the limitation of any single branched-chain amino acid. Pantothenate, which stimulated growth of this auxotroph, had no effect on the enzyme levels. It became evident from these results that in S. marcescens isoleucine-valine biosynthetic enzymes are subject to multivalent repression by three branched-chain amino acids. The abu-r mutants had high enzyme levels in minimal medium, with or without three branched-chain amino acids. Therefore, in abu-r mutants, isoleucine-valine biosynthetic enzymes are genetically derepressed. This derepression was considered to be the primary cause for valine accumulation and increased isoleucine accumulation.     

Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli

Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.     

Regulation of the formation of isoleucyl-tRNA-synthetase and the level of isoleucine biosynthetic enzymes in E. coli K12

Summary During derepression of threonine deaminase and acetolactate synthetase due to valine deficiency—initiated by -aminobutyric acid limited growth of E. coli K12 or by limited valine supply to an ilv/leu auxotroph of E. coli K12—no alteration of the specific activity of isoleucyl-tRNA-synthetase occurs. Leucine limited growth of the auxotroph, leading to an even higher derepression of the isoleucine biosynthetic enzymes, also does not affect the specific activity of isoleucyl-tRNA-synthetase. However, under growth conditions where the same degree of derepression of threonine deaminase is due to isoleucine deficiency, as in E. coli K12B or two valine resistant mutants thereof grown in the presence of valine, or in the auxotroph during growth-limiting isoleucine supply, a specific two- to three-fold derepression of the isoleucyl-tRNA-synthetase takes place. But there is no strict correlation between the degree of derepression of threonine deaminase due to isoleucine deficiency and the degree of derepression of isoleucyl-tRNA-synthetase, as especially shown in case of the valine resistant mutant Val R4 and Val R5 grown in the presence of valine.These results demonstrate that the rate of formation of isoleucyl-tRNA-synthetase and of threonine deaminase are not regulated by the same molecular devices and that a certain degree of isoleucine deficiency is a prerequisite for a derepression of isoleucyl-tRNA-synthetase.     

Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes

Engineering of Saccharomyces cerevisiae to produce advanced biofuels such as isobutanol has received much attention because this yeast has a natural capacity to produce higher alcohols. In this study, construction of isobutanol production systems was attempted by overexpression of effective 2-keto acid decarboxylase (KDC) and combinatorial overexpression of valine biosynthetic enzymes in S. cerevisiae D452-2. Among the six putative KDC enzymes from various microorganisms, 2-ketoisovalerate decarboxylase (Kivd) from L. lactis subsp. lactis KACC 13877 was identified as the most suitable KDC for isobutanol production in the yeast. Isobutanol production by the engineered S. cerevisiae was assessed in micro-aerobic batch fermentations using glucose as a sole carbon source. 93?mg/L isobutanol was produced in the Kivd overexpressing strain, which corresponds to a fourfold improvement as compared with the control strain. Isobutanol production was further enhanced to 151?mg/L by additional overexpression of acetolactate synthase (Ilv2p), acetohydroxyacid reductoisomerase (Ilv5p), and dihydroxyacid dehydratase (Ilv3p) in the cytosol.