Construction and characterization of Salmonella typhimurium strains that accumulate and excrete alpha- and beta-isopropylmalate.
Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates alpha- and beta-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Delta(leuD-ara)798 fol-162], accumulated and excreted alpha-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted alpha- and beta-isopropylmalate. The yield of alpha-isopropylmalate isolated from the culture medium of JK527 was more than five times the amount obtained from a comparable volume of medium in which Neurospora crassa strain FLR(92)-1-216 (normally used as the source for alpha- and beta-isopropylmalate) was grown. Not only was the yield greater, but S. typhimurium strains are much easier to handle and grow to saturation much faster than N. crassa strains. The combination of the two regulatory mutations flr-19, which results in constitutive expression of the leucine operon, and leuA2010, which renders the first leucine-specific biosynthetic enzyme insensitive to feedback inhibition by leucine, generated limitations in the production of valine and pantothenic acid. The efficient, irreversible, and unregulated conversion of alpha-ketoisovaleric acid into alpha-isopropylmalate (alpha-isopropylmalate synthetase K(m) for alpha-ketoisovaleric acid, 6 x 10(-5) M) severely restricted the amount of alpha-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase K(m) for alpha-ketoisovaleric acid, 1.1 x 10(-3) M; transaminase B K(m) for alpha-ketoisovaleric acid, 2 x 10(-3) M).
D015079:2-Isopropylmalate Synthase / Q000378:metabolism; D005809:Genes, Regulator; D045782:Hemiterpenes; D007651:Keto Acids / Q000378:metabolism; D007930:Leucine / Q000096:biosynthesis; D008293:Malates / Q000378:metabolism*; D009154:Mutation; D009876:Operon; D011407:Propane / Q000031:analogs & derivatives / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014161:Transduction, Genetic
D045782:Hemiterpenes; D007651:Keto Acids; D008293:Malates; C005906:alpha-isopropylmalate; C001505:alpha-ketoisovalerate; C025313:beta-isopropylmalate; D015079:2-Isopropylmalate Synthase; D007930:Leucine; D011407:Propane