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. 1980 May;142(2):513–520. doi: 10.1128/jb.142.2.513-520.1980

Construction and Characterization of Salmonella typhimurium Strains That Accumulate and Excrete α- and β- Isopropylmalate

Patricia N Fultz 1,†, Kyung K L Choung 1, Jost Kemper 1
PMCID: PMC294015  PMID: 6991477

Abstract

Two Salmonella typhimurium strains, which could be used as sources for the leucine biosynthetic intermediates α- and β-isopropylmalate were constructed by a series of P22-mediated transductions. One strain, JK527 [flr-19 leuA2010 Δ(leuD-ara)798 fol-162], accumulated and excreted α-isopropylmalate, whereas the second strain, JK553 (flr-19 leuA2010 leuB698), accumulated and excreted α- and β-isopropylmalate. The yield of α-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 FLR92-1-216 (normally used as the source for α- and β-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 α-ketoisovaleric acid into α-isopropylmalate (α-isopropylmalate synthetase Km for α-ketoisovaleric acid, 6 × 10−5 M) severely restricted the amount of α-ketoisovaleric acid available for conversion into valine and pantothenic acid (ketopantoate hydroxymethyltransferase Km for α-ketoisovaleric acid, 1.1 × 10−3 M; transaminase B Km for α-ketoisovaleric acid, 2 × 10−3 M).

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Selected References

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