Correlation of biochemical blocks and genetic lesions in leucine auxotrophic strains of the imperfect yeast Candida maltosa

Abstract

The four enzymatic steps in the conversion of alpha-ketoisovaleriate to leucine were examined in the wild type and in 13 leucine auxotrophic strains of Candida maltosa. The genetic lesions in the auxotrophs, involve at least five different loci and are correlated with three enzymatic steps. This was confirmed by gene cloning, protoplast fusion, and enzyme assays. The pathway for leucine biosynthesis in C. maltosa shows general similarity to that of other lower eukaryotes but there are individual differences in the numbers of genes responsible for single enzymatic steps. A disomic state of the chromosomes carrying genes coding for alpha-isopropylmalate synthase and beta-isopropyl-malate dehydrogenase was elucidated.

Candida maltosa

“…An additional novel control step exists, the inhibition of 3-IPM dehydrogenase by L-valine (Bode 1991; Birnbaum 1991c). The enzyme biosynthesis is regulated by at least three different control mechanisms of gene regulation -leucine repression, valine induced derepression, and general control of amino acid biosynthesis (Becher et al 1991;Bode 1991;Bode et al 1991). The isolated leu2 mutants were used to establish hostvector systems for C. maltosa using the cloned LEU2 gene (Takagi et al 1988a;Wedler et al 1990;Becher et al 1991;cf. Tables 14 and 15).…”

“…The enzyme biosynthesis is regulated by at least three different control mechanisms of gene regulation -leucine repression, valine induced derepression, and general control of amino acid biosynthesis (Becher et al 1991;Bode 1991;Bode et al 1991). The isolated leu2 mutants were used to establish hostvector systems for C. maltosa using the cloned LEU2 gene (Takagi et al 1988a;Wedler et al 1990;Becher et al 1991;cf. Tables 14 and 15). The potential application of leul and leu2 mutants of C. maltosa for the production of2-IPM and 3-IPM, respectively, was recently reported see Sect.…”

“…4.2 and 4.4). The enzymes could be purified at least partially (Table 4) and their catalytic properties were determined, showing some differences to the enzymes of other yeast and fungi (Bode andBirnbaum 1988, 1991c;Becher et al 1991;Bode 1991). The pathway is metabolically controlled mainly by feedback inhibition of the first enzyme 2-IPM synthase by leucine.…”

“…The four enzymatic steps in the conversion of a-ketoisovalerate to leucine, including 2-IPM synthase (EC 4.1.3.12, coded by the LEU4 gene), IPM dehydratase (EC 4.2.1.33, LEUl gene), 3-IPM dehydrogenase (EC 1.1.1.85, LEU2 gene) and L-Ieucine (branched-chain) aminotransferase (EC 2.6.1.42), were examined in the wild-type and leucine auxotrophic strains of C. maltosa (Takagi et al 1986a;Bode andBirnbaum 1988, 1991c;Wedler et al 1990;Bode 1991;Bode et al 1991;Becher et al 1991; see also Table 6 and Sects. 4.2 and 4.4).…”

Leucine Biosynthesis in Fungi: Entering Metabolism through the Back Door

“…However, the regulation seems to differ in at least some respects from that seen in S. cerevisiae and N. crassa, where very little formation of isomerase and dehydrogenase occurs in synthase-minus strains (see above). Another study of leucine biosynthesis was carried out with Candida maltosa, an imperfect yeast that is able to assimilate liquid hydrocarbons for biomass production (9). In this case, 13 leucine auxotrophs were found to constitute five complementation groups (determined by protoplast fusion since C. maltosa lacks a sexual cycle), one affecting ␣-isopropylmalate synthase, three affecting isopropylmalate isomerase, and one affecting ␤-isopropylmalate dehydrogenase.…”