K-042. Evidence for Two Pathways for Isoleucine Biosynthesis in Geobacter sulfurreducens

M. V. Coppi¹, C. Risso¹, S. J. VanDien², A. Orloff¹, P. C. Moe¹, D. R. Lovley¹;
¹Univ. of Massachusetts, Amherst, MA, ²Genomatica Inc., San Diego, CA.

Reconstruction of central metabolic network of Geobacter sulfurreducens suggested that biosynthesis of isoleucine occurred via a pathway similar to that of Escherichia coli, in which the isoleucine precursor, 2-oxobutanoate, is generated from threonine. However, ¹³C labeling studies conducted in G. sulfurreducens suggested that threonine was not the major precursor for isoleucine biosynthesis. Based on the similarity of the mass isotopomer distributions of leucine and isoleucine, the majority of isoleucine appeared to be derived from acetyl-CoA and/or pyruvate, possibly via the citramalate pathway. Analysis of the G. sulfurreducens genome revealed candidate genes for the enzymes citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate. Single and double knockout mutants of these genes were constructed and characterized. The double mutant was an isoleucine auxotroph, whereas both single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single knockout mutants revealed the expected enzymatic deficiencies. ¹³C-labeling studies revealed clear differences in the isoleucine flux ratios of the single knockout mutants, indicative of primary of the threonine-dependent pathway in the citramalate synthase knockout mutant vs. primary use of the citramalate-dependent pathway in the threonine ammonia-lyase knockout mutant. Collectively these data indicate that in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases. This clade contains representatives from a wide variety of microorganisms, suggesting that the citramalate pathway for isoleucine biosynthesis may be more common than previously suspected.