Poster: Metabolic engineering

Abs # 296: The novel Arabidopsis phenylalanine insensitive growth (pig) mutant displays a deregulated homeostasis of free amino acids

Presenter:	Voll, Lars M, voll@msu.edu
Authors	Voll, Lars M (A)   Fiene, Gabi  (B)   Weber, Andreas PM (A)
Affiliations:	(A): Michigan State University (B): University of Cologne

Branched amino acid biosynthesis pathways are frequently feedback-regulated at the committed steps of the respective branches and several feedback-insensitive mutants of amino acid biosynthesis have been isolated to date. The feedback-regulated enzymes at the committed steps of the shikimate pathway are anthranilate synthase on the branch leading to tryptophan and chorismate mutase on the branch leading to phenylalanine and tyrosine. Whereas mutants in the anthranilate synthase branch have been reported previously, no mutants of the chorismate mutase branch are identified. We have isolated a phenylalanine insensitive growth (pig) mutant, pig1-1 , from an EMS-mutagenized population by a forward genetic screen, employing phenylalanine concentrations that completely abrogate the germination of the wild-type. The corresponding nuclear mutation is recessive. Furthermore, pig1-1 does not phenocopy mutants of the anthranilate synthase branch. However, the pig-mutant was also cross-tolerant to tyrosine and tryptophan. In leaves of soil-grown pig1-1, free amino acids were elevated by 1.3- to 2-fold compared to the wild-type. When supplemented with permissive phenylalanine concentrations, free amino acid contents of pig1-1 were reduced by 90% with respect to the wild-type, while protein contents remained unaffected in all conditions applied. The observed differences between phenylalanine deplete and replete growth suggest that the metabolism of free amino acids is generally deregulated in pig1-1. Contrary to our initial expectation, the PIG1 locus is unlikely to positively affect the shikimate pathway, because phenylpropanoid contents in pig1-1 were lower than in the wild-type. The molecular characterization of the mutation is currently in progress.