Title

CLONING GENERAL TRANSAMINASE GENES IN ESCHERICHIA COLI K-12 AND MULTICOPY SUPPRESSION

Date of Completion

January 1987

Keywords

Biology, Microbiology

Degree

Ph.D.

Abstract

Four general transaminase genes aspC, ilvE, avtA and tyrB, which encode transaminase A, transaminase B, transaminase C and transaminase D, respectively, were cloned in vivo using a miniMu plasmid element. It was shown in this study that null activity mutations in two different transaminase genes (ilvE and tyrB) and one probable alanine biosynthetic gene (alaA) were suppressed when avtA, which encodes the alanine-valine transaminase, transaminase C, is present on a multicopy plasmid. This suppression is due to the synthesis of isoleucine, leucine and alanine at physiologically significant levels by cells that have elevated alanine-valine transaminase levels. Suppression of a mutation in one gene by amplified copies of a nonallelic, wild-type gene is termed multicopy suppression. Further enzyme kinetic studies showed that, in the case of isoleucine synthesis, the biochemical basis of multicopy suppression of ilvE is due to a low Vmax of transaminase C towards 2-keto-3-methylvalerate (the precursor of isoleucine), which makes the ability of transaminase C to participate significantly in isoleucine synthesis so dependent on gene dosage.^ Multicopy suppression was used as a genetic tool to clone the genes involved in L-alanine biosynthesis. MiniMu plasmid transductants of an auxotroph that has a partial requirement for either alanine or valine were selected in the absence of these amino acids. Three different classes of miniMu plasmids were obtained: avtA, which encodes the alanine-valine transaminase; tyrB, which encodes the aromatic amino acid transaminase (transaminase D); and a previously undescribed gene, called alaB; which encodes an alanine-glutamate transaminase. Although the basis of multicopy suppression in alanine biosynthesis is only partially understood, multicopy suppression is useful for detecting secondary functions of the gene product and for identifying genes involved in a certain biosynthetic pathway.^ The feasibility of cloning transaminase genes on a multicopy plasmid by selecting for 3-chloro-L-alanine resistance was tested. Results showed an unknown gene was cloned, instead of transaminase genes. The inability to identify this unknown gene which probably codes for a 3-chloro-L-alanine degradative enzyme, but not for O-acetylserine sulfhydrylase, has hindered the further analyses. ^