Cloning of an aroF allele encoding a tyrosine-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

Abstract

In Escherichia coli, genes aroF+, aroG+, and aroH+ encode isoenzymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. A single base pair change in aroF causes a Pro-148-to-Leu-148 substitution and results in a tyrosine-insensitive enzyme.

Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

“…The genes, aroF, aroH, and aroG, which code for the enzymes for this step, are feedback inhibited by the aromatic amino acids Tyr, Trp, and Phe, respectively (1,22,23). Deregulation of the feedback inhibition is essential to divert glucose to the aromatic amino acid pathway, and mutations were reported in the literature for the aroF and aroG genes that reduce or eliminate the feedback inhibition (12,24). In this study, the single amino acid mutant aroF gene, renamed as aroF FBR , was overexpressed.…”

“…The remaining two sequences were for native genes ubiC (EcoCyc accession number b4039/ ECK4031) and aroF FBR (GenBank accession number NC_000913), which encoded chorismate pyruvate-lyase and mutant phospho-2-dehydro-3-deoxyheptonate aldolase, respectively. The mutant gene aroF FBR encodes an amino acid replacement (Pro-148 is replaced with Leu), and this replacement has been proven to be resistant to Tyr amino acid feedback inhibition (12).…”

“…The plasmid with these three nonnative genes also expressed two native genes, ubiC and aroF FBR , which encoded chorismate pyruvate-lyase and 3-deoxy-arabino-heptulosonate 7-phosphate (DAHP) synthase, respectively. The aroF FBR gene was a mutated aroF gene (P148L) which encoded a mutant DAHP synthase which was resistant to feedback inhibition (FBR) (12). In the proposed MA production pathway, chorismate was first cleaved by native chorismate pyruvate-lyase (encoded by ubiC) to PHB, which was converted to PCA by PHB hydrolyase (encoded by pobA), along with the conversion of NADPH to NADP.…”

Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis

“…A single coding mutation (Pro148Leu) was introduced into E. coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase gene, by overlapping PCR, to make the Eco_aroF FBR gene. 22) In the first step, two separate PCRs were used to generate the primary PCR products, designated PCR Notes: The pathway of CoQ biosynthesis in S. pombe is shown. S. cerevisiae protein names are shown in parantheses.…”

“…5). The genes encoded the following proteins: truncated HMG-CoA reductase from S. cerevisiae that had no inhibitory regulation in the mevalonate pathway (Sce_thmgr1), 25) feedback-inhibition-resistant mevalonate kinase from S. cerevisiae (Sce_mvk FBR ), 23) phosphomevalonate kinase from S. pombe (Spo_erg8, SPAC343.01c), diphosphomevalonate decarboxylase from S. pombe (Spo_mvd1, SPAC24C9.03), feedbackinhibition-resistant DAHP synthase from E. coli (Eco_aroF FBR ), 22) chorismate synthase from S. pombe (Spo_aro2, SPCC1223.14), chorismate mutase from S. pombe (Spo_aro7, SPAC16E8.04c), phosphoenolpyruvate synthase from E. coli (Eco_ppsA), and chorismate lyase from E. coli (Eco_ubiC). Plasmids were constructed by inserting the appropriate PCR amplicon into pREP1.…”