Expression in Escherichia coli K-12 of the structural gene for catabolic dehydroquinase of Neurospora crassa

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Abstract
The inducible quinic acid catabolic pathway of Neurospora crassa is controlled by four genes, the qa cluster which includes structural genes qa-2, qa-3, qa-4 for three enzymes and a regulatory gene, qa-1 . In this paper we report the molecular cloning of at least the qa-2 gene which encodes the catabolic dehydroquinase (5-dehydroquinate hydro-lyase, EC 4.2.1.10). Endo·R· Hin dIII restriction endonuclease fragments of N. crassa DNA from a qa-1 c (constitutive) mutant and of Escherichia coli plasmid pBR322 DNA were ligated in vitro and used to transform an aroD6 (5-dehydroquinate hydrolyase deficient) strain of E. coli K12. The recombinant plasmid (pVK55) isolated from one AroD + transformant (SK1518) contained, in addition to pBR322, two N. crassa Hin dIII fragments with molecular weights of 2.3 × 10 6 and 1.9 × 10 6 . Derivatives of SK1518 cured of plasmid DNA were phenotypically Amp s and AroD - . These cured strains, retransformed with pVK55, were phenotypically Amp R and AroD + . Strains transformed with pVK55 possessed 5-dehydroquinate hydrolyase activity but no activity was present in any AroD - strain. The enzyme extracted from strains containing the recombinant plasmid was identical to N. crassa catabolic dehydroquinase by the criteria of heat stability, ammonium sulfate fractionation, immunological crossreactivity, molecular weight, and purification characteristics. This identity demonstrates that the N. crassa qa-2 + gene is carried by the recombinant plasmid and is apparently transcribed and translated with complete fidelity. Furthermore, subunit assembly of the N. crassa polypeptides also occurs in E. coli , because the catabolic dehydroquinase is a multimer composed of approximately 20 identical subunits.