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We have constructed an RNA molecule containing a hammerhead ribozyme that is under allosteric control. In the inactive state, the RNA enzyme is unable to cleave a suitable substrate.The formation of the active state of the ribozyme is triggered by a specific interaction with a DNA oligonucleotide effector that is complementary to a single-stranded loop in the RNA enzyme molecule.Other DNA or RNA molecules containing unrelated nucleotide sequences do not function as allosteric effectors. This work demonstrates the feasibility of designing RNA enzymes that are specifically activated in response to an artificially designed molecular recognition event. Such enzymes may have practical applications.
We demonstrate a novel and efficient bioprocess for production of the cephalosporin intermediates, 7-aminocephalosporank acid (7-ACA) or 7-amino deacetoxycephalosporanic acid (7-ADCA). The Streptomyces clavuligerus expandase gene or the Cephalosporium acremonium expandase-hydroxylase gene, with and without the acetyltransferase gene, were expressed in a penicillin production strain of Penicillium chrysogenum. Growth of these transformants in media containing adipic acid as the side chain precursor resulted in efficient production of cephalosporins having an adipyl side chain, proving that adipyl-6-APA is a substrate for either enzyme in vivo. Strains expressing expandase produced adipyl-7-ADCA, whereas strains expressing expandase-hydroxylase produced both adipyl-7-ADCA and adipyl-7-ADAC (aminodeace-tylcephalosporanic acid). Strains expressing expandase-hydroxylase and acetyltransferase produced adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. The adipyl side chain of these cephalosporins was easily removed with a Pseudomonas-derived amidase to yield the cephalosporin intermediates.