The use of directed evolution towards altering the substrate specificity of acyl-coenzyme A : isopenicillin N acyl transferase and transforming it from generalist to specialist

摘要

Acyl Coenzyme A: Isopenicillin N Acyl Transferase (AT) is a key enzyme in the biosynthesis of β-lactam antibiotics in penicillin producing organisms such as P. chrysogenum and A. nidulans. Its natural activity is to exchange the side chain of the low activity antibiotic IPN [18] for the phenylacetyl side chain resulting in the more active antibiotic Penicillin G [5]. The biosynthesis of β-lactams has been exploited towards producing these compounds for therapeutic use. However, increasing bacterial resistance means new analogues in this compound class are constantly sought.As well as improving current production methods of β-lactam antibiotics, AT's broad substrate specificity means it could potentially play a role in the development and production of alternative β-lactam antibiotics that are able to overcome resistance.This thesis describes the identification of an AT mutant with improved acylation activity (AAT activity) via screening of an AT library using a previously developed screening method. Approaches towards the development of a method for the identification of AT mutants with improved hydrolysis activity were also explored. The main problem to overcome in developing such a screen is the inhibitory effect of 6-APA [1], the product of hydrolysis, on AT's IAT activity. The first approach investigated the potential of increasing the sensitivity of an assay by targeting AT to the periplasm. A second approach using β-lactamases to hydrolyse 6-APA [1] thus freeing up the active site of AT was also investigated.