Construction of Streptomyces jumonjinensis isopenicillin N synthase double mutants, Ser119Ala/Glu120Gly and Thr308Ala/Thr309Val, overcomes insoluble expression in Escherichia coli

Abstract

Of the isopenicillin N synthase isozymes, Streptomyces jumonjinensis isopenicillin N synthase (sjIPNS) is the only insoluble isozyme produced in Escherichia coli during heterologous expression. Hence, this study aims to optimise the soluble expression of sjIPNS in E. coli. By lowering the cultivation temperature from 37 to 25°C, previous studies have shown that the solubility of IPNS from S. clavuligerus (scIPNS), S. lipmanii (slIPNS) and Nocardia lactamdurans (nIPNS) in E. coli was much improved but the same could not be achieved for sjIPNS in this study. To decipher the uniqueness of the elusively insoluble sjIPNS, its amino acid sequence was compared to three other soluble isozymes, scIPNS, slIPNS and nIPNS. The computational analyses revealed two positions with adjacent sites at 119/120 and 308/309, where by sjIPNS differs significantly in terms of amino acid identities and properties. Site-directed mutagenesis was then used to alter these sites to investigate their influence on solubility. The two double mutants constructed, Ser119Ala/Glu120Gly and Thr308Ala/Thr309Val sjIPNS, were found to be soluble at 28 and 25°C, with optimal production up to 25.3 and 23.0%, respectively, of the total soluble proteins. Furthermore, both mutants were found to be catalytically active. The results of this study constitute the first report on the use of site-directed mutagenesis to successfully transform the solubility of sjIPNS to closely resemble that of other soluble bacterial IPNS isozymes. Copyright © 2001 Elsevier Science B.V.