Using structure-guided library design combined with an innovative ultrahigh throughput antibiotic screen,we have successfully engineered hLYZ variants that evade the E.coli inhibitory protein Ivyc.Surprisingly, however, the topperforming engineered enzymes acquired an unexpected sensitivity to the E. coli MliC inhibitor,the P.aeruginosa Ivyp
inhibitor,or both. Efforts to rationalize these observations via molecular modeling of the various enzyme−inhibitor complexes proved inconclusive, and in general these results
highlight the complexity of molecular determinants that underlie lysozyme recognition by proteinaceous inhibitors. Thus, it will be a challenge to develop hLYZ variants that are broadly evasive toward different classes of inhibitors or even similar orthologs from different pathogens. However, the diversity of our current Ivyc-resistant variants suggests that there are many alternative solutions to this general molecular engineering problem. Alternative library designs and appropriate application of high throughput screening technologies should enable efficient mining of higher performance enzymes.
For example, one reasonable next step would be construction of
an error-prone PCR library and more aggressive screening
against cocktails of inhibitory proteins. Regardless, the results
reported here represent an important proof-of-concept for
developing inhibitor-resistant lysozyme variants. We anticipate
that future efforts will capitalize on the lessons learned to
produce highly active enzymes able to more efficiently attack
and kill pathogens that currently subvert nature’s repertoire of
lysozyme antibiotics