(143d) Repurposing Bacterial Quality Control for the Engineering of Proteins With Superior Traits

In this study, we have repurposed the intrinsic quality control of the twin-arginine translocation (Tat) pathway to enhance the traits of target proteins using directed evolution.  As a proof-of-concept, we applied this method to the endocellulase Cel5A from the fungal plant pathogen Fusarium graminearum to improve its production and function.  Our approach is based on a novel two-tiered genetic selection and screening method.  First, a Tat-based genetic selection is applied that links protein translocation with resistance to beta-lactam antibiotics.  Since the quality control mechanism of the Tat pathway only permits the export of folded proteins, this genetic selection allows for the rapid and high-throughput isolation of well-folded, stable Cel5A library members while eliminating those that are poorly folded.  A second screening step is imposed to ensure that the proteins that pass the Tat quality control retain high activity.   For Cel5A, this involves a screen of enzyme activity using the soluble cellulose substrate carboxymethyl cellulose (CMC).  Following two iterations through our two-tiered selection and screen, we isolated a Cel5A variant whose production is increased 10-fold over the parent enzyme.  The gain in production is achieved without any loss in activity, underscoring the value in this two-step evolution approach.  Further, we have characterized several of the improved variants to determine which biophysical properties are selected by the Tat quality control mechanism.  The results of these experiments are helping to shed light on the poorly understood quality control mechanism and should guide future protein engineering attempts that exploit this pathway.