Expanding The Capabilities Of A Bacterial Quality Control Mechanism For Engineering Enzymes
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. Following two iterations through our dual selection and screen, we isolated a Cel5A variant whose production is increased 30-fold over the parent enzyme. The gain in production is achieved without any loss in activity on soluble or insoluble cellulose substrates, underscoring the value in this two-step evolution approach. Further, we have characterized several of the improved variants to begin determining which biophysical properties are selected through the directed evolution process. Additionally, we have discovered the chaperone-like activity of a component of the Tat pathway. The results of these experiments are helping to shed light on the poorly understood quality control mechanism and will guide future protein engineering attempts that exploit this pathway. ii
Protein engineering; Cellular quality control; Heterologous cellulase production
Lin, Hening; Putnam, David A.; Varner, Jeffrey D.
Ph.D. of Chemical Engineering
Doctor of Philosophy
dissertation or thesis