(17g) Employing Bacterial Secretion for a High-Throughput Biomaterials Production and Screening Platform

Secretion is emerging as a useful strategy for exporting proteins of biotechnological interest from bacteria. The type III secretion system (T3SS) in Salmonella enterica is an ideal path to protein export because it is non-essential for bacterial metabolism and allows for target proteins to cross both bacterial membranes in one step, via characteristic needle-like protein structures. We engineered a super-secreting strain of Salmonella for the high-titer production of a variety of biochemically challenging heterologous proteins, including degradation-prone biopolymer proteins such as resilin, tropoelastin, spider silk, and mussel adhesive proteins. We achieve titers of approximately 50 mg/l for a variety of elastomeric proteins – a 100-fold improvement on wild type levels – at relative purity and with minimal truncation. We used our secretion production platform for several applications to date: 1) to create soluble biopolymer-forming proteins that were cross-linked to create biomaterials with unique properties; 2) to template metals in a wire-like configuration with nanoscale control over composition; and 3) to engineer adhesive proteins with desired properties by coupling with a high-throughput adhesion test. With this talk, we will provide details on the construction and analysis of these distinct types of materials and their features, and discuss future directions for this versatile protein secretion platform.