Engineering Extracellular Secretion Pathways In Escherichia Coli

Abstract

Extracellular secretion is highly desirable in preparative protein production. The bacterium Escherichia coli is a commonly used for both laboratory- and industrial- scale biosynthesis of proteins, but it lacks many of the pathways for exporting proteins out of cells. This lack of a dedicated extracellular secretion system represents a major bottleneck across many biotechnology disciplines, in particular the bioprocessing of plant biomass where extracellular secretion of cellulase is required. Furthermore, the study and engineering of extracellular secretion systems is limited due to a lack of high-throughput screen to identify rare genetic conditions that affect secretion activity. Recently, it was discovered in E. coli that the YebF protein is secreted efficiently into the supernatant when over expressed, and YebF has been employed to carry heterologous proteins into the supernatant via C-terminal genetic fusions. Here, we harness the YebF pathway to simultaneously co-secrete active cellulases into the culture medium, which enabled non-cellulolytic E. coli cells to utilize and convert cellulose to bioenergy products. We also developed a universal approach to study and engineer YebF and other extracellular secretion pathways. This high-throughput screening platform was used to screen a genome-wide transposon insertion library for the isolation of gene deletions that upregulate the secretion of YebF and YebF fusions. We also developed an alternative strategy for engineering extracellular secretion systems by way of a genetic selection where the nonsecretory phenotype is lethal. Finally, we describe some of the physiological consequences to the bacterial host caused by heterologous protein secretion, in particular an envelope stress response that triggers CRISPR RNA-mediated DNA silencing. iii