Directed Evolution of an Escherichia coli Secretor Strain Using the Curli Pathway

A key goal of many bacterial engineering projects is to produce protein, due to their potent chemical and material functions. However, recombinant proteins are usually produced intracellularly, and secreting arbitrary proteins directly into an extracellular environment is challenging. In this work, we explored recombinant protein secretion using the curli secretion pathway in the probiotic Escherichia coli Nissle 1917 strain (EcN). We developed a Green Fluorescent Protein (GFP) secretion system by fusing GFP of the N-terminus sequence from the curli monomer protein while co-expressing the curli export machinery. We characterized and optimized our designs, obtaining ∼60 μg/mL extracellular GFP protein from plasmid pSecGFP1h. To further increase protein yield, we performed a directed evolutionary process using chemical mutagenesis and selecting for both high GFP yield and secretion of a protein toxic to E. coli. We performed several rounds of mutagenesis and selection to generate two distinct lineages, which both secreted ∼120 μg/mL extracellular GFP. We then sequenced and analyzed the genomes of strains making up the two lineages, assembling genome sequences for 8 EcN-derived strains, finding ∼50 point mutations per round of mutagenesis, and identifying the genes affected. We further assessed the capability of our evolved E. coli strains to secrete diverse proteins, including proteinaceous materials, enzymes, and therapeutic peptides. We obtained successful secretion for many proteins and found that secretion was sequence-specific, although no simple metric could predict success. The methodology we developed for microbial strain engineering and protein secretion has the potential to be generalizable to many biotechnological applications to take advantage of the abilities of proteins.