(629e) A Novel Technique for Transiently Permeablizing E. coli to Enable Extracellular Protein Engineering

While many intracellular biological processes have been commercialized at scale via fermentation, many natively extracellular enzymes with potential industrial applications (e.g. – digestive oxidases that can break down large substrates or enzymes with high steric specificity for building pharmaceuticals) remain understudied due to limitations in laboratory engineering. Bacillus subtilis is an effective host organism for industrial scale production of some extracellular enzymes as it is naturally secretory, but lacks the convenience and broad suite of genetic tools available for protein engineering in Escherichia coli. Unfortunately, E. coli and its close relatives in Pseudomonadota are not naturally secretory and current engineering for extracellular proteins is generally performed in low-throughput by purifying and characterizing mutant enzymes directly. In recent years, work has been done to develop E. coli strains with a permeable outer membrane phenotype that improves titers of recombinant proteins during fermentation and simplifies purification. This is usually accomplished by deleting or otherwise down regulating key outer membrane proteins, such as lipoproteins lpp and pal, peptidoglycan synthetases mrcA/B, and embedded porins like Omp A/C/F, resulting in a more porous membrane.

To enable better extracellular protein engineering in E. coli, we are utilizing optimized inner membrane secretion, which is a native activity of E. coli, in combination with the permeable phenotype approach. Inner membrane secretion in E. coli is highly protein specific and generally directed by N-terminal fusion tags that are removed upon translocation to the periplasm. We are developing iterations of these tags to optimize this transport step in conjunction with a novel approach to outer membrane permeabilization; controlling expression of a small inhibiting RNA (siRNA) factor, MicL, which is involved in suppressing metabolically expensive production of proteins like lpp during cell stress. The intended effect is a transiently induced permeable outer membrane state that allows a bacterial culture to grow as normal until protein expression and export is desired. The siRNA mediated approach will be validated against previously tested genome knockouts of lpp and mrcA using quantitatively measured fluorescence of GFP recovered from different cell and media fractions to determine secretion efficiency. The permeable E. coli strains will be used to express variants of Proteinase K and other highly active proteases which are normally toxic within the cytoplasm. Mutant libraries of these proteases will be screened to isolate improved variants and establish this approach for high-throughput engineering of secretory proteins.