Production of industrially relevant proteins using cell-wall deficient cells

Abstract

Over the last few decades, E. coli cells have been increasingly used in industrial biotechnology to produce products of high economic value. This includes enzymes, therapeutics, and dietary supplements. However, currently used E. coli systems have drawbacks, making them sub-optimal for producing certain types of products. L-form E. coli could target these issues and be established as an industrially viable strain for specific commercial products. L-forms are cell wall deficient strains of bacteria that can be created using mutations, spacial constraints, or antibiotic treatments. Their property of L-form switching could provide robustness in industrial settings. This project deals with understanding the properties of an E. coli L-form strain and how it responds to standard DNA manipulation and transformation techniques. The project also looks at this strain’s ability to produce simple fluorescent proteins and goes on to scale up to more complex proteins like enzymes. Using two enzymes, ɑ-amylase and β-galactosidase, we show that the L-form strain can also produce functional enzymes. We were able to come up with a new protocol for reliably transforming L-form E. coli and were also able to determine the optimal conditions for protein expression on solid media. Scaling up experimentation with bioreactors and testing the enzymes in a liquid assay format would allow for a better understanding of L-form E. coli. Creating a stable L-form from an industrial strain would also aid in comparing protein production between various strains.