Engineering Incorporation of Non-Standard Amino Acids in a Gram-Positive Bacterium

The incorporation of non-standard amino acids (nsAA) within protein sequences offers the benefits of an expanded repertoire of chemistry for protein products and a means to control protein expression at the translational level. For live organisms, nsAA incorporation can also be used to achieve biological containment by synthetic auxotrophy. However, the effectiveness of synthetic auxotrophy outside of batch mono-cultures is unknown, as is the ease of transferring nsAA incorporation technologies to other industrially relevant bacteria. In this talk, I will highlight our latest characterization of Escherichia coli synthetic auxotrophs when added directly to mammalian cell cultures and our successful efforts to engineer nsAA incorporation in the gram-positive microbe Bacillus subtilis. B. subtilis is a model system for both the study of bacterial cell biology and for industrial uses as a probiotic, rhizobacterium, efficient protein secretor, and spore-former. To enable greater understanding and control of proteins in B. subtilis, we demonstrated broad and efficient genetic code expansion in B. subtilis by incorporating many distinct non-standard amino acids within proteins using multiple genetic code expansion systems and two choices of codons. We used these systems to achieve click-labelling, photo-crosslinking, and translational titration. Our work sets the stage for a B. subtilis system that could be augmented in chemical capabilities as a live cell factory and safeguarded effectively in contexts outside the reactor.