Improving Genomically Recoded Escherichia coli for the Production of Proteins Containing Non-Standard Amino Acids

The expansion of the genetic code to co-translationally incorporate non-standard amino acids is a rapidly growing field in synthetic biology. This is due to its potential to transform how we synthesize materials and therapeutics, investigate protein structure, and understand the evolution of the translation system. A pioneering effort has recently developed an Escherichia coli strain lacking all TAG codons and release factor 1 which allows for genetic encoding of an additional, non-standard amino acid. This strain however has not previously been optimized for protein production. Here we describe the construction of a series of genomically recoded organisms that are optimized for protein production and have the additional ability to tune protein expression through a T7 RNA polymerase (T7RNAP). Using multiplex automated genome engineering (MAGE), we targeted the individual deletion of 5 putative negative effector genes as well as all double and several triple mutants. We found that reduction of nuclease and protease activity increases wildtype sfGFP production by 260% and sfGFP containing 2TAGs production by 225% and 560% with p-azidophenylalanine and N6-(propargyloxycarbonyl)-L-Lysine, respectively. Next, we designed and optimized an IPTG and a Tetracycline inducible T7RNAP cassette for reduced background expression and high final cell density. These cassettes were genomically inserted into the top mutant strains. We envision that our library of strains will provide the community with multiple strain options for expression of proteins containing non-standard amino acids with increased protein yield and cell density.