Selective and Site-Specific Incorporation of Nonstandard Amino Acids Within Proteins for Therapeutic Applications.

The incorporation of nonstandard amino acids (nsAAs) within protein sequences has broadened the chemical functionalities available for use in the study, prevention, or treatment of disease. The ability to genetically encode the introduction of nsAAs at precise sites of target recombinant proteins has enabled numerous applications such as bioorthogonal conjugation, thrombin inhibition, intrinsic biological containment of live organisms, and immunochemical termination of self-tolerance. Genetic systems that perform critical steps in enabling nsAA incorporation are known as orthogonal translation systems or orthogonal aminoacyl-tRNA synthetase/tRNA pairs.

Discovery of L-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids.

Beta-hydroxy non-standard amino acids (β-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for β-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities.

A platform for distributed production of synthetic nitrated proteins in live bacteria.

The incorporation of the nonstandard amino acid para-nitro-L-phenylalanine (pN-Phe) within proteins has been used for diverse applications, including the termination of immune self-tolerance. However, the requirement for the provision of chemically synthesized pN-Phe to cells limits the contexts where this technology can be harnessed. Here we report the construction of a live bacterial producer of synthetic nitrated proteins by coupling metabolic engineering and genetic code expansion.

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells.

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C-C bond formation. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E.