Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.

Improving genomically recoded Escherichia coli to produce proteins containing non-canonical amino acids.

A genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.∆A) enables efficient genetic encoding of chemically diverse non-canonical amino acids (ncAAs) into proteins. While C321.∆A has opened new opportunities in chemical and synthetic biology, this strain has not been optimized for protein production, limiting its utility in widespread industrial and academic applications. To address this limitation, the construction of a series of genomically recoded organisms that are optimized for cellular protein production is described. It is demonstrated that the functional deactivation of nucleases (e.g., rne, endA) and proteases (e.g., lon) increases production of wild-type superfolder green fluorescent protein (sfGFP) and sfGFP containing two ncAAs up to ≈5-fold. Additionally, a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains is introduced. Using an optimized platform, the ability to introduce two identical N6 -(propargyloxycarbonyl)-L -Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent strain is demonstrated. The authors envision that their library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries.

Incorporation of Nonproteinogenic Amino Acids in Class I and II Lantibiotics.

Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. These methods complement recent alternative approaches to incorporate nonproteinogenic amino acids into lantibiotics.

Cell-Free Synthetic Biology: Engineering Beyond the Cell.

Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biology applications. In this review, we focus on the ability of CFPS to expand our understanding of biological systems and its applications in the synthetic biology field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biology as well as a protein production technology in years to come.