Therapeutic promise of engineered nonsense suppressor tRNAs.

Nonsense mutations change an amino acid codon to a premature termination codon (PTC) generally through a single-nucleotide substitution. The generation of a PTC results in a defective truncated protein and often in severe forms of disease. Because of the exceedingly high prevalence of nonsense-associated diseases and a unifying mechanism, there has been a concerted effort to identify PTC therapeutics. Most clinical trials for PTC therapeutics have been conducted with small molecules that promote PTC read through and incorporation of a near-cognate amino acid. However, there is a need for PTC suppression agents that recode PTCs with the correct amino acid while being applicable to PTC mutations in many different genomic landscapes. With these characteristics, a single therapeutic will be able to treat several disease-causing PTCs. In this review, we will focus on the use of nonsense suppression technologies, in particular, suppressor tRNAs (sup-tRNAs), as possible therapeutics for correcting PTCs. Sup-tRNAs have many attractive qualities as possible therapeutic agents although there are knowledge gaps on their function in mammalian cells and technical hurdles that need to be overcome before their promise is realized. This article is categorized under: RNA Processing > tRNA Processing Translation > Translation Regulation.

Cell-Free Synthesis of Natural Compounds from Genomic DNA of Biosynthetic Gene Clusters.

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.

Fatty Acid Biosynthesis: Chain-Length Regulation and Control.

De novo biosynthesis of fatty acids is an iterative process requiring strict regulation of the lengths of the produced fatty acids. In this review, we focus on the factors determining chain lengths in fatty acid biosynthesis. In a nutshell, the process of chain-length regulation can be understood as the output of a chain-elongating C-C bond forming reaction competing with a terminating fatty acid release function. At the end of each cycle in the iterative process, the synthesizing enzymes need to "decide" whether the growing chain is to be elongated through another cycle or released as the "mature" fatty acid. Recent research has shed light on the factors determining fatty acid chain length and has also achieved control over chain length for the production of the technologically interesting short-chain (C4 -C8 ) and medium-chain (C10 -C14 ) fatty acids.

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression.

The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.