A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology
Simon J Moore, Hung-En Lai, Soo-Mei Chee, Ming Toh, Seth Coode, Kameshwari Chengan, Patrick Capel, Christophe Corre, Emmanuel LC de los Santos and Paul S Freemont
Prokaryotic cell-free coupled transcription–translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test tube. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool, and an active background metabolism. However, our initial yields were low (36 μg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 μg/mL of expressed recombinant protein. To complement this, we rapidly characterize a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes, and demonstrate an initial proof of concept for combined transcription, translation, and biosynthesis of Streptomyces metabolic pathways in a single “one-pot” reaction.
Engineering bacteria to produce pure phage-like particles for gene delivery
Tridgett Matthew, Abab, Maria, Osgerby Alexander, Ramirez Garcia Robert and Jaramillo Alfonso
Natural and engineered phages have been used in many applications, but their use to deliver user-defined genetic cargoes has been hampered by contamination with replicative phage, restricting use of the technology beyond the laboratory. Here we present a method to produce transducing particles without contamination. In addition, we demonstrate the use of a helper phage-free transducing particle preparation as an antimicrobial agent. This will pave the way for the development of new phage-based technologies with greater scope than lytic phage therapy.
Orkun Soyer publications
Inhibiting the reproduction SARS-CoV-2 through perturbations in human cell metabolic network
Hadrien Delattre, Kalesh Sasidharan, Orkun S Soyer
Here, we made use of genomic and structural information to create a biomass function capturing the amino and nucleic acid requirements of SARS-CoV-2. Incorporating this biomass function into a stoichiometric metabolic model of the human lung cell and applying metabolic flux balance analysis, we identified host-based metabolic perturbations inhibiting SARS-CoV-2 reproduction. Our results highlight reactions in the central metabolism, as well as amino acid and nucleotide biosynthesis pathways. By incorporating host cellular maintenance into the model based on available protein expression data from human lung cells, we find that only few of these metabolic perturbations are able to selectively inhibit virus reproduction.
Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity
Helen Morcrette, Andrea Kovacs-Simon, Richard K Tennant, John Love, Sariqa Wagley, Zheng R Yang, David J Studholme, Orkun S Soyer, Olivia L Champion, Clive S Butler and Richard W Titball
The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.
Engineering isoprenoid quinone production in yeast
Isoprenoid quinones are bioactive molecules that are traditionhetic Biolally found to be involved in primary metabolism, where they act as electron transporters, but specialized isoprenoid quinones are also produced by all domains of life. Here, we report the engineering of a baker’s yeast strain, Saccharomyces cerevisiae EPYFA3, for the production of isoprenoid quinones. As a proof of concept, our new host strain was used to overproduce the endogenous isoprenoid quinone coenzyme Q6, resulting in a nearly three-fold production increase. EPYFA3 represents a valuable platform for the heterologous production of high value isoprenoid quinones, and facilitates the elucidation of isoprenoid quinone biosynthetic pathways
Pattern Engineering of Living Bacterial Colonies Using Meniscus Driven Fluidic Channels
Vasily Kantsler, Elena Ontañón-McDonald, Cansu Kuey, Manjari J. Ghanshyam, Maria Chiara Roffin, and Munehiro Asally
Transcription factors control gene expression in all life. This raises the question of what is the smallest protein that can support such activity. In nature, Cro from bacteriophage l is one of the smallest known repressors (66 amino acids), and activators are typically much larger (e.g., l cI, 237 amino acids). In this study, we show that directed evolution results in a new Cro activator-repressor that functions as efficiently as l cI in vivo. This is the smallest protein activator that enables polymerase recruitment, highlighting the capacity of transcription factors to evolve from very short peptide sequences.