6 Search Results

Integrases serve as powerful biotechnology tools that catalyze recombination at specific DNA sequences (att sites) and facilitate chromosomal integration of gene cargos transferred into cells. Given that genomes often lack the attB integration sites recognized by frequently utilized integrases, integrase technology has largely been restricted to genetic engineering of model organisms into which attB sites can be synthetically introduced. To enable single-step site-specific integrase-mediated genome editing in a broad spectrum of prokaryotes, we have devised the Integrase-On-Demand (IOD) method. IOD systematically identifies integrases, within bacteria and archaea, that can integrate into available attB sites in any target prokaryote. Computational resultsmore » show that diverse bacteria generally have multiple potentially useable native attB sites for novel integrases. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973, measuring efficiency of integration using nonreplicating vectors. By eliminating the requirement to introduce non-native attB sites into the target genome, IOD may, when suitable transformation methods exist, allow facile genome integration of large constructs in nonmodel and possibly nonculturable bacteria.« less

Cupriavidus necator H16 is a facultative chemolithotroph capable of using CO₂ as a carbon source, making it a promising organism for carbon-negative biomanufacturing of petroleum-based product alternatives. In contrast to model microbes, genetic engineering technologies are limited in C. necator, constraining its utility in basic and applied research. Here, we developed a genome engineering technology to efficiently mobilize, integrate, and express synthetic genetic elements (SGEs) in C. necator. We tested the chromosomal expression of four inducible promoters to optimize an engineered genetic landing pad for tunable gene expression. To demonstrate utility, we employed the SGE system to design, mobilize, andmore » express eight heterologous inorganic carbon uptake pathways in C. necator. We demonstrated all inorganic carbon uptake systems’ upregulated intracellular bicarbonate concentrations under heterotrophic conditions. This work establishes the utility of the SGE strategy for expedited integration and tunable expression of heterologous pathways, and enhances intracellular bicarbonate concentrations in C. necator.« less

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using amore » high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications.« less

Small molecules encoded by biosynthetic pathways mediate cross-species interactions and harbor untapped potential, which has provided valuable compounds for medicine and biotechnology. Since studying biosynthetic gene clusters in their native context is often difficult, alternative efforts rely on heterologous expression, which is limited by host-specific metabolic capacity and regulation. Here, in this work, we describe a computational-experimental technology to redesign genes and their regulatory regions with hybrid elements for cross-species expression in Gram-negative and -positive bacteria and eukaryotes, decoupling biosynthetic capacity from host-range constraints to activate silenced pathways. These synthetic genetic elements enabled the discovery of a class of microbiome-derivedmore » nucleotide metabolites—tyrocitabines—from Lactobacillus iners. Tyrocitabines feature a remarkable orthoester-phosphate, inhibit translational activity, and invoke unexpected biosynthetic machinery, including a class of “Amadori synthases” and “abortive” tRNA synthetases. Our approach establishes a general strategy for the redesign, expression, mobilization, and characterization of genetic elements in diverse organisms and communities.« less

Starting from our previous finding of 14 known drugs as inhibitors of the main protease (M^pro) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC₅₀ values in a kinetic assay. Free-energy perturbation (FEP) calculations for M^pro-ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to M^pro. Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment ofmore » COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization.« less

Organisms possessing genetic codes with unassigned codons raise the question of how cellular machinery resolves such codons and how this could impact horizontal gene transfer. Here, we use a genomically recoded Escherichia coli to examine how organisms address translation at unassigned UAG codons, which obstruct propagation of UAG-containing viruses and plasmids. Using mass spectrometry, we show that recoded organisms resolve translation at unassigned UAG codons via near-cognate suppression, dramatic frameshifting from at least −3 to +19 nucleotides, and rescue by ssrA-encoded tmRNA, ArfA, and ArfB. We then demonstrate that deleting tmRNA restores expression of UAG-ending proteins and propagation of UAG-containingmore » viruses and plasmids in the recoded strain, indicating that tmRNA rescue and nascent peptide degradation is the cause of impaired virus and plasmid propagation. The ubiquity of tmRNA homologs suggests that genomic recoding is a promising path for impairing horizontal gene transfer and conferring genetic isolation in diverse organisms.« less