Synthetic genomes unveil the effects of synonymous recoding.

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers. Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement-recoding-is frequently lethal, and how recoding impacts fitness remains poorly explored.

Strategies to identify and edit improvements in synthetic genome segments episomally.

Genome engineering projects often utilize bacterial artificial chromosomes (BACs) to carry multi-kilobase DNA segments at low copy number. However, all stages of whole-genome engineering have the potential to impose mutations on the synthetic genome that can reduce or eliminate the fitness of the final strain. Here, we describe improvements to a multiplex automated genome engineering (MAGE) protocol to improve recombineering frequency and multiplexability.

A swapped genetic code prevents viral infections and gene transfer.

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer. However, numerous viruses and mobile genetic elements encode parts of the translational apparatus, potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes.

Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE).

inPOSE: A Flexible Toolbox for Chromosomal Cloning and Amplification of Bacterial Transgenes.

Cloning the genes and operons encoding heterologous functions in bacterial hosts is now almost exclusively carried out using plasmid vectors. This has multiple drawbacks, including the need for constant selection and variation in copy numbers. The chromosomal integration of transgenes has always offered a viable alternative; however, to date, it has been of limited use due to its tedious nature and often being limited to a single copy.

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces.

ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E.

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal.

Recombineering and MAGE.

Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes. Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE). MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies.

Hybrid Inhibitors of DNA Gyrase A and B: Design, Synthesis and Evaluation.

The discovery of multi-targeting ligands of bacterial enzymes is an important strategy to combat rapidly spreading antimicrobial resistance. Bacterial DNA gyrase and topoisomerase IV are validated targets for the development of antibiotics. They can be inhibited at their catalytic sites or at their ATP binding sites.

Rational design of balanced dual-targeting antibiotics with limited resistance.

Antibiotics that inhibit multiple bacterial targets offer a promising therapeutic strategy against resistance evolution, but developing such antibiotics is challenging. Here we demonstrate that a rational design of balanced multitargeting antibiotics is feasible by using a medicinal chemistry workflow. The resultant lead compounds, ULD1 and ULD2, belonging to a novel chemical class, almost equipotently inhibit bacterial DNA gyrase and topoisomerase IV complexes and interact with multiple evolutionary conserved amino acids in the ATP-binding pockets of their target proteins.

Targeted mutagenesis of multiple chromosomal regions in microbes.

Directed evolution allows the effective engineering of proteins, biosynthetic pathways, and cellular functions. Traditional plasmid-based methods generally subject one or occasionally multiple genes-of-interest to mutagenesis, require time-consuming manual interventions, and the genes that are subjected to mutagenesis are outside of their native genomic context. Other methods mutagenize the whole genome unselectively which may distort the outcome.

Improved bacterial recombineering by parallelized protein discovery.

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, multiplex automated genome engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however, currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, efficient SSAPs must first be found.

Second-generation 4,5,6,7-tetrahydrobenzo[]thiazoles as novel DNA gyrase inhibitors.

DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel antibacterial drugs. Building from our first generation of 4,5,6,7-tetrahydrobenzo[]thiazole-based DNA gyrase inhibitors, we designed and prepared an optimized series of analogs that show improved inhibition of DNA gyrase and topoisomerase IV from and , with IC values in the nanomolar range. Importantly, these inhibitors also show improved antibacterial activity against Gram-positive strains.

Multiple-Site Diversification of Regulatory Sequences Enables Interspecies Operability of Genetic Devices.

The features of the light-responsive cyanobacterial CcaSR regulatory module that determine interoperability of this optogenetic device between and have been examined. For this, all structural parts (, and genes for synthesis of phycocyanobilin, the / system from , and its cognate downstream promoter) were maintained but their expression levels and stoichiometry diversified by (i) reassembling them together in a single broad host range, standardized vector and (ii) subjecting the noncoding regulatory sequences to multiple cycles of directed evolution with random genomic mutations (DIvERGE), a recombineering method that intensifies mutation rates within discrete DNA segments. Once passed to , various clones displayed a wide dynamic range, insignificant leakiness, and excellent capacity in response to green light.

Dual Escherichia coli DNA Gyrase A and B Inhibitors with Antibacterial Activity.

The emergence of multidrug-resistant bacteria is a global health threat necessitating the discovery of new antibacterials and novel strategies for fighting bacterial infections. We report first-in-class DNA gyrase B (GyrB) inhibitor/ciprofloxacin hybrids that display antibacterial activity against Escherichia coli. Whereas DNA gyrase ATPase inhibition experiments, DNA gyrase supercoiling assays, and in vitro antibacterial assays suggest binding of the hybrids to the E.

Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene.

The mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown.

Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations.

Multitargeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets, are generally considered to be a promising therapeutic strategy against resistance evolution.

Limited Evolutionary Conservation of the Phenotypic Effects of Antibiotic Resistance Mutations.

Multidrug-resistant clinical isolates are common in certain pathogens, but rare in others. This pattern may be due to the fact that mutations shaping resistance have species-specific effects. To investigate this issue, we transferred a range of resistance-conferring mutations and a full resistance gene into Escherichia coli and closely related bacteria.

CRISPR-interference-based modulation of mobile genetic elements in bacteria.

Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of engineered functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes.

Enzyme promiscuity shapes adaptation to novel growth substrates.

Evidence suggests that novel enzyme functions evolved from low-level promiscuous activities in ancestral enzymes. Yet, the evolutionary dynamics and physiological mechanisms of how such side activities contribute to systems-level adaptations are not well characterized. Furthermore, it remains untested whether knowledge of an organism's promiscuous reaction set, or underground metabolism, can aid in forecasting the genetic basis of metabolic adaptations.

An optimised series of substituted N-phenylpyrrolamides as DNA gyrase B inhibitors.

ATP competitive inhibitors of DNA gyrase and topoisomerase IV have great therapeutic potential, but none of the described synthetic compounds has so far reached the market. To optimise the activities and physicochemical properties of our previously reported N-phenylpyrrolamide inhibitors, we have synthesized an improved, chemically variegated selection of compounds and evaluated them against DNA gyrase and topoisomerase IV enzymes, and against selected Gram-positive and Gram-negative bacteria. The most potent compound displayed IC values of 6.

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts.

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof.