Genom-evolúció és-stabilitás vizsgálata az Esherichia coli genom redukciójával = Studies on genome evolution and stability by engineering a reduced Escherichia coli genome

A project célja egy minimál genomú Escherichia coli sejt létrehozása a genom tervszerű redukciójával. A laboratóriumi környezetben nélkülözhető gének eliminálásával egyszerűsített sejt "javított" modellszervezetként és biotechnológiai eszközként szolgálhat. Precíz deléciókkal 18,8%-kal csökkentettük a K-12 törzs genomját. Összehasonlító genomikai elemzés alapján kiejtettük az összes mobil genetikai elemet, számos virulenciagént, ismeretlen funkciójú és nem-esszenciális gént. A genomméret 4 639 675 bp-ról 3 768 583 bp-ra csökkent. A deléciókon túl egyes metabolikus defekteket (pirimidin és izoleucin bioszintézis) korrigáltunk. A 62 deléciós törzs (MDS62) növekedési rátája eléri a szülői törzsét. Az MDS törzsek hatékonyan transzformálhatók, és magas plazmid és rekombináns fehérje produkcióra képesek. A mobilis genetikai elemek eliminációja megnövekedett genomi stabilitást eredményezett. A deléciós (MDS) sejtek mutációs rátája -különösen stresszkörülmények között - alacsonyabb a szülői törzsénél. A mobilis elemek stresszhatásra bekövetkező indukciója egyfajta - a sejt integritását megőrző - védekezési mechanizmusra utal. | We have initiated the rational reduction of the Escherichia coli genome in order to obtain a minimal E. coli cell. Our primary goal is to construct an "improved" model organism and biotechnological tool by the rational elimination of genes unnecessary under laboratory conditions. The K-12 genome was reduced by precise scarless deletions. The new strains, with genomes up to 18.8% smaller, were designed by bioinformatic comparative genomics of four E. coli strains to identify non-essential genes and recombinogenic, mobile or cryptic virulence sequences, as well as genes with unknown functions for elimination. Besides genome reduction from 4,639,675 to 3,768,583 bp, certain metabolic deficiencies of the parent strain, e.g. pyrimidine or isoleucine biosynthesis were also corrected. The multiple deletion strain harboring 62 deletions (MDS62) displays no decrease in growth-rate. Additional useful features of the strains include efficient transformability and high yield of plasmid and protein production. Removal of all mobile genetic elements resulted in an increased genomic and plasmid stability. MDS cells display a decreased mutation rate, especially under stressful conditions. Stress-induced mobilisation of IS elements can serve as a defense mechanism protecting the integrity of the cell

Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications

<p>Abstract</p> <p>Background</p> <p>Evolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome <it>E. coli </it>MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential.</p> <p>Results</p> <p>We analyze an extreme case of toxic plasmid clone instability, and show that random host IS element hopping, causing inactivation of the toxic cloned sequences, followed by automatic selection of the fast-growing mutants, can prevent the maintenance of a clone developed for vaccine production. Analyzing the molecular details, we identify a hydrophobic protein as the toxic byproduct of the clone, and show that IS elements spontaneously landing in the cloned fragment relieve the cell from the stress by blocking transcription of the toxic gene. Bioinformatics analysis of sequence reads from early shotgun genome sequencing projects, where clone libraries were constructed and maintained in <it>E. coli</it>, suggests that such IS-mediated inactivation of ectopic genes inhibiting the growth of the <it>E. coli </it>cloning host might happen more frequently than generally anticipated, leading to genomic instability and selection of altered clones.</p> <p>Conclusions</p> <p>Delayed genetic adaptation of clean-genome, IS-free MDS42 host improves maintenance of unstable genetic constructs, and is suggested to be beneficial in both laboratory and industrial settings.</p

Low-mutation-rate, reduced-genome Escherichia coli: an improved host for faithful maintenance of engineered genetic constructs

<p>Abstract</p> <p>Background</p> <p>Molecular mechanisms generating genetic variation provide the basis for evolution and long-term survival of a population in a changing environment. In stable, laboratory conditions, the variation-generating mechanisms are dispensable, as there is limited need for the cell to adapt to adverse conditions. In fact, newly emerging, evolved features might be undesirable when working on highly refined, precise molecular and synthetic biological tasks.</p> <p>Results</p> <p>By constructing low-mutation-rate variants, we reduced the evolutionary capacity of MDS42, a reduced-genome <it>E. coli </it>strain engineered to lack most genes irrelevant for laboratory/industrial applications. Elimination of diversity-generating, error-prone DNA polymerase enzymes involved in induced mutagenesis achieved a significant stabilization of the genome. The resulting strain, while retaining normal growth, showed a significant decrease in overall mutation rates, most notably under various stress conditions. Moreover, the error-prone polymerase-free host allowed relatively stable maintenance of a toxic methyltransferase-expressing clone. In contrast, the parental strain produced mutant clones, unable to produce functional methyltransferase, which quickly overgrew the culture to a high ratio (50% of clones in a 24-h induction period lacked functional methyltransferase activity). The surprisingly large stability-difference observed between the strains was due to the combined effects of high stress-induced mutagenesis in the parental strain, growth inhibition by expression of the toxic protein, and selection/outgrowth of mutants no longer producing an active, toxic enzyme.</p> <p>Conclusions</p> <p>By eliminating stress-inducible error-prone DNA-polymerases, the genome of the mobile genetic element-free <it>E. coli </it>strain MDS42 was further stabilized. The resulting strain represents an improved host in various synthetic and molecular biological applications, allowing more stable production of growth-inhibiting biomolecules.</p

Conditional DNA repair mutants enable highly precise genome engineering

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement

Exploring the fitness benefits of genome reduction in Escherichia coli by a selection-driven approach

Artificial simplification of bacterial genomes is thought to have the potential to yield cells with reduced complexity, enhanced genetic stability, and improved cellular economy. Of these goals, economical gains, supposedly due to the elimination of superfluous genetic material, and manifested in elevated growth parameters in selected niches, have not yet been convincingly achieved. This failure might stem from limitations of the targeted genome reduction approach that assumes full knowledge of gene functions and interactions, and allows only a limited number of reduction trajectories to interrogate. To explore the potential fitness benefits of genome reduction, we generated successive random deletions in E. coli by a novel, selection-driven, iterative streamlining process. The approach allows the exploration of multiple streamlining trajectories, and growth periods inherent in the procedure ensure selection of the fittest variants of the population. By generating single- and multiple-deletion strains and reconstructing the deletions in the parental genetic background, we showed that favourable deletions can be obtained and accumulated by the procedure. The most reduced multiple-deletion strain, obtained in five deletion cycles (2.5% genome reduction), outcompeted the wild-type, and showed elevated biomass yield. The spectrum of advantageous deletions, however, affecting only a few genomic regions, appears to be limited

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias

Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid

Bacterial evolution of antibiotic hypersensitivity.

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large-scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole-genome sequencing of laboratory-evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton-motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF-dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade-offs under antibiotic selection