Low Copy Number Plasmid Research Articles

Multiple Displacement Amplification as a Solution for Low Copy Number Plasmid Sequencing.

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probiotic Lactobacillus plantarum WCFS1.

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as potential protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against WCFS1 (pTRKH2) as the indicator strain, showing that functional expression of plantacyclin B21AG had been achieved. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 ± 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 ± 0.05 and 25.19 ± 2.68 copies respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 results in similar killing activity being observed in both the wild type and recombinant hosts despite the lower copy number of pCycB21.

Expression, purification and antimicrobial activity of recombinant pediocin PA-1 M31L, a PA-1 derivative with enhanced stability

Pediocin PA −1 is class IIa bacteriocin that displays efficient antimicrobial activity against pathogenic Listeriaspp. This bacteriocin is known to lose activity during long periods of storage especially at non optimal pH, thus reducing its usefulness for the pharmaceutical and food industries. Loss of activity has been attributed to oxidation of the methionine residue at position 31, however, replacement of this residue by leucine results in a peptide with activity equivalent to that of the native peptide. In this work, the heterologous expression of the structural (with Met31 to Leu substitution), accessory and transport genes from pediocin PA-1 operon was carried out in Escherichia coli TunerTM (DE3) cells. The sequences of all genes were redesigned using codon bias for the host and were cloned into an expression vector that allows control of plasmid copy number. The heterologous expression of pediocin Met31Leu was optimized for temperature, induction time, IPTG concentration and plasmid copy number and was evaluated via antimicrobial activity assays against Listeria innocua DPC3572. Maximum activity (2560 AU mL−1) was achieved using low plasmid copy number and 6 h of induction at 37 °C with 1 mM of IPTG. Recombinant pediocin PA-1M31L was successfully purified in 5 steps (>95 % purity) as confirmed by mass spectrometry (4606.27 Da) with a yield of 0.725 mg per liter of culture. This variant showed a similar spectrum of activity to the native pediocin PA-1 and is an interesting alternative for industrial applications due to its greater stability.

CopySwitch-in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. While there are numerous ways of doing so on protein or mRNA level, our approach aims for the underlying number of coding sequences. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host's intrinsic natural competence and homologous recombination capabilities. Strains are transformed with a linearized, low copy number plasmid containing an antibiotic resistance marker and homology regions up- and downstream of the gene of interest. Said gene is copied onto the vector, rendering it circular and replicative and thus selectable. We could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful transformation. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher. A major benefit of this method is the ability to work with very large regions of interest, since all relevant steps are carried out in vivo and are thus far less prone to mechanical DNA damage.

Improved Thermotolerance of Genome-Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL /cI857 System.

The higher intracellular ATP levels of genome-edited strains of P. putida that result from deleting various energy-consuming functions has been exploited for expanding the window of thermal tolerance of this bacterium. Unlike instant growth halt and eventual death of the naturally occurring strain P. putida KT2440 at 42 °C, the EM42 variant maintained growth and viability of most of the population at the higher temperature for at least 6 h. The authors took advantage of this quality for implementing a robust thermo-inducible heterologous expression device in this species. To this end, the cI857/PL pair of the lambda phage of Escherichia coli was reshaped as a functional cargo that followed the SEVA (Standard European Vector Architecture) format. Quantitation of the transcriptional output of the resulting expression device with GFP reporter technology in various gene dosages identified conditions of unprecedented induced/uninduced ratios (>300 folds) and very high total transcriptional capacity in this bacterial host. The broad-host range nature of the cognate replication origins makes expression vectors pSEVA2214 (low plasmid copy number), pSEVA2314 (medium), and pSEVA2514 (high) to cover a wide range of heterologous expression needs in P. putida and possibly other Gram-negative species.

Adaptive response of yeast cells to triggered toxicity of phosphoribulokinase

Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.

Copy number of ArsR reporter plasmid determines its arsenite response and metal specificity.

The key component in bacteria-based biosensors is a transcriptional reporter employed to monitor induction or repression of a reporter gene corresponding to environmental change. In this study, we made a series of reporters in order to achieve highly sensitive detection of arsenite. From these reporters, two biosensors were developed by transformation of Escherichia coli DH5α with pLHPars9 and pLLPars9, consisting of either a high or low copy number plasmid, along with common elements of ArsR-luciferase fusion and addition of two binding sequences, one each from E. coli and Acidithiobacillus ferrooxidans chromosome, in front of the R773 ArsR operon. Both of them were highly sensitive to arsenite, with a low detection limit of 0.04μM arsenite (~ 5μg/L). They showed a wide dynamic range of detection up to 50μM using high copy number pLHPars9 and 100μM using low copy number pLLPars9. Significantly, they differ in metal specificity, pLLPars9 more specific to arsenite, while pLHPars9 to both arsenite and antimonite. The only difference between pLHPars9 and pLLPars9 is their copy numbers of plasmid and corresponding ratios of ArsR to its binding promoter/operator sequence. Therefore, we propose a working model in which DNA bound-ArsR is different from its free form in metal specificity.

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4-Å resolution

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.

Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli.

The engineering of synthetic circuits in cells relies on the use of well-characterized biological parts that would perform predicted functions under the situation considered, and many efforts have been taken to set biological standards that could define the basic features of these parts. However, since most synthetic biology projects usually require a particular cellular chassis and set of growth conditions, defining standards in the field is not a simple task as gene expression measurements could be affected severely by genetic background and culture conditions. In this study, we addressed promoter parameterization in bacteria in different genetic backgrounds and growth conditions. We found that a small set of constitutive promoters of different strengths controlling a short-lived GFP reporter placed in a low-copy number plasmid produces remarkably reproducible results that allow for the calibration of promoter activity over different genetic backgrounds and physiological conditions, thus providing a simple way to set standards of promoter activity in bacteria. Based on these results, we proposed the utilization of synthetic constitutive promoters as tools for calibration for the standardization of biological parts, in a way similar to the use of DNA and protein ladders in molecular biology as references for comparison with samples of interest.

A Highly Promiscuous Integron, Plasmids, Extended Spectrum Beta Lactamases and Efflux Pumps as Factors Governing Multidrug Resistance in a Highly Drug Resistant Vibrio fluvialis Isolate BD146 from Kolkata, India.

In an earlier study from this laboratory, Vibrio fluvialis BD146, a clinical isolate from Kolkata, India, 2002, was found to be resistant to all the fourteen antibiotics tested. It harboured a high copy number plasmid pBD146 and a low copy number plasmid. In the present study, a more detailed analysis was carried out to unravel different resistance mechanisms in this isolate. Sequencing showed that variable region of class 1 integron located on low copy number plasmid harbored arr3-cmlA-blaOXA10-aadA1 gene cassettes. Analysis for extended spectrum beta lactamases (ESBLs) revealed that BD146 was ESBL positive. Efflux pumps were involved in the drug resistance phenotype for chloramphenicol, kanamycin, streptomycin and tetracycline. Sequence analysis of pBD146 revealed the presence of genes encoding BDint an integrase with a unique sequence having little similarity to other known integrases, toxin-antitoxin (parE/parD), a replicase, trimethoprim resistance (dfrVI) and quinolone resistance (qnrVC5). Presence of cmlA, putative novel integrase and toxin-antitoxin system in V. fluvialis has been documented for the first time in this report. pBD146 showed 99% sequence similarity with pVN84 from V. cholerae O1 of Vietnam, 2004 and a plasmid from V. parahaemolyticus v110 of Hong Kong, 2010. Conjugation experiments proved the ability of pBD146 and the low copy number plasmid, to get transferred to another host imparting their antibiotic resistance traits to the transconjugants. Therefore, present study has indicated that plasmids played an important role for dissemination of drug resistance.

The development and analyses of several Gram-negative arsenic biosensors using a synthetic biology approach

Microbial biosensors hold promise as a method to detect bioavailable heavy metals in the environment. However, applications are limited by use of bacterial species with little environmental relevance. In this study, six arsenic biosensors were developed and comparatively analysed. The genetic element consisted of an arsR transcriptional regulator and its cognate operator/promoter region, upstream of a promoterless gfp gene. This allowed for a quantifiable fluorescence output which is regulated by ArsR, in response to arsenic. The genetic element was cloned into a narrow host range, high-copy number plasmid and broad host range, low-copy number plasmid. Preliminary analyses were performed in Escherichia coli whereby microbial biosensor functionality was not effected by plasmid copy number or the stage of bacterial growth. Following, the broad-host range arsenic biosensor construct was transferred into Pseudomonas aeruginosa, Shewanella oneidensis and wild-type Enterobacter spp. Comparative analyses were used to determine the detection limit, induction time and specificity of the arsenic biosensors. The detection limits and induction times varied between bacterial species however, all arsenic biosensors were specific to arsenite. The variations between the microbial biosensors were hypothesized to be related to the genetic element of the biosensor construct, nutrient availability, native transcriptional machinery and presence of endogenous heavy metal resistance pathways

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation.

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.

Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells

Many plasmids used for gene cloning and heterologous protein expression in Escherichia coli cells are low copy number or single copy number plasmids. The extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study, we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB, and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. By contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.

Localization of Low Copy Number Plasmid pRC4 in Replicating Rod and Non-Replicating Cocci Cells of Rhodococcus erythropolis PR4.

Rhodococcus are gram-positive bacteria, which can exist in two different shapes rod and cocci. A number of studies have been done in the past on replication and stability of small plasmids in this bacterium; however, there are no reports on spatial localization and segregation of these plasmids. In the present study, a low copy number plasmid pDS3 containing pRC4 replicon was visualized in growing cells of Rhodococcus erythropolis PR4 (NBRC100887) using P1 parS-ParB-GFP system. Cells were initially cocci and then became rod shaped in exponential phase. Cocci cells were found to be non-replicating as evident by the presence of single fluorescence focus corresponding to the plasmid and diffuse fluorescence of DnaB-GFP. Rod shaped cells contained plasmid either present as one fluorescent focus observed at the cell center or two foci localized at quarter positions. The results suggest that the plasmid is replicated at the cell center and then it goes to quarter position. In order to observe the localization of plasmid with respect to nucleoid, plasmid segregation was also studied in filaments where it was found to be replicated at the cell center in a nucleoid free region. To the best of our knowledge, this is the first report on segregation of small plasmids in R. erythropolis.

Segrosome Complex Formation during DNA Trafficking in Bacterial Cell Division.

Bacterial extrachromosomal DNAs often contribute to virulence in pathogenic organisms or facilitate adaptation to particular environments. The transmission of genetic information from one generation to the next requires sufficient partitioning of DNA molecules to ensure that at least one copy reaches each side of the division plane and is inherited by the daughter cells. Segregation of the bacterial chromosome occurs during or after replication and probably involves a strategy in which several protein complexes participate to modify the folding pattern and distribution first of the origin domain and then of the rest of the chromosome. Low-copy number plasmids rely on specialized partitioning systems, which in some cases use a mechanism that show striking similarity to eukaryotic DNA segregation. Overall, there have been multiple systems implicated in the dynamic transport of DNA cargo to a new cellular position during the cell cycle but most seem to share a common initial DNA partitioning step, involving the formation of a nucleoprotein complex called the segrosome. The particular features and complex topologies of individual segrosomes depend on both the nature of the DNA binding protein involved and on the recognized centromeric DNA sequence, both of which vary across systems. The combination of in vivo and in vitro approaches, with structural biology has significantly furthered our understanding of the mechanisms underlying DNA trafficking in bacteria. Here, I discuss recent advances and the molecular details of the DNA segregation machinery, focusing on the formation of the segrosome complex.

Quantitative Localization Microscopy Reveals a Novel Organization of a High-Copy Number Plasmid

The maintenance of high-copy number plasmids within bacteria had been commonly thought to result from free diffusion and random segregation. Recent microscopy experiments, however, observed high-copy number plasmids clustering into discrete foci, which seemed to contradict this model, and hinted at an undiscovered active mechanism, as often found in low-copy number plasmids. We recently investigated the cellular organization of a ColE1-derivative plasmid in Escherichia coli bacteria using quantitative superresolved microscopy based on single-molecule localization in combination with single-molecule fluorescence in situ hybridization (smFISH). We observed that many of the plasmids aggregated into large clusters, although most of the plasmids were randomly distributed throughout the bacteria, minus an excluded volume about the chromosomal DNA. Our results indicate that neither of the previous models completely encompasses the behavior of high-copy number plasmids. We also found many plasmids within the chromosomal volume, providing further evidence that the nucleoid does not fully exclude DNA and RNA.

Salmonella enterica serovar Typhimurium utilizes the ClpPX and Lon proteases for optimal fitness in the ceca of chickens

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a leading cause of salmonellosis. Poultry and poultry products are implicated in transmission of Salmonella to humans. In 2013, an outbreak of S. Typhimurium occurred that comprised 39 states within the United States and was associated with backyard flocks of chickens. Colonization of the avian host by S. Typhimurium requires numerous genetic factors encoded within the bacterium. Of particular interest are genetic factors induced by alternative sigma factors within S. Typhimurium since these genetic elements are important for adaptation to different environmental stresses. The heat shock response is a dedicated change in gene regulation within bacteria in response to several stresses, specifically growth at 42°C. Because chickens have a higher body temperature than other animals (42°C) the hypothesis was tested that components of the heat shock response are important for optimal fitness within the chicken. To this end, deletion of the heat shock proteases clpPX (BTNC0022) or lon (BTNC0021) was accomplished and the bacterial fitness in vivo was compared to the “wild-type” strain (NC1040) using a competition assay. One-day-old chicks were orally gavaged with an equal mixture of NC1040 and either BTNC0022 or BTNC0021. Quantification of viable bacteria over time by using plate counts indicated that deletion of either heat shock protease resulted in significantly reduced colonization of the chicken ceca compared to the wild-type strain. To satisfy the molecular Koch's postulates, clpPX and lon mutants were complemented in trans using a low-copy number plasmid for additional in vivo experiments. Complementation studies confirmed the importance of either heat shock protease to colonization of the chicken ceca. This report demonstrated that both ClpPX and Lon were important for optimal fitness within chickens. Moreover, these results suggested that components of the heat shock may be critical factors used by S. Typhimurium for colonization of poultry. The use of feed additives or other treatments that inactivate or inhibit ClpPX or Lon may reduce the bacterial burden of S. Typhimurium in poultry.

Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening.

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%-92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (Mw) of 2.7-6.8 × 106 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8±0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation.