Plasmid Research Articles

Identification of plasmids from thermophilic Streptomyces strains and development of a gene cloning system for thermophilic Streptomyces species.

To develop a host-vector system for use in thermophilic Streptomyces, multi-copy plasmids were screened for thermophilic Streptomyces species using data from public bioresource centers (JCM and NBRC). Of 27 thermophilic Streptomyces strains, three harbored plasmids. One plasmid (pSTVU1), derived from S. thermovulgaris NBRC 16615 (= JCM 4520, ATCC 19284, DSM 40444, ISP 5444, NRRL B-12375, NCIMB 10078), was multi-copy and relatively small in size. Analysis of the sequence of this multi-copy plasmid revealed that it was 7 838bp and contained at least 10 predicted open reading frames (ORFs). The plasmid was introduced into 14 thermophilic Streptomyces strains (of 18 strains examined) and several mesophilic Streptomyces strains (S. lividans, S. parvulus, and S. avermitilis). pSTVU1 can be transferred by mixed culture because the plasmid encodes the ORF that regulates the transfer function. Plasmid transfer was observed not only between strains within the same species but also between mesophilic Streptomyces and thermophilic Streptomyces (and vice versa); however, the efficiency of this transfer was extremely low. We also confirmed that a derivative of pSTVU1 can be used as a multi-copy vector in the gene expression system that is expected to exhibit gene-dosage effects, establishing a method for efficient production of thermophilic α-amylase.

Improving the Z3EV promoter system to create the strongest yeast promoter.

Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast S. cerevisiae, a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been no attempts to construct the highest-strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z3EV, we were able to construct a promoter (P36) with approximately 1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by estradiol is maintained. When combined with a multicopy plasmid, it can express up to approximately 50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.

Stable Platform for Mevalonate Bioproduction from CO2.

Stable production of value-added products using a microbial chassis is pivotal for determining the industrial suitability of the engineered biocatalyst. Microbial cells often lose the multicopy expression plasmids during long-term cultivations. Owing to the advantages related to titers, yields, and productivities when using a multicopy expression system compared with genomic integrations, plasmid stability is essential for industrially relevant biobased processes. Cupriavidus necator H16, a facultative chemolithoautotrophic bacterium, has been successfully engineered to convert inorganic carbon obtained from CO2 fixation into value-added products. The application of this unique capability in the biotech industry has been hindered by C. necator H16 inability to stably maintain multicopy plasmids. In this study, we designed and tested plasmid addiction systems based on the complementation of essential genes. Among these, implementation of a plasmid addiction tool based on the complementation of mutants lacking RubisCO, which is essential for CO2 fixation, successfully stabilized a multicopy plasmid. Expressing the mevalonate pathway operon (MvaES) using this addiction system resulted in the production of ∼10 g/L mevalonate with carbon yields of ∼25%. The mevalonate titers and yields obtained here using CO2 are the highest achieved to date for the production of C6 compounds from C1 feedstocks.

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgos) and the DNA defense module DdmDE system. Through biochemical analysis, structural determination, and invivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

Multilevel metabolic engineering for enhanced synthesis of S-adenosylmethionine by Bacillus amyloliquefaciens.

To enhance the de novo synthesis of SAM, the effects of several key genes on SAM synthesis were examined based on modular strategy, and the key genes were manipulated to obtain an engineered strain with high SAM production. In Bacillus amyloliquefaciens HSAM6, the deletion of argG gene to block aspartic acid branching degradation increased SAM titer to 254.78 ± 15.91 mg/L, up 18% from HSAM6. Subsequently, deleting the moaA gene to boost the supply of 5-methyltetrahydrofolate led to the stunted growth and the plummeting yield of SAM. Further improvement of strain growth by overexpression of the citA gene, while SAM synthesis was not significantly enhanced. Finally, the maximum SAM titer (452.89 ± 13.42mg/L) was obtained by overexpression SAM2 gene using the multicopy plasmid. The deletion of argG gene and the overexpression of SAM2 gene significantly improved SAM synthesis in B. amyloliquefaciens.

DdmDE eliminates plasmid invasion by DNA-guided DNA targeting.

Horizontal gene transfer is a key driver of bacterial evolution, but it also presents severe risks to bacteria by introducing invasive mobile genetic elements. To counter these threats, bacteria have developed various defense systems, including prokaryotic Argonautes (pAgo) and the D NA D efense M odule DdmDE system. Through biochemical analysis, structural determination, and in vivo plasmid clearance assays, we elucidate the assembly and activation mechanisms of DdmDE, which eliminates small, multicopy plasmids. We demonstrate that DdmE, a pAgo-like protein, acts as a catalytically inactive, DNA-guided, DNA-targeting defense module. In the presence of guide DNA, DdmE targets plasmids and recruits a dimeric DdmD, which contains nuclease and helicase domains. Upon binding to DNA substrates, DdmD transitions from an autoinhibited dimer to an active monomer, which then translocates along and cleaves the plasmids. Together, our findings reveal the intricate mechanisms underlying DdmDE-mediated plasmid clearance, offering fundamental insights into bacterial defense systems against plasmid invasions.

The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

Plasmid targeting and destruction by the DdmDE bacterial defence system.

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation1. Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET)2. Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe thatthe helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to amonomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria.

Copper-based grape pest management has impacted wine aroma

Despite the high energetic cost of the reduction of sulfate to H2S, required for the synthesis of sulfur-containing amino acids, some wine Saccharomyces cerevisiae strains have been reported to produce excessive amounts of H2S during alcoholic fermentation, which is detrimental to wine quality. Surprisingly, in the presence of sulfite, used as a preservative, wine strains produce more H2S than wild (oak) or wine velum (flor) isolates during fermentation. Since copper resistance caused by the amplification of the sulfur rich protein Cup1p is a specific adaptation trait of wine strains, we analyzed the link between copper resistance mechanism, sulfur metabolism and H2S production. We show that a higher content of copper in the must increases the production of H2S, and that SO2 increases the resistance to copper. Using a set of 51 strains we observed a positive and then negative relation between the number of copies of CUP1 and H2S production during fermentation. This complex pattern could be mimicked using a multicopy plasmid carrying CUP1, confirming the relation between copper resistance and H2S production. The massive use of copper for vine sanitary management has led to the selection of resistant strains at the cost of a metabolic tradeoff: the overproduction of H2S, resulting in a decrease in wine quality.

Plasmid-mediated phenotypic noise leads to transient antibiotic resistance in bacteria

The rise of antibiotic resistance is a critical public health concern, requiring an understanding of mechanisms that enable bacteria to tolerate antimicrobial agents. Bacteria use diverse strategies, including the amplification of drug-resistance genes. In this paper, we showed that multicopy plasmids, often carrying antibiotic resistance genes in clinical bacteria, can rapidly amplify genes, leading to plasmid-mediated phenotypic noise and transient antibiotic resistance. By combining stochastic simulations of a computational model with high-throughput single-cell measurements of blaTEM-1 expression in Escherichia coli MG1655, we showed that plasmid copy number variability stably maintains populations composed of cells with both low and high plasmid copy numbers. This diversity in plasmid copy number enhances the probability of bacterial survival in the presence of antibiotics, while also rapidly reducing the burden of carrying multiple plasmids in drug-free environments. Our results further support the tenet that multicopy plasmids not only act as vehicles for the horizontal transfer of genetic information between cells but also as drivers of bacterial adaptation, enabling rapid modulation of gene copy numbers. Understanding the role of multicopy plasmids in antibiotic resistance is critical, and our study provides insights into how bacteria can transiently survive lethal concentrations of antibiotics.

A dual fluorescence protein expression system detects cell cycle dependent protein noise

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, ‘noisy’, gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

Pyoverdine-antibiotic combination treatment: its efficacy and effects on resistance evolution in Escherichia coli.

Antibiotic resistance is a growing concern for global health, demanding innovative and effective strategies to combat pathogenic bacteria. Pyoverdines, iron-chelating siderophores produced by environmental Pseudomonas spp., present a novel class of promising compounds to induce growth arrest in pathogens through iron starvation. While we previously demonstrated the efficacy of pyoverdines as antibacterials, our understanding of how these molecules interact with antibiotics and impact resistance evolution remains unknown. Here, we investigated the propensity of three Escherichia coli strains to evolve resistance against pyoverdine, the cephalosporin antibiotic ceftazidime, and their combination. We used a naive E. coli wildtype strain and two isogenic variants carrying the bla TEM-1 β-lactamase gene on either the chromosome or a costly multicopy plasmid to explore the influence of genetic background on selection for resistance. We found that strong resistance against ceftazidime and weak resistance against pyoverdine evolved in all E. coli variants under single treatment. Ceftazidime resistance was linked to mutations in outer membrane porin genes (envZ and ompF), whereas pyoverdine resistance was associated with mutations in the oligopeptide permease (opp) operon. In contrast, ceftazidime resistance phenotypes were attenuated under combination treatment, especially for the E. coli variant carrying bla TEM-1 on the multicopy plasmid. Altogether, our results show that ceftazidime and pyoverdine interact neutrally and that pyoverdine as an antibacterial is particularly potent against plasmid-carrying E. coli strains, presumably because iron starvation compromises both cellular metabolism and plasmid replication.

A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris

In recent years, nanobodies have played an increasingly crucial role in virus neutralization, ELISA detection, and medical imaging. This study aimed to explore a universal expression strategy in Pichia pastoris using three nanobodies, denoted Va, Vb, and Vc, as model proteins. Initially, plasmids pLD-AOXα and pLD-AOX were engineered to minimize the risk of antibiotic resistance gene drift. Optimization of promoters and signal peptides resulted in a 1.38-fold and 1.89-fold increase in Va production. Further optimization of gene dosage led to an additional 1.39-fold enhancement in Va yield. Subsequently, 25 molecular chaperones were co-expressed with Va under the control of the wild-type AOX1 promoter, with HAC1 further increasing Va yield by 1.5-fold. By fine-tuning the promoter strength for HAC1, Va production was increased by 2.41-fold under the control of the 55p promoter. Finally, through high-density fermentation, the Va yield reached 2.13 g/L, representing a 49.8-fold increase compared to the initial strain 1-AOXα-Va in shake-flask culture. Integration of pLD-55p-HAC1 into the GS115 genome resulted in the H55 host, and the transformation of multicopy plasmids into this host led to a 1.98-fold increase in Vb yield and a 2.34-fold increase in Vc yield, respectively. The engineering of antibiotic-free parental plasmids, modification of expression components, gene dosage optimization, and the H55 host are regarded as a composite strategy which will pave the way for efficient expression of nanobodies in the future.

Sensing chemical-induced genotoxicity and oxidative stress via yeast-based reporter assays using NanoLuc luciferase.

Mutagens and oxidative agents damage biomolecules, such as DNA; therefore, detecting genotoxic and oxidative chemicals is crucial for maintaining human health. To address this, we have developed several types of yeast-based reporter assays designed to detect DNA damage and oxidative stress. This study aimed to develop a novel yeast-based assay using a codon-optimized stable or unstable NanoLuc luciferase (yNluc and yNluCP) gene linked to a DNA damage- or oxidative stress-responsive promoter, enabling convenient sensing genotoxicity or oxidative stress, respectively. End-point luciferase assays using yeasts with a chromosomally integrated RNR3 promoter (PRNR3)-driven yNluc gene exhibited high levels of chemiluminescence via NanoLuc luciferase and higher fold induction by hydroxyurea than a multi-copy plasmid-based assay. Additionally, the integrated reporter system detected genotoxicity caused by four different types of chemicals. Oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and menadione) were successfully detected through transient expressions of luciferase activity in real-time luciferase assay using yeasts with a chromosomally integrated TRX2 promoter (PTRX2)-linked yNlucCP gene. However, the luciferase activity was gradually induced in yeasts with a multi-copy reporter plasmid, and their expression profiles were notably distinct from those observed in chromosomally integrated yeasts. The responses of yNlucCP gene against three oxidative chemicals, but not diamide and zinc oxide suspension, were observed using chromosomally integrated reporter yeasts. Given that yeast cells with chromosomally integrated PRNR3-linked yNluc and PTRX2-linked yNlucCP genes express strong chemiluminescence signals and are easily maintained and handled without restrictive nutrient medium, these yeast strains with NanoLuc reporters may prove useful for screening potential genotoxic and oxidative chemicals.

Massive integration of large gene libraries in the chromosome of Escherichia coli.

Large gene libraries are frequently created in Escherichia coli plasmids, which can induce cell toxicity and expression instability due to the high gene dosage. To address these limitations, gene libraries can be integrated in a single copy into the bacterial chromosome. Here, we describe an efficient system for the massive integration (MAIN) of large gene libraries in the E. coli chromosome that generates in-frame gene fusions that are expressed stably. MAIN uses a thermosensitive integrative plasmid that is linearized invivo to promote extensive integration of the gene library via homologous recombination. Positive and negative selections efficiently remove bacteria lacking gene integration in the target site. We tested MAIN with a library of 107 VHH genes that encode nanobodies (Nbs). The integration of VHH genes into a custom target locus of the E. coli chromosome enabled stable expression and surface display of the Nbs. Next-generation DNA sequencing confirmed that MAIN preserved the diversity of the gene library after integration. Finally, we screened the integrated library to select Nbs that bind a specific antigen using magnetic and fluorescence-activated cell sorting. This allowed us to identify Nbs binding the epidermal growth factor receptor that were not previously isolated in a similar screening of a multicopy plasmid library. Our results demonstrate that MAIN enables large gene library integration into the E. coli chromosome, creating stably expressed in-frame fusions for functional screening.

Single-Copy Gene Editing of a Cell Wall-Anchored Pilin in Actinomyces oris.

The Gram-positive bacterium Actinomyces oris expresses a unique cell wall-anchored fimbria comprised of the fimbrial shaft FimA and the tip fimbrillin CafA, whose gene is not genetically linked to the fimA locus, unlike many other fimbrial gene loci in Gram-positive bacteria. Mutational analyses of individual fimbrillins, FimA and CafA, in A. oris often rely on multi-copy plasmids that may alter the stoichiometry of fimbrillins in vivo, hence fimbrial assembly. Here, we provide a robust method for single-copy gene expression and mutagenesis in A. oris, using CafA as an experimental model. This method can be applied for single-copy gene editing in various bacterial systems.

MetaCC allows scalable and integrative analyses of both long-read and short-read metagenomic Hi-C data

Metagenomic Hi-C (metaHi-C) can identify contig-to-contig relationships with respect to their proximity within the same physical cell. Shotgun libraries in metaHi-C experiments can be constructed by next-generation sequencing (short-read metaHi-C) or more recent third-generation sequencing (long-read metaHi-C). However, all existing metaHi-C analysis methods are developed and benchmarked on short-read metaHi-C datasets and there exists much room for improvement in terms of more scalable and stable analyses, especially for long-read metaHi-C data. Here we report MetaCC, an efficient and integrative framework for analyzing both short-read and long-read metaHi-C datasets. MetaCC outperforms existing methods on normalization and binning. In particular, the MetaCC normalization module, named NormCC, is more than 3000 times faster than the current state-of-the-art method HiCzin on a complex wastewater dataset. When applied to one sheep gut long-read metaHi-C dataset, MetaCC binning module can retrieve 709 high-quality genomes with the largest species diversity using one single sample, including an expansion of five uncultured members from the order Erysipelotrichales, and is the only binner that can recover the genome of one important species Bacteroides vulgatus. Further plasmid analyses reveal that MetaCC binning is able to capture multi-copy plasmids.

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Hijacking a small plasmid to confer high-level resistance to aztreonam–avibactam and ceftazidime–avibactam

Acquired β-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all β-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of β-lactamase gene blaCMY-2, encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of blaCMY-2R were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of blaCMY-2R on the IncP1 plasmid, but possessed anther blaCMY-2R on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of blaCMY-2R significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.

High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination.

Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development. Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication. Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication. This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.