Use Of Plasmids Research Articles

Sequencing Strategy to Ensure Accurate Plasmid Assembly.

Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.

Development of a small shuttle plasmid for use in oral Veillonella and initial appraisal of potential for fluorescence-based applications.

Oral Veillonella species are among the early colonizers of the human oral cavity. We constructed a small, single-selectable-marker shuttle plasmid, examined its ability to be transformed into diverse oral Veillonella strains, and assessed its potential use for expressing a gene encoding an oxygen-independent fluorescent protein, thus generating a fluorescent Veillonella parvula strain. Because tetracycline resistance is common in Veillonella, we replaced genes encoding ampicillin- and tetracycline-resistance in a previously described shuttle plasmid (pBSJL2) with a chloramphenicol acetyltransferase gene. The resulting plasmid pCF1135 was successfully introduced into four strains representing V. parvula and V. atypica by either natural transformation or electroporation. We then modified this plasmid to express a gene encoding an oxygen-independent fluorescent protein in V. parvula SKV38. The resulting strain yielded a fluorescence signal intensity ∼16 times higher than the wild type in microplate-based fluorimetry experiments. While fluorescence microscopy demonstrated that planktonic cells, colonies, and biofilms of fluorescent V. parvula could also be imaged, photobleaching was a significant issue. In conclusion, we anticipate this genetic system and information provided here will facilitate expanded studies of oral Veillonella species' properties and behavior.

Sequencing Strategy to Ensure Accurate Plasmid Assembly.

Despite the wide use of plasmids in research and clinical production, the need to verify plasmid sequences is a bottleneck that is too often underestimated in the manufacturing process. Although sequencing platforms continue to improve, the method and assembly pipeline chosen still influence the final plasmid assembly sequence. Furthermore, few dedicated tools exist for plasmid assembly, especially for de novo assembly. Here, we evaluated short-read, long-read, and hybrid (both short and long reads) de novo assembly pipelines across three replicates of a 24-plasmid library. Consistent with previous characterizations of each sequencing technology, short-read assemblies had issues resolving GC-rich regions, and long-read assemblies commonly had small insertions and deletions, especially in repetitive regions. The hybrid approach facilitated the most accurate, consistent assembly generation and identified mutations relative to the reference sequence. Although Sanger sequencing can be used to verify specific regions, some GC-rich and repetitive regions were difficult to resolve using any method, suggesting that easily sequenced genetic parts should be prioritized in the design of new genetic constructs.

Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange

Historically, mutagenesis in the non-model enteropathogenic bacterium Clostridioides difficile has been challenging. Developing a versatile and reliable method of generating targeted mutations in C. difficile is important to further our understanding of its pathogenesis. Some of the most common targeted mutagenesis systems rely on allelic exchange mediated by either uracil auxotrophy combined with a toxic uracil precursor, a toxin/anti-toxin system, group II introns, or CRISPR/Cas mutagenesis. However, each of these methods suffers from its own issues. Here, we develop and test an allelic exchange strategy which better facilitates screening for integration and selecting for excision than previous systems. This is achieved by controlling plasmid replication with a theophylline-dependent riboswitch cloned upstream of repA, the gene whose product controls plasmid replication. This allows efficient mutant generation, can be performed in a wild-type strain of C. difficile, does not have the off-target effects inherent to group II introns, and alleviates the problem of testing multiple gRNA targets in CRISPR mutagenesis.

Transgene-free genome editing of plants

The presence of foreign DNA is the main obstacle to the application of biotechnological plant varieties. However, transgene-free technologies in the field of genome editing make it possible to overcome this problem. In most countries that already have legislation in this area, plants without foreign DNA do not require field trials and safety tests.
 The easiest way to avoid integration of transgenes is the delivery of RNP complexes directly into the cell without the use of plasmids. However subsequent selection of edited cells in the absence of a selective marker and plant regeneration are quite difficult. Therefore, traditional genetic constructs are used more often, despite that the elements of the CRISPR system are integrated into the genome. Backcrossing and cross-pollination are used to get rid of unwanted inserts. There are opportunities to accelerate the selection process, such as the Transgene Killer CRISPR system, which ensures the death of plants carrying transgenes in the early embryonic stages [1].
 Constructs based on viral replicons integrated into T-DNA are an alternative option. They provide a high level of transient expression of CRISPR elements which are not integrated into the genome. Such vectors were created on the basis of geminiviruses, rhabdoviruses, potexviruses, potyviruses, bunyaviruses [2]. The ability of viruses to move between cells can be both preserved and lost due to the removal of the corresponding proteins.
 One of the newest approaches is grafting of shoots onto roots expressing Cas and guide RNA [3]. The addition of tRNA-like motifs to the transcripts ensured their mobility and dispersal along the shoot. Heritable edits were observed in the progeny of grafted plants.
 Thus, for transgene-free editing technologies of plant genomes are rapidly developing, which will accelerate the commercialization of new varieties with economically valuable traits.

Gene-Activated Materials in Regenerative Dentistry: Narrative Review of Technology and Study Results.

Treatment of a wide variety of defects in the oral and maxillofacial regions requires the use of innovative approaches to achieve best outcomes. One of the promising directions is the use of gene-activated materials (GAMs) that represent a combination of tissue engineering and gene therapy. This approach implies that biocompatible materials will be enriched with gene-carrying vectors and implanted into the defect site resulting in transfection of the recipient's cells and secretion of encoded therapeutic protein in situ. GAMs may be presented in various designs depending on the type of material, encoded protein, vector, and way of connecting the vector and the material. Thus, it is possible to choose the most suitable GAM design for the treatment of a particular pathology. The use of plasmids for delivery of therapeutic genes is of particular interest. In the present review, we aimed to delineate the principle of work and various designs of plasmid-based GAMs and to highlight results of experimental and clinical studies devoted to the treatment of periodontitis, jaw bone defects, teeth avulsion, and other pathologies in the oral and maxillofacial regions.

Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system

BackgroundAromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes.ResultsTo facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L.ConclusionsWe developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.

Super-Resolution Spinning-Disk Confocal Microscopy Using Optical Photon Reassignment (SoRa) to Visualize the Actin Cytoskeleton in Macrophages.

Macrophages are motile, morphologically plastic cells that undergo substantial cytoskeletal remodeling to facilitate their roles in innate and adaptive immunity. Macrophages are adept at producing a variety of specialized actin-driven structures and processes including the formation of podosomes and the ability to engulf particles through phagocytosis and sample large amounts of extracellular fluid via micropinocytosis. Here, we describe techniques for immunostaining proteins and transfecting macrophages with plasmids for use with either fixed or live cell imaging. Furthermore, we discuss the use of spinning-disk super-resolution using optical reassignment to generate sub-diffraction limited structures using this type of confocal microscope.

Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile Virus Mosquito Vector, Culex quinquefasciatus.

Simple SummaryCulex mosquitoes are responsible for many established and emerging mosquito-borne diseases worldwide. These mosquitoes serve as the principal global transmission vector of West Nile virus, which is the leading cause of mosquito-borne disease in humans in the United States. Unfortunately, effective therapeutic drugs and vaccines are still lacking for most mosquito-borne diseases, and current vector controls (e.g., insecticide use) have fallen short of eradicating the disease burden. These concerns highlight the importance of developing novel strategies to prevent arbovirus transmission by Culex mosquitoes, especially as the prevalence of Culex-borne diseases increases globally. Underlying such approaches is often an improved understanding of virus–mosquito interactions. To investigate the putative antiviral role of Culex immune genes, we developed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Culex quinquefasciatus-derived (Hsu) cell cultures. We showed that this newly constructed plasmid allows for efficient and reliable codelivery of all CRISPR reagents in vitro in a single plasmid system. These findings suggest that this tool may serve as a valuable resource for the establishment of mutant Hsu-derived cell populations, enabling the identification of mosquito host genes involved in antiviral response. Elucidating gene functions supports the development of alternative gene-based vector control strategies for Culex mosquitoes.Culex quinquefasciatus mosquitoes are a globally widespread vector of multiple human and animal pathogens, including West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. Since the introduction of West Nile virus to the United States in 1999, a cumulative 52,532 cases have been reported to the CDC, including 25,849 (49.2%) neuroinvasive cases and 2456 (5%) deaths. Viral infections elicit immune responses in their mosquito vectors, including the RNA interference (RNAi) pathway considered to be the cornerstone antiviral response in insects. To investigate mosquito host genes involved in pathogen interactions, CRISPR/Cas9-mediated gene-editing can be used for functional studies of mosquito-derived cell lines. Yet, the tools available for the study of Cx. quinquefasciatus-derived (Hsu) cell lines remain largely underdeveloped compared to other mosquito species. In this study, we constructed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Hsu cell cultures. By comparing it to the original Drosophila melanogaster CRISPR/Cas9 plasmid, we showed that the Culex-optimized plasmid demonstrated highly efficient editing of the genomic loci of the RNAi proteins Dicer-2 and PIWI4 in Hsu cells. These new tools support our ability to investigate gene targets involved in mosquito antiviral response, and thus the future development of gene-based vector control strategies.

Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs

The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.

Regulatory & supply chain implications for plasmids as critical starting materials in the manufacture of viral vector gene therapy products

Plasmid DNA is actively being used as starting material/intermediates, drug substance, and/or drug product in the manufacture of several therapeutics including DNA vaccines, viral-vector and non-viral-vector gene therapies, and mRNA vaccines. Since plasmids have a large range of uses, there have been various plasmid guidelines released from the different agencies. Specifically for gene therapy, recent guidelines have tried to provide clearer guidance, however there is still ambiguity around quality requirements. For example, the specifications for the release of plasmids from a plasmid manufacturer and the facility requirements for accepting these plasmids for use in viral vector manufacturing are not always aligned and can cause disconnect in the supply chain. For a sponsor managing plasmid manufacturing for use as starting material in viral vector manufacturing, it is important to be aware of these differences in requirements ahead of time. The current manuscript highlights these regulatory differences and calls out the importance for a sponsor to build a robust supply chain between the plasmid manufacturer and the viral vector manufacturer well in advance to minimize bottlenecks due to quality requirements and timelines.

Tools of the trade: plasmid repositories and standardized plasmid manipulation for molecular and synthetic biology.

SummaryPlasmids are extrachromosomal genetic elements capable of autonomous replication within a host cell. They play a key role in bacterial ecology and evolution, facilitating the mobilization of accessory genes by horizontal gene transfer. Crucially, plasmids also serve as valuable tools in modern molecular biology. Here, we highlight recent articles aimed at implementing standardized plasmid assembly techniques and plasmid repositories to promote open science as well as to improve experimental reproducibility across laboratories. Research focused on assisting these fundamental aims is a further step towards improving standardization in molecular and synthetic biology.

Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants.

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples.

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) are a heterogeneous group of disorders causing progressive loss of vision, affecting approximately one in 1000 people worldwide. Gene augmentation therapy, which typically involves using adeno-associated viral vectors for delivery of healthy gene copies to affected tissues, has shown great promise as a strategy for the treatment of IRDs. However, the use of viruses is associated with several limitations, including harmful immune responses, genome integration, and limited gene carrying capacity. Here, we review the advances in non-viral gene augmentation strategies, such as the use of plasmids with minimal bacterial backbones and scaffold/matrix attachment region (S/MAR) sequences, that have the capability to overcome these weaknesses by accommodating genes of any size and maintaining episomal transgene expression with a lower risk of eliciting an immune response. Low retinal transfection rates remain a limitation, but various strategies, including coupling the DNA with different types of chemical vehicles (nanoparticles) and the use of electrical methods such as iontophoresis and electrotransfection to aid cell entry, have shown promise in preclinical studies. Non-viral gene therapy may offer a safer and effective option for future treatment of IRDs.

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1

BackgroundIndustrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism.ResultsWe identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5 × 103 CFU/μg (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA.ConclusionsPlasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

Identifying Direct Interactions Between the Rho GTPase TCL and Coronin CRIB Motifs

TCL is a Rho GTPase that contributes to tumor‐associated angiogenesis and metastatic melanoma; however, less is known about its basic cell biology and biochemistry. Currently, our lab has found that GTP‐loaded TCL tends to localize to the plasma membrane, while GDP‐loaded TCL localizes to intracellular vesicles. Our data also suggests that vesicle localization of GDP‐loaded TCL occurs through a protein/protein interaction. Coronins are a logical partner for this interaction as they bind to other Rho GTPases when they are in a GDP‐loaded form. To test for a direct interaction between Coronins and TCL, the CRIB domains from the seven human Coronin isoforms have been cloned into plasmids for use in interaction assays using both traditional GST‐fusion protein precipitation and protein complementation using an in vitro split luciferase assay system. Given the importance of TCL in angiogenesis and melanoma, delineation of its interactions with partner proteins is essential for understanding its role in pathology‐related processes.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

Use of plasmids for expression of proteins from the genus Leishmania in Escherichia coli: current state and perspectives.

Leishmaniosis is caused by the protozoa of the genus Leishmania with a wide spectrum of clinical and epidemiological manifestations which are characterized into four clinical groups: cutaneous, mucocutaneous, diffuse cutaneous, and visceral. American visceral leishmaniosis (AVL) or visceral leishmaniosis (VL) has been known as the most severe form of the disease. However, despite the growing number of people exposed to the infection risk and the great effort done by the scientific community worldwide to significantly increase the knowledge about these diseases, there is no vaccine capable of preventing VL in humans. In this short review, we present some of the plasmids used for the expression of recombinant protein by Escherichia coli strains used mainly for the second generation of vaccines for leishmaniosis. It can be emphasized that currently, these vectors and hosts play an important role in developing vaccine strategies against the disease. Indeed, use of the E. coli BL21 (DE) strain is remarkable mainly due to its characteristics for being a stable protein producer as well as the use of histidine tags for antigen purification. KEY POINTS: • Plasmid vectors and E. coli will continue being important for studies about leishmaniosis. • Protein purification exploiting histidine tags is a key technique.

Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control.

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.

Strategies for achieving high‐level and stable production of toxic Streptomyces phospholipase D in Escherichia coli

AbstractBACKGROUNDPhospholipase D (PLD) has great potential in the pharmaceutical and food industries for manufacturing bioactive phospholipids. Overproduction of PLD in Escherichia coli is difficult due to its high cytotoxicity which causes plasmid instability, short‐term PLD synthesis, cell lysis and PLD leakage. To address this issue, integrated optimization of PLD production in upstream and downstream processing was performed.RESULTSEffective strategies in upstream processing include the use of plasmids containing the pSC101 par region in recA− E. coli, the use of moderately strong and tightly regulated promoter PBAD, optimization of codon usage and amino acid supplementation, maintaining the best cellular state by supplementing nutrition, and saturation induction at low temperature. Using these strategies, a large amount of PLD (1.2 × 105 U L−1, 81.5 mg L−1) was obtained in the batch culture and PLD synthesis lasted 9 h. Additionally, cell lysis did not occur –99% of the recombinant PLD remained in the periplasm. Ultimately, the PLD was purified 327‐fold by a four‐step downstream process comprising extraction with 10 mmol L−1 Ethylenediaminetetraacetic acid and 0.05% (w/v) sodium deoxycholate, ammonium sulfate precipitation, anion exchange and Ni2+‐affinity chromatography.CONCLUSIONThis study provides the basis for large‐scale production of PLD in E. coli. © 2018 Society of Chemical Industry