Combining a Base Deaminase Mutator with Phage-Assisted Evolution.

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

Enhanced single-base mutation diversity by the combination of cytidine deaminase with DNA-repairing enzymes in yeast.

The occurrence of random mutations can increase the diversity of the genome and promote the evolutionary process of organisms. High efficiency mutagenesis techniques significantly accelerate the evolutionary process. In this work, we describe a targeted mutagenesis system named MutaT7trans to significantly increase mutation rate and generate mutations across all four nucleotides in yeast. We constructed different DNA-repairing enzyme-PmCDA1-T7 RNA polymerase (T7 RNAP) fusion proteins, achieved targeted mutagenesis by flanking the target gene with T7 promoters, and tuned the mutation spectra by introducing different DNA-repairing enzymes. With this mutagenesis tool, the proportion of non-C â†’ T mutations was 10-11-fold higher than the cytidine deaminase-based evolutionary tools, and the transversion mutation frequency was also elevated. The mutation rate of the target gene was significantly increased to 5.25 × 10-3 substitutions per base (s. p. b.). We also demonstrated that MutaT7trans could be used to evolve the CrtE, CrtI, and CrtYB gene in the ß-carotene biosynthesis process and generate different types of mutations.

Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning.

Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter of Escherichia coli K207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L-1 (627 mg L-1 isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L-1 to 100 mg L-1, in the titer of its tetraketide product was achieved.

High-Throughput Regulatory Part Prototyping and Analysis by Cell-Free Protein Synthesis and Droplet Microfluidics.

Engineering regulatory parts for improved performance in genetic programs has played a pivotal role in the development of the synthetic biology cell programming toolbox. Here, we report the development of a novel high-throughput platform for regulatory part prototyping and analysis that leverages the advantages of engineered DNA libraries, cell-free protein synthesis (CFPS), high-throughput emulsion droplet microfluidics, standard flow sorting adapted to screen droplet reactions, and next-generation sequencing (NGS). With this integrated platform, we screened the activity of millions of genetic parts within hours, followed by NGS retrieval of the improved designs. This in vitro platform is particularly valuable for engineering regulatory parts of nonmodel organisms, where in vivo high-throughput screening methods are not readily available. The platform can be extended to multipart screening of complete genetic programs to optimize yield and stability.

Split T7 promoter-based isothermal transcription amplification for one-step fluorescence detection of SARS-CoV-2 and emerging variants.

The negative global impact of the coronavirus disease pandemic has highlighted the crucial need for a rapid and convenient method of viral RNA detection. In this study, we report a novel method, termed as the split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR), for one-pot detection of viral RNA. STAR uses a split T7 promoter that is applied to a three-way junction to mediate the selective transcription by the T7 RNA polymerase in the presence of target RNA. In addition, a light-up RNA aptamer is used for signal amplification. STAR can detect viral RNA in less than 30 min with high specificity and sensitivity. By testing of 60 nasopharyngeal SARS-CoV-2 samples, the STAR assay demonstrates an excellent sensitivity and specificity of 96.7% and 100%, respectively. Moreover, we provide experimental evidence of the broad applicability of this assay through the multiplex detection of SARS-CoV-2 variants (D614G mutation) and direct detection of bacterial 16S rRNA.

In Vitro Method for Synthesis of Large-Scale dsRNA Molecule as a Novel Plant Protection Strategy.

Double-stranded RNA (dsRNAs) molecules are the precursors and effective triggers of RNAi in most organisms. RNAi can be induced by the direct introduction of dsRNAs in plants, fungi, insects, and nematodes. Until now RNAi is usually established by transformation of the plant with a construct that produces hairpin RNAs. Alternatively, advances in RNA biology demonstrated efficiently the in vitro method of large-scale synthesis of dsRNA molecule. Here we describe the de novo synthesis of dsRNA molecule targeting the specific gene of interest for functional application. Selection of off-target effective siRNA regions, flanking of T7 promoter sequences, T7 polymerase reaction, and maintenance of the stability of dsRNA molecules are the main criteria of this method to obtain pure and effective yield for functional applications. IPTG (isopropyl-ß-D-thiogalactopyranoside) induced, T7 express E. coli cells, could be used for large scale synthesis of dsRNA molecule are also described in this method.

T7-lac promoter vectors spontaneous derepression caused by plant-derived growth media may lead to serious expression problems: a systematic evaluation.

BACKGROUND: The widespread usage of protein expression systems in Escherichia coli (E. coli) is a workhorse of molecular biology research that has practical applications in biotechnology industry, including the production of pharmaceutical drugs. Various factors can strongly affect the successful construction and stable maintenance of clones and the resulting biosynthesis levels. These include an appropriate selection of recombinant hosts, expression systems, regulation of promoters, the repression level at an uninduced state, growth temperature, codon usage, codon context, mRNA secondary structure, translation kinetics, the presence/absence of chaperons and others. However, optimization of the growth medium's composition is often overlooked. We systematically evaluate this factor, which can have a dramatic effect on the expression of recombinant proteins, especially those which are toxic to a recombinant host. RESULTS: Commonly used animal tissue- and plant-based media were evaluated using a series of clones in pET vector, containing expressed Open Reading Frames (ORFs) with a wide spectrum of toxicity to the recombinant E. coli: (i) gfpuv (nontoxic); (ii) tp84_28-which codes for thermophilic endolysin (moderately toxic); and (iii) tthHB27IRM-which codes for thermophilic restriction endonuclease-methyltransferase (REase-MTase)-RM.TthHB27I (very toxic). The use of plant-derived peptones (soy peptone and malt extract) in a culture medium causes the T7-lac expression system to leak. We show that the presence of raffinose and stachyose (galactoside derivatives) in those peptones causes premature and uncontrolled induction of gene expression, which affects the course of the culture, the stability of clones and biosynthesis levels. CONCLUSIONS: The use of plant-derived peptones in a culture medium when using T7-lac hybrid promoter expression systems, such as Tabor-Studier, can lead to uncontrolled production of a recombinant protein. These conclusions also extend to other, lac operator-controlled promoters. In the case of proteins which are toxic to a recombinant host, this can result in mutations or deletions in the expression vector and/or cloned gene, the death of the host or highly decreased expression levels. This phenomenon is caused by the content of certain saccharides in plant peptones, some of which (galactosides) may act as T7-lac promoter inducer by interacting with a Lac repressor. Thus, when attempting to overexpress toxic proteins, it is recommended to either not use plant-derived media or to use them with caution and perform a pilot-scale evaluation of the derepression effect on a case-by-case basis.

Development of an expression-tunable multiple protein synthesis system in cell-free reactions using T7-promoter-variant series.

New materials with a low environmental load are expected to be generated through synthetic biology. To widely utilize this technology, it is important to create cells with designed biological functions and to control the expression of multiple enzymes. In this study, we constructed a cell-free evaluation system for multiple protein expression, in which synthesis is controlled by T7 promoter variants. The expression of a single protein using the T7 promoter variants showed the expected variety in expression levels, as previously reported. We then examined the expression levels of multiple proteins that are simultaneously produced in a single well to determine whether they can be predicted from the promoter activity values, which were defined from the isolated protein expression levels. When the sum of messenger ribonucleic acid (mRNA) species is small, the experimental protein expression levels can be predicted from the promoter activities (graphical abstract (a)) due to low competition for ribosomes. In other words, by using combinations of T7 promoter variants, we successfully developed a cell-free multiple protein synthesis system with tunable expression. In the presence of large amounts of mRNA, competition for ribosomes becomes an issue (graphical abstract (b)). Accordingly, the translation level of each protein cannot be directly predicted from the promoter activities and is biased by the strength of the ribosome binding site (RBS); a weaker RBS is more affected by competition. Our study provides information regarding the regulated expression of multiple enzymes in synthetic biology.

Repressor-Like On-Off Regulation of Protein Expression by the DNA-Binding Transcription Activator-Like Effector in T7 Promoter-Based Cell-Free Protein Synthesis.

The aim of this study was to develop a transcription activator-like effector (TALE)-based technology to regulate protein synthesis in cell-free systems. We attempted to regulate the T7 promoter system, which has no natural mechanism of expression control, and sought to arbitrarily induce protein expression through the formation and dissociation of TALE and target DNA complexes. Protein synthesis was performed in a cell-free system in the presence of TALE, which recognized and bound to a sequence upstream of the T7 promoter, and protein expression was suppressed by approximately 80 % compared to in the absence of TALE. This suggests that masking part of the promoter region strongly suppresses protein synthesis. Additionally, competitive inhibition of TALE binding to the target DNA template led to protein synthesis levels that were equivalent to the levels in the absence of TALE. Our results demonstrate that DNA recognition by TALE can regulate the expression of the T7 promoter system.

[Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF].

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins.

BACKGROUND: Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or pDuet series. RESULTS: A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. CONCLUSION: We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the pDuet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

Design and validation of a transposon that promotes expression of genes in episomal DNA.

Functional metagenomics, or the cloning and expression of DNA isolated directly from environmental samples, represents a source of novel compounds with biotechnological potential. However, attempts to identify such compounds in metagenomic libraries are generally inefficient in part due to lack of expression of heterologous DNA. In this research, the TnC_T7 transposon was developed to supply transcriptional machinery during functional analysis of metagenomic libraries. TnC_T7 contains bidirectional T7 promoters, the gene encoding the T7 RNA polymerase (T7RNAP), and a kanamycin resistance gene. The T7 RNA polymerase gene is regulated by the inducible arabinose promoter (PBAD), thereby facilitating inducible expression of genes adjacent to the randomly integrating transposon. The high processivity of T7RNAP should make this tool particularly useful for obtaining gene expression in long inserts. TnC_T7 functionality was validated by conducting in vitro transposition of pKR-C12 or fosmid pF076_GFPmut3*, carrying metagenomic DNA from soil. We identified transposon insertions that enhanced GFP expression in both vectors, including insertions in which the promoter delivered by the transposon was located as far as 8.7 kb from the GFP gene, indicating the power of the high processivity of the T7 polymerase. The results gathered in this research demonstrate the potential of TnC_T7 to enhance gene expression in functional metagenomic studies.

High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli.

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.

Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Induction of T7 Promoter at Higher Temperatures May Be Counterproductive.

Bacterial expression of recombinant proteins is the most popular and convenient method for obtaining large quantities of pure protein. The induction of T7 promoter with isopropyl-ß-d-thiogalactopyranoside (IPTG) is widely used for expression of large quantities of proteins in Escherichia coli. It has been reported that basic T7 promoter is leaky and expresses cloned genes without induction. The effect of T7 promoter induction on expression of proteins at different temperature using flow cytometry has not yet been investigated. Green fluorescent protein (GFP) as a non-peptide tag can be used for protein solubility screening and for high-throughput optimization of expression conditions using flow cytometry. Therefore, flow cytometry was used to study the effect of induction on the expression of T7 promoter driven emerald GFP (emGFP) at various temperatures. We noticed that percentage of emGFP positive cells decreased instead of increasing upon induction at higher temperatures. Western blot analysis confirmed that the amount of total and soluble emGFP decreased in induced cells compared uninduced cells at higher temperatures. Our results indicate that induction of basic T7 promoter at higher temperature may not necessarily increase protein expression. While using a basic T7 promoter it is highly recommended to analyze the effect of induction on protein expression at various temperatures.

Building an Inducible T7 RNA Polymerase/T7 Promoter Circuit in Synechocystis sp. PCC6803.

To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc1O and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.

Bacteriophage T7 transcription system: an enabling tool in synthetic biology.

Since its discovery in the 1970s, the T7 RNA polymerase (T7 RNAP) transcription system has been applied extensively as an effective tool in molecular biology because of its robust function in various hosts, including prokaryotic, eukaryotic and cell free systems. Recently, the T7 RNAP transcription system has emerged as a critical component for synthetic biology. The present paper summarizes the advances of the T7 RNAP transcription system in synthetic biology, including the recent progress of T7 RNAP structure and its cognate promoter and terminator and its application in cell free systems, logic gates and orthogonal genetic circuits.

Escherichia coli chromosome-based T7-dependent constitutive overexpression system and its application to generating a phenylalanine producing strain.

Many metabolic engineering approaches have been attempted to generate strains capable of producing valuable compounds. One of main goals is industrial application of these strains. Integration of synthetic pathway genes into the Escherichia coli chromosome enables generation of a plasmid-free strain that is stable and useful for industrial applications. Strains that do not require induction are advantageous in terms of cost. In the present study, we constructed a constitutive overexpression system in E. coli to generate plasmid-free and inducer-free strains. The T7 RNA polymerase/T7 promoter overexpression system, which is an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible gene overexpression system (T7-dependent inducible overexpression system), was modified to be a constitutive overexpression system. The constructed overexpression system, a "chromosome-based T7-dependent constitutive overexpression system", was applied in a metabolic engineering study to generate a plasmid-free and inducer-free phenylalanine producing strain of E. coli.

Expression of T7-based constructs in tobacco cells.

Bacteriophage T7 promoter and RNA polymerase (T7-Pol) are widely used for recombinant protein expression in bacteria. In plants, there exists conflicting results regarding the efficacy of protein expression from T7-Pol-derived mRNAs. To reconcile these contradictory observations, the expression of green fluorescent protein (GFP) from T7 constructs was evaluated in tobacco protoplasts. T7 constructs transcribed by a nuclearly targeted T7-Pol did not express GFP in plant protoplasts, however T7-Pol lacking a nuclear targeting signal was able to translate cytosolically transcribed mRNAs, but only if the messages contained a viral translation enhancer. GFP expression was further evaluated at the plant level by using agroinfiltration-mediated transient expression system. Unlike for cytosolic expression, nuclear T7 transcripts containing a viral translation enhancer element did not express GFP, and modifications designed to stabilize and facilitate export of T7 transcripts to the cytosol did not improve the expression. We conclude that expression of nuclear T7 constructs is not feasible in tobacco cells, but cytosolic transcription provides an alternative means to over-express RNAs directly in the cytosol.

CRISPR-Cas Expands Dynamic Range of Gene Expression From T7RNAP Promoters.

Reducing leaky gene expression is critical for improving protein yield of recombinant bacteria and stability of engineered cellular circuits in synthetic biology. Leaky gene expression occurs when a genetic promoter is not fully repressed, leading to unintended protein synthesis in the absence of stimuli. Existing work have devised specific molecular strategies for reducing leaky gene expression of each promoter. In contrast, we describe a repurposed, modular CRISPRi system that attenuates leaky gene expression using a series of single-guide RNAs targeting the PT7/LacO1 promoter. Furthermore, we demonstrate the efficacy of CRISPRi to significantly increase the dynamic range of T7 RNA Polymerase (T7RNAP) promoters. In addition, we demonstrate that the CRISPRi system can be applied to enhance growth of bacteria that suffer from leaky expression of a toxic protein. Our work establishes a new application of CRISPRi in genomic engineering to improve the control of recombinant gene expression. The approach is potentially generalizable to other gene expression system by changing the single-guide RNAs.