Constitutive Promoter Research Articles

Creating a Genetic Toolbox for the Carbon-Fixing, Nitrogen-Fixing and Dehalogenating Bacterium Xanthobacter autotrophicus.

Xanthobacter autotrophicus is a metabolically flexible microorganism with two key features: (1) The organism has adapted to grow on a wide variety of carbon sources including CO2, methanol, formate, propylene, haloalkanes and haloacids; and (2) X. autotrophicus was the first chemoautotroph identified that could also simultaneously fix N2, meaning the organism can utilize CO2, N2, and H2 for growth. This metabolic flexibility has enabled use of X. autotrophicus for gas fixation, the creation of fertilizers and foods from gases, and the dehalogenation of environmental contaminants. Despite the wide variety of applications that have already been demonstrated for this organism, there are few genetic tools available to explore and exploit its metabolism. Here, we report a genetic toolbox for use in X. autotrophicus. We first identified suitable origins of replication and quantified their copy number, and identified antibiotic resistance cassettes that could be used as selectable markers. We then tested several constitutive and inducible promoters and terminators and quantified their promoter strengths and termination efficiencies. Finally, we demonstrated that gene expression tools remain effective under both autotrophic and dehalogenative metabolic conditions to show that these tools can be used in the environments that make X. autotrophicus unique. Our extensive characterization of these tools in X. autotrophicus will enable genetic and metabolic engineering to optimize production of fertilizers and foods from gases, and enable bioremediation of halogenated environmental contaminants.

The Construction of an Extreme Radiation-Resistant Perchlorate-Reducing Bacterium Using Deinococcus deserti Promoters.

Perchlorate is one of the major inorganic pollutants in the natural environment and the living environment, which is toxic to organisms and difficult to degrade due to its special structure. As previously reported, the Phoenix Mars lander detected approximately 0.6% perchlorate in the Martian soil, indicating challenges for Earth-based life to survive there. Currently, biological approaches using dissimilatory perchlorate-reducing bacteria (DPRB) are the most promising methods for perchlorate degradation. However, the majority of DPRB exhibit limited radiation resistance, rendering them unsuitable for survival on Mars. In this study, we obtained the transcriptome data of Deinococcus deserti, and predicted and identified multiple constitutive expression promoters of D. deserti with varying activities. The top-five most active promoters were separately fused to specific genes involved in the degradation of perchlorate from DPRB Dechloromonas agitata CKB, and transformed into Deinococcus radiodurans R1, forming a novel dissimilatory perchlorate-reducing bacterium, R1-CKB. It exhibited both efficient perchlorate degradation capability and strong radiation resistance, potentially offering a valuable tool for the further enhancement of the Martian atmosphere in the future.

Conditional Knockdown of Endogenous MicroRNAs in CHO Cells Using TET-ON-SanDI Sponge Vectors.

MicroRNAs (miRNAs) are small, noncoding RNAs of about 22 nucleotides in length and have proven to be useful targets for genetic modifications for desirable phenotypes in the biotech industry. The use of constitutively expressed "miRNA sponge" vectors in which multiple, tandem miRNA-binding sites containing transcripts are transcriptionally regulated by a constitutive promoter for downregulating the levels of endogenous microRNAs in Chinese hamster ovary (CHO) cells has shown to be more advantageous than using synthetic antisense oligonucleotides. The application of miRNA sponges in biotechnological processes, however, could be more effective, if the expression of miRNA sponges could be tuned. In this chapter, we present a method for the generation of stable CHO cell lines expressing a TET-ON-SanDI-miRNA sponge which is in theory expressed only in the presence of an inducer.

Engineering of tnaC-Based Tryptophan Biosensors for Dynamic Control of Violacein Production.

Tryptophan not only serves as a fundamental building block for protein synthesis but also acts as a metabolic precursor for a diverse array of high-value chemicals. Although a few tryptophan-responsive biosensors are currently available, there is a growing interest in developing high-performance biosensors. In this study, we create a miniature toolkit of tryptophan biosensors based upon the leader regulatory region of the tnaCAB operon, which is responsible for tryptophan catabolism in Escherichia coli. Four variants are generated by engineering the tnaC leader sequence, which encodes a leader peptide composed of 24 amino acid residues. Subsequently, the performance of both the natural tnaC sequence and its engineered variants is assessed in a reporter strain based on the MazEF toxin-antitoxin system. The results demonstrate that two engineered variants exhibit increased sensitivity to low levels of tryptophan. Moreover, the engineered biosensors are further optimized by replacing the native promoter of tnaC with a phage-derived constitutive promoter. Intriguingly, the engineered biosensors can be reconstructed for extended application in Pseudomonas putida, a robust microbial chassis for metabolic engineering. In summary, our study expands the toolkit of tryptophan biosensors that can be broadly used for the bioproduction of many other high-value tryptophan-derived products.

Combining culture optimization and synthetic biology to improve production and detection of secondary metabolites in Myxococcus xanthus: application to myxoprincomide.

Microbial secondary metabolites play crucial ecological roles in governing species interactions and contributing to their defense strategies. Their unique structures and potent bioactivities have been key in discovering antibiotics and anticancer drugs. Genome sequencing has undoubtedly revealed that myxobacteria constitute a huge reservoir of secondary metabolites as the well-known producers, actinomycetes. However, because most secondary metabolites are not produced in the laboratory context, the natural products from myxobacteria characterized to date represent only the tip of the iceberg. By combining the engineering of a dedicated Myxococcus xanthus DZ2 chassis strain with a two-step growth medium protocol, we provide a new approach called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) for the production of non-ribosomal peptides synthetases (NRPS)/polyketides synthases (PKS) secondary metabolites from myxobacteria. We further show that the 2PRIM-BOOST strategy will facilitate the screening of secondary metabolites for biological activities of medical interest. As proof of concept, using a constitutive strong promoter, the myxoprincomide from M. xanthus DZ2 has been efficiently produced and its biosynthesis has been enhanced using the 2PRIM-BOOST approach, allowing the identification of new features of myxoprincomide. This strategy should allow the chances to produce and discover new NRPS, PKS, and mixed NRPS/PKS hybrid natural metabolites that are currently considered as cryptic and are the most represented in myxobacteria.IMPORTANCEMicrobial secondary metabolites are important in species interactions and are also a prolific source of drugs. Myxobacteria are ubiquitous soil-dwelling bacteria constituting a huge reservoir of secondary metabolites. However, because most of these molecules are not produced in the laboratory context, one can estimate that only one-tenth have been characterized to date. Here, we developed a new strategy called two-step Protocol for Resource Integration and Maximization-Biomolecules Overproduction and Optimal Screening Therapeutics (2PRIM-BOOST) that combines the engineering of a dedicated Myxococcus xanthus chassis strain together with growth medium optimization. By combining these strategies with the insertion of a constitutive promoter upstream the biosynthetic gene cluster (BGC), the production of myxoprincomide, a characterized low-produced secondary metabolite, was successfully and significantly increased. The 2PRIM-BOOST enriches the toolbox used to produce previously cryptic metabolites, unveil their ecological role, and provide new molecules of medical interest.

A Novel Biosynthetic Strategy for Ginsenoside Ro: Construction of a Metabolically Engineered Saccharomyces cerevisiae Strain Using a Newly Identified UGAT Gene from Panax ginseng as the Key Enzyme Gene and Optimization of Fermentation Conditions.

Ginsenoside Ro, as one of the few oleanane-type ginsenosides, is well known for its unique molecular structure and biological activities. Currently, research on the biosynthesis of ginsenoside Ro is still in its early stages. Therefore, the establishment of a new ginsenoside Ro cell factory is of great significance for the in-depth development and utilization of genes related to ginsenoside Ro synthesis, as well as for the exploration of pathways to obtain ginsenoside Ro. In this study, we cloned endogenous constitutive promoters, terminators, and other genetic elements from S. cerevisiae BY4741. These elements were then sequentially assembled with the uridine diphosphate glucuronic acid transferase gene identified in our previously study (PgUGAT252645) and several other reported key enzyme genes, to construct DNA fragments used for integration into the genome of S. cerevisiae BY4741. By sequentially transferring these DNA fragments into chemically competent cells of engineering strains and conducting screening and target product detection, we successfully constructed an engineered S. cerevisiae strain (BY-Ro) for ginsenoside Ro biosynthesis using S. cerevisiae BY4741 as the host cell. Strain BY-Ro produced 253.32 μg/L of ginsenoside Ro under optimal fermentation conditions. According to subsequent measurements and calculations, this equates to 0.033 mg/g DCW, corresponding to approximately 31% of the ginsenoside Ro content found in plant samples. This study not only included a deeper investigation into the function of PgUGAT252645 but also provides a novel engineering platform for ginsenoside Ro biosynthesis.

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.

Development of genetically modified rust resistant wheat: A breakthrough by dinted introgression of novel DREB2C and HSFA2 genes under stress induced expression

Wheat is a major staple food worldwide yet numerous yield limiting agents affect its productivity. Stripe rust is a major culprit in this context and efforts have been made to culminate this pathogen using conventional as well as advanced innovative techniques. Transgenic technology is of significant importance in this context and numerous success stories are evident to prove its worth. In the current study, two novel genes HSFA2 and DREB2C were expressed in an elite wheat genotype Akbar, Fakhre-e-Bhakhar under constitutive CAMV35S promoter and stress inducible rd29 Promoters. The shoot cut method was used for the Agrobacterium-mediated transformation and putative transformants were selected on kanamycin 50 mg/L. The resultant transformants were tested through PCR for transgene integration whereas expression analysis was carried out through realtime qPCR. Expression of both of the aforementioned genes was found to be higher under rd29 promoter as compared with transgene(s) expression under CAMV35S promoter. In the bioassay, transgenic wheat plants demonstrated significant tolerance to stress, exhibiting only minor spotting under constitutive expression conditions. Upon exposure to stress, these plants showed exceptional resistance to stripe rust, producing large, bold grains compared to individual trait expressions and negative controls. Subsequently, the DREB2C gene was knocked out to determine if stripe rust control was specifically attributed to this gene. Following the knockout, the onset of stripe rust was comparable to that of the negative control. This led to the conclusion that pyramiding the DREB2C gene with HSFA2 through dual expression represents a novel and highly effective strategy for controlling the widespread stripe rust in wheat. This approach also offers resistance to high temperatures (above 32 °C) from the pollination stage through to maturity.

Compensatory reactions of B cells in response to chronic HIV-1 Tat exposure.

Patients infected with human immunodeficiency virus-1 (HIV-1) have an increased incidence of B-cell lymphoma, even though HIV-1 does not infect B cells. The development of B-cell lymphomas appears to be related to the action of the HIV-1 transactivator protein (Tat), which is released from HIV-infected cells and penetrates uninfected B cells, affecting host cell gene expression. Upon chronic HIV-1 infection, Tat acts on the cells for a long time, probably allowing the cells to adapt to the presence of the viral protein. The aim of this work was to identify and study the mechanism of adaptation of cells to prolonged (chronic) exposure to HIV-1 Tat. We performed a comparative analysis of cells expressing Tat under the action of either an inducible promoter or a constitutive promoter, allowing us to model acute and chronic Tat effects, respectively. We found that the acute action of Tat leads to the suppression of cell proliferation, probably due to the downregulation of genes associated with replication and protein synthesis. In the case of chronic action of Tat, cell proliferation was restored and the expression of genes associated with the implementation of protective (antiviral) functions of the cell was increased. Analysis using proteasome inhibitors showed that in the case of chronic action, intense Tat proteolysis occurred, which could be the main mechanism of B-cell adaptation. Thus, B cells have a powerful mechanism to adapt to the entry of HIV-1 Tat, the efficiency of which may determine the frequency of lymphomagenesis in HIV-1-infected patients.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Genetic Transformation of Triticum dicoccum and Triticum aestivum with Genes of Jasmonate Biosynthesis Pathway Affects Growth and Productivity Characteristics.

The transformation protocol based on the dual selection approach (fluorescent protein and herbicide resistance) has been applied here to produce transgenic plants of two cereal species, emmer wheat and bread wheat, with the goal of activating the synthesis of the stress hormone jasmonates by overexpressing ALLENE OXIDE SYNTHASE from Arabidopsis thaliana (AtAOS) and bread wheat (TaAOS) and OXOPHYTODIENOATE REDUCTASE 3 from A. thaliana (AtOPR3) under the strong constitutive promoter (ZmUbi1), either individually or both genes simultaneously. The delivery of the expression cassette encoding AOS was found to affect morphogenesis in both wheat species negatively. The effect of transgene expression on the accumulation of individual jasmonates in hexaploid and tetraploid wheat was observed. Among the introduced genes, overexpression of TaAOS was the most successful in increasing stress-inducible phytohormone levels in transgenic plants, resulting in higher accumulations of JA and JA-Ile in emmer wheat and 12-OPDA in bread wheat. In general, overexpression of AOS, alone or together with AtOPR3, negatively affected leaf lamina length and grain numbers per spike in both wheat species. Double (AtAOS + AtOPR3) transgenic wheat plants were characterized by significantly reduced plant height and seed numbers, especially in emmer wheat, where several primary plants failed to produce seeds.

Development of CadR-based cadmium whole cell biosensor for visual detection of environmental Cd2+

BackgroundAs a threat to human health and public health, cadmium (Cd) pollution has received widespread social concern. Our previously constructed CadR-based bacterial whole cell biosensor (WCB) epCadR5 showed high sensitivity and specificity in cadmium detection. However, the application of the sensor is still hindered by the need for laboratory equipment to read the fluorescence signal output. In this study, we aimed to optimizing the sensor to make it available for visual detection of environmental cadmium and simplify the detection process to advance practical application of the sensor. ResultsBy replacing the constitutive promoter with J110, the fluorescence signal output of the sensor was significantly increased and the fluorescence leakage was decreased. In addition, the fluorescence signal output of green fluorescence protein (GFP) was enhanced by the addition of a 5′ untranslated region (5′-UTR) mlcR10. The fluorescence signal output of the WCB is sufficiently robust to be visible and distinguishable to the naked eye, which is of paramount importance for visual detection. The sensor readout can be conveniently recorded by mobile phone camera and quantified. For ease of on-site application, the WCB's visual detection procedures and conditions were further optimized and simplified. The WCB demonstrated good linearity and detection limit (1.81 μg/L) for visual detection of Cd2+ without the assistance of bulky laboratory equipment. For the detection of real environmental samples, the WCB visual detection results were close to those of WCB-flow cytometry (FACS) and graphite furnace atomic absorption spectroscopy (GFAAS). SignificanceIn this work, we developed an easy-to-use, on-site and visual detection biosensor for monitoring environmental Cd2+. It will advance the utilization of cadmium WCBs in practical settings. The optimization and simplification strategy in the study also provide new insights into the visualization of other bacterial biosensors, and will advance the practical application of WCBs.

Continuous Process Verification 4.0 application in upstream: adaptiveness implementation managed by AI in the hypoxic bioprocess of the Pichia pastoris cell factory.

The experimental approach developed in this research demonstrated how the cloud, the Internet of Things (IoT), edge computing, and Artificial Intelligence (AI), considered key technologies in Industry 4.0, provide the expected horizon for adaptive vision in Continued Process Verification (CPV), the final stage of Process Validation (PV). Pichia pastoris producing Candida rugosa lipase 1 under the regulation of the constitutive GAP promoter was selected as an experimental bioprocess. The bioprocess worked under hypoxic conditions in carbon-limited fed-batch cultures through a physiological control based on the respiratory quotient (RQ). In this novel bioprocess, a digital twin (DT) was built and successfully tested. The implementation of online sensors worked as a bridge between the microorganism and AI models, to provide predictions from the edge and the cloud. AI models emulated the metabolism of Pichia based on critical process parameters and actionable factors to achieve the expected quality attributes. This innovative AI-aided Adaptive-Proportional Control strategy (AI-APC) improved the reproducibility comparing to a Manual-Heuristic Control strategy (MHC), showing better performance than the Boolean-Logic-Controller (BLC) tested. The accuracy, indicated by the Mean Relative Error (MRE), was for the AI-APC lower than 4%, better than the obtained for MHC (10%) and BLC (5%). Moreover, in terms of precision, the same trend was observed when comparing the Root Mean Square Deviation (RMSD) values, becoming lower as the complexity of the controller increases. The successful automatic real time control of the bioprocess orchestrated by AI models proved the 4.0 capabilities brought by the adaptive concept and its validity in biopharmaceutical upstream operations.

Overexpression of Gossypium arboreum 3-ketoacyl-CoA synthase 6 (GaKCS6) gene enhanced leaf epicuticle wax in Gossypium hirsutum L. and improved tolerance against whitefly

Cotton Leaf Curl Virus (CLCuV) is a significant threat to cotton production, as it causes Cotton Leaf Curl Disease (CLCuD). Whitefly serves as a vector for the transmission of this virus. It can be controlled by developing barriers against whitefly infestation. The leaf epicuticle wax acts as a protective barrier against whitefly attacks. Research into wax biosynthesis and the fatty acid elongation pathway has highlighted the role of the 3-ketoacyl-CoA synthase (KCS) gene family in producing very-long-chain fatty acids (VLCFAs) in plants. The 3-ketoacyl-CoA synthase 6 (GaKCS6) gene, isolated from the CLCuV-resistant FDH-170 variety of Gossypium arboreum, was cloned under the control of the CaMV35S constitutive promoter and transformed into the CLCuV-susceptible Gossypium hirsutum variety CKC-3 resulting in significantly higher leaf epicuticle wax deposition. Overexpression of GaKCS6 in the transgenic cotton plants was confirmed through quantitative real-time PCR. The transgenic plants not only exhibited average growth but also showed improvements in agronomic traits. Scanning Electron Microscope (SEM) analysis further validated the enhanced leaf epicuticle wax deposition in transgenic plants compared to non-transgenic (control). A free-choice bioassay against whiteflies demonstrated that the transgenic plants remained free of viral infection, as confirmed by real-time PCR. These findings indicate that increased leaf epicuticle wax deposition in transgenic cotton effectively prevents whitefly attacks and the transmission of CLCuV. It suggests that the GaKCS6 gene plays a crucial role in producing leaf epicuticle wax through the VLCFAs biosynthesis pathway.

Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter PgpmA* from six candidate promoters for expressing ilvBN. PgpmA* showcased the expression strength 23.3-fold that of the native promoter PilvBN. Moreover, three synthetic RBS libraries based on the promoter PgpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that "R(9)N(6)" was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of PilvBN. Finally, the promoter PgpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBNS155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Enhanced heterologous gene expression in Trichoderma reesei by promoting multicopy integration

Trichoderma reesei displays a high capability to produce extracellular proteins and therefore is used as a platform for the expression of heterologous genes. In a previous study, an expression cassette with the constitutive tef1 promoter and the cbh1 terminator compatible with flow cytometry analysis was developed. Independent transformants obtained by a random integration into the genome of a circular plasmid containing the expression cassette showed a wide range of fluorescence levels. Whole genome sequencing was conducted on eight of the transformed strains using two next-generation sequencing (NGS) platforms: Illumina paired-end sequencing and Oxford Nanopore. In all strains, the expression plasmid was inserted at the same position in the genome, i.e., upstream of the tef1 gene, indicating an integration by homologous recombination. The different levels of fluorescence observed correspond to different copy numbers of the plasmid. Overall, the integration of a circular plasmid with the green fluorescence protein (egfp) transgene under the control of tef1 promoter favors multicopy integration and allows over-production of this heterologous protein on glucose. In conclusion, an expression system based on using the tef1 promotor could be one of the building blocks for improving high-value heterologous protein production by increasing the copy number of the encoding genes into the genome of the platform strain.Key points• Varied eGFP levels from tef1 promoter and cbh1 terminator expression.• Whole genome sequencing on short and long reads platforms reveals various plasmid copy numbers in strains.• Plasmids integrate at the same genomic site by homologous recombination in all strains.

Modified Tn7 transposon vectors for controlled chromosomal gene expression.

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing differential transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted.IMPORTANCEChromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology.

De Novo Synthesis of Perdeuterated Phosphoinositide by Installing a Non-native Phospholipid Biopathway in E. coli.

Phosphatidylinositol (PI) and its phosphorylated derivatives are of paramount importance in cellular functions and diseases. Understanding their diverse roles is, however, challenged by difficulties in synthesis and labeling techniques. In this proof-of-concept study, we demonstrate that PI can be straightforwardly de novo-synthesized and deuterium (2H)-labeled in Escherichia coli by genomic insertion of PI synthase from Trypanosoma brucei under constitutive synthetic promoter proD. Insertion into loci atpi-gidB and ybb revealed PI accumulation of 41% and 34% (mol/mol), respectively, when cultivated with glycerol as the sole carbon source. Growth of the atpi-gidB-PIS strain in deuterium-labeled (2H) substrates D2O, D8-glycerol, and D6-myo-inositol achieved PI deuteration of 90%, PE deuteration of 95%, and total fatty acids|fatty acid (FA) deuteration of 97%. This study offers an alternative convenient route to chemical and enzymatic labeling synthesis of PI; more excitingly, this work also, in principle, opens a door for tailoring the FA profile of deuterated PI/PE for task-specific application by repurposing FA biosynthesis pathways.

Detection of Short-Chain Chlorinated Aliphatic Hydrocarbons through an Engineered Biosensor with Tailored Ligand Specificity.

Short-chain chlorinated aliphatic hydrocarbons (SCAHs), commonly used as industrial reagents and solvents, pose a significant threat to ecosystems and human health as they infiltrate aquatic environments due to extensive usage and accidental spills. Whole-cell biosensors have emerged as cost-effective, rapid, and real-time analytical tools for environmental monitoring and remediation. While the broad ligand specificity of transcriptional factors (TFs) often prohibits the application of such biosensors. Herein, we exploited a semirational transition ligand approach in conjunction with a positive/negative fluorescence-activated cell sorting (FACS) strategy to develop a biosensor based on the TF AlkS, which is highly specific for SCAHs. Furthermore, through promoter-directed evolution, the performance of the biosensor was further enhanced. Mutation in the -10 region of constitutive promoter PalkS resulted in reduced AlkS leakage expression, while mutation in the -10 region of inducible promoter PalkB increased its accessibility to the AlkS-SCAHs complex. This led to an 89% reduction in background fluorescence leakage of the optimized biosensor, M2-463, further enhancing its response to SCAHs. The optimized biosensor was highly sensitive and exhibited a broader dynamic response range with a 150-fold increase in fluorescence output after 1 h of induction. The detection limit (LOD) reached 0.03 ppm, and the average recovery rate of SCAHs in actual water samples ranged from 95.87 to 101.20%. The accuracy and precision of the proposed biosensor were validated using gas chromatography-mass spectrometry (GC-MS), demonstrating the promising application for SCAH detection in an actual environment sample.

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.