Advances in Synthetic-Biology-Based Whole-Cell Biosensors: Principles, Genetic Modules, and Applications in Food Safety.

A whole-cell biosensor based on synthetic biology provides a promising new method for the on-site detection of food contaminants. The basic components of whole-cell biosensors include the sensing elements, such as transcription factors and riboswitches, and reporting elements, such as fluorescence, gas, etc. The sensing and reporting elements are coupled through gene expression regulation to form a simple gene circuit for the detection of target substances. Additionally, a more complex gene circuit can involve other functional elements or modules such as signal amplification, multiple detection, and delay reporting. With the help of synthetic biology, whole-cell biosensors are becoming more versatile and integrated, that is, integrating pre-detection sample processing, detection processes, and post-detection signal calculation and storage processes into cells. Due to the relative stability of the intracellular environment, whole-cell biosensors are highly resistant to interference without the need of complex sample preprocessing. Due to the reproduction of chassis cells, whole-cell biosensors replicate all elements automatically without the need for purification processing. Therefore, whole-cell biosensors are easy to operate and simple to produce. Based on the above advantages, whole-cell biosensors are more suitable for on-site detection than other rapid detection methods. Whole-cell biosensors have been applied in various forms such as test strips and kits, with the latest reported forms being wearable devices such as masks, hand rings, and clothing. This paper examines the composition, construction methods, and types of the fundamental components of synthetic biological whole-cell biosensors. We also introduce the prospect and development trend of whole-cell biosensors in commercial applications.

Applications of the Whole-Cell System in the Efficient Biosynthesis of Heme.

Heme has a variety of functions, from electronic reactions to binding gases, which makes it useful in medical treatments, dietary supplements, and food processing. In recent years, whole-cell system-based heme biosynthesis methods have been continuously explored and optimized as an alternative to the low-yield, lasting, and adverse ecological environment of chemical synthesis methods. This method relies on two biosynthetic pathways of microbial precursor 5-aminolevulinic acid (C4, C5) and three known downstream biosynthetic pathways of heme. This paper reviews the genetic and metabolic engineering strategies for heme production in recent years by optimizing culture conditions and techniques from different microorganisms. Specifically, we summarized and analyzed the possibility of using biosensors to explore new strategies for the biosynthesis of heme from the perspective of synthetic biology, providing a new direction for future exploration.

Discovery of 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety as novel ALK inhibitors.

To overcome drug resistance caused by ALK kinase mutations especially G1202R, two series of novel 2,4-diarylaminopyrimidine derivatives bearing dithiocarbamate moiety were designed, synthesized and evaluated for their biological activities. Among all the target compounds, B10 efficiently inhibited the proliferation of ALK-positive Karpas299 and H2228 cells both with IC50 values of 0.07 µM. In addition, B10 exhibited remarkable enzymatic inhibitory potency with IC50 values of 4.59 nM, 2.07 nM and 5.95 nM toward ALKWT, ALKL1196M and ALKG1202R, respectively. Furthermore, B10 induced apoptosis in H2228 cell and caused cell cycle arrest in G2/M phase. Ultimately, the binding modes of B10 with ALKWT and ALKG1202R were ideally established, which further confirmed the structural basis in accordance with the SARs analysis. These results indicated that B10 was a potent ALK inhibitor for ALKG1202R mutation treatment and deserved for further investigation.

A 90-day subchronic toxicology screen of genetically modified rice Lac-3 and its effects on the gut microbiota in Sprague-Dawley rats.

A 90-day subchronic toxicology screen of genetically modified (GM) rice Lac-3 expressing human lactoferrin (hLF) and its effects on the gut microbiota were studied in comparison to non-GM rice fed to Sprague-Dawley (SD) rats. Three different dietary concentrations (17.5%, 35% and 70%, w/w) of the GM rice or its corresponding non-GM rice were used. Additionally, the phylotypes of gut microbiota in the control group, the 70% GM rice diet group and the 70% non-GM rice diet group on day 90 were determined by 16S rRNA sequencing. The results of the 90-day subchronic feeding study demonstrated that the GM rice Lac-3 containing human lactoferrin (LF) gene is considered as safe as the non-GM rice. The results of bacterial 16S rRNA sequencing showed that the structure of gut microbiota in the 70% GM group slightly changed when compared with the control group and the 70% non-GM group. There were no significant differences in the microbiota diversity among the three groups.

Safety assessment of transgenic canola RF3 with bar and barstar gene on Sprague-Dawley (SD) rats by 90-day feeding test.

Canola is one of the most important plant oilseed crops. To avoid the threat of herbicides, the RF3 line with bar gene and barstar gene was developed, which can act as glufosinate resistance resources and restore fertility in hybrid lines. To assess the food safety of transgenic canola RF3, 2.5%, 5% and 10% GM canola RF3 and its non-GM isogenic line Drakkar were formulated into diet to feed Spragure-Dawley (SD) rats for 90 days. The effects on the general growth and toxicological parameters, as well as gut microbiota of rats, were evaluated. Several significant differences on body weight, feed consumption, relative organ weight, hematology and serum biochemistry were observed among rats in the 90-day feeding test. However, these statistical differences were randomly observed among different groups and were not dose-related, which were not considered to be biologically significant. Furthermore, the results of bacterial 16S rRNA sequencing of fecal samples showed that the diets containing GM canola did not disturb the balance of gut microbiota. In conclusion, the canola RF3 is considered as safe and wholesome as the non-GM canola based on this 90-day feeding test and gut microbiota analysis.

Rice- or pork-based diets with similar calorie and content result in different rat gut microbiota.

Rice is the most important food crop, and pork is the most widely eaten meat in the world. In this study, we compared the gut microbiota of the rats fed with rice or pork mixed diets, which have similar caloric contents. The physiological indices (body weights, hematology, serum chemistry, organ weights and histopathology) of two groups were all within the normal range. Two diets did not induce difference in the diversity of gut bacteria. However, Firmicutes were significantly higher in rice diet group, while Bacteroidetes were enriched in pork diet group. Butyrate and the bacteria enzymes ß-glucuronidase, ß-glucosidase and nitroreductase in the feces were all drastically higher in pork diet group. This study indicates that different diets with similar calorie and nutritional composition could change the community structure but not the diversity of rat fecal microbiota.

Effects of neutrophils peptide-1 transgenic Chlorella ellipsoidea on the gut microbiota of male Sprague-Dawley rats, as revealed by high-throughput 16S rRNA sequencing.

Rabbit neutrophils peptide-1 (NP-1) is a type of defensin that possesses a broad spectrum of antimicrobial activity. Chlorella ellipsoidea is a new eukaryotic expression system for exogenously producing NP-1. The NP-1 transgenic C. ellipsoidea can be directly added into feed as antimicrobial agent without any purification procedure for the NP-1 peptide. However, the effects of C. ellipsoidea and NP-1 on the host gut microbiota should be explored before application. In this study, diets containing different concentrations (1.25, 2.5, and 5%) of C. ellipsoidea and NP-1 transgenic C. ellipsoidea were administered to male Sprague-Dawley rats. Compared with the chow diet control group, none of the experimental groups showed any significant differences in their growth indices, and no histopathological damage was observed. The phylotypes of gut microbiota in the control group, the 5% C. ellipsoidea diet group and the 5% NP-1 transgenic C. ellipsoidea diet group were determined by 16S rRNA sequencing. The results showed that both 5% experimental groups had shifted community memberships of gut microbiota. In particular, the 5% NP-1 transgenic C. ellipsoidea diet exhibited an increased abundance of most Gram-positive bacterial taxa and a reduced abundance of most Gram-negative bacterial taxa, and it promoted the growth of some lactic acid bacterial genera. Lactic acid bacteria, especially the Bifidobacterium and Lactobacillus, have been widely reported to be benefic effects on the host. Thus NP-1 transgenic C. ellipsoidea is promising feed additive and gut regulator, as it have the potential to increase the abundance of Bifidobacterium and Lactobacillus in gut microbiota of animal.

MicroRNA profiling of rats with ochratoxin A nephrotoxicity.

BACKGROUND: Nephrotoxicity is the most prominent one among the various toxicities of ochratoxin A (OTA). MicroRNAs (miRNAs) are small non-coding RNAs that have an impact on a wide range of biological processes by regulating gene expression at post-transcriptional level or protein systhesis level. The objective of this study is to analyze miRNA profiling in the kidneys of rats gavaged with OTA. RESULTS: To profile miRNAs in the kidneys of rats with OTA nephrotoxicity, high-throughput sequencing and bioinformatics approaches were applied to analyze the miRNAs in the kidney of rats following OTA treatment. A total of 409 known miRNAs and 8 novel miRNAs were identified in the kidney and the levels of the novel miRNAs were varied in response to different doses of OTA. Expression of miR-129, miR-130a, miR-130b, miR-141, miR-218b and miR-3588 were uniquely suppressed in mid dose but then elevated in high dose, with opposite expression to their target genes. The expression pattern was closely related with the "MAPK signaling pathway". Dicer1 and Drosha were significantly suppressed, indicating an impairment of miRNA biogenesis in response to OTA. CONCLUSIONS: The abrogation of miRNA maturation process suggests a new target of OTA toxicity. Moreover, the identification of the differentially expressed miRNAs provides us a molecular insight into the nephrtoxicity of OTA.

Research status and hotspots on the mechanisms of liver X receptor in cancer progression: A bibliometric analysis.

BACKGROUND: The mechanism of liver X receptor in cancer has been gradually revealed in recent years. This study is committed to analyzing the current research status of the mechanism of liver × receptor in cancer progression by using bibliometric methods and to explore the development trend of liver × receptor related research in the future, in order to provide some reference for further exploration in this field. METHODS: The Web of Science core collection database was used to carry out the original data retrieval. Excel software was used for data statistics. Vosviewer and CiteSpace software were used to analyze the publication situation, cooperation network, reference co-citation, keyword and term co-occurrence, term bursts, and cluster analysis, and draw visual maps. RESULTS: A total of 631 publications meeting the research criteria were included by December 2022, with an average of 32.5 citations per paper. The main research fields were molecular biology, oncology and cell biology, and the papers were mainly published in journals about molecular, biology and immunology. Cell is the journal with the highest citation. The United States is the most influential country, the University of California, Los Angeles is the main research institution, and Gustafsson, Jan-ake is the author with the highest output. In reference co-citation clustering, cluster#2 "cancer development" is the main cluster, and the period from 2014 to 2018 is an important stage of relevant theoretical progress. "Tumor microenvironment" with high burst and novelty became the most noteworthy term in term burst. CONCLUSION: Using bibliometric methods to reveal the current status of LXR and cancer mechanisms, and making predictions of possible future hotspots based on the analysis of the current situation, the translation of LXR anti-cancer research to clinical applications, the impact on the tumor microenvironment as a whole and more immune pathways, and the formation of a systematic cognition of the effects of more cancer cell lines and oncogenic signaling crosstalk, which is a possible direction for future research.

High-throughput visualization mutation screening technology to enhance the specificity of CadR based whole-cell cadmium biosensor.

As a heavy metal pollutant, Cd2+ often enters the human body through the food chain causing great harm to human health. Whole-cell biosensor is an emerging technology for rapid on-site detection of heavy metals with the advantages of inexpensive, fast to mass-produce, and strong in anti-interference resistance, but suffering from insatisfactory specificity. In this study, a strategy of Adjacent Site Saturation Mutation (ASSM) was designed to improve the specificity of transcription factor CadR, which acted as the recognition element and determined the specificity of whole cell Cd2+ biosensors. A specific saturated library was constructed using the strategy of adjacent mutation. After two rounds of high-throughput visual screening, a whole-cell biosensor with good response to Cd2+, and with significant weakened Hg2+ interference was obtained. The optimized whole-cell biosensor showed a linear dynamic concentration range from 500 nM to 100 µM, a detection limit of 0.079 µM, and has satisfactory specificity and anti-interference. The ASSM strategy proposed in this study can provide a new method for the application of synthetic biology in food safety detection, indicating the importance of whole-cell biosensors for the detection of heavy metals.

Replacing manual operation with bio-automation: A high-throughput evolution strategy to construct an integrated whole-cell biosensor for the simultaneous detection of methylmercury and mercury ions without manual sample digestion.

Methylmercury is primarily responsible for most food mercury pollution cases. However, most biosensors developed for mercury pollution analysis can only detect mercury ions. Although oxidative strong-acid digestion or microwave-assisted digestion can convert methylmercury into mercury ions, it is unsuitable for on-site detection. This study designed a bio-digestion gene circuit and integrated it into a mercury ion whole-cell biosensor，creating a novel on-site methylmercury detection method. Five alkyl mercury lyases from different bacterial genomes were screened via bioinformatics analysis, of which goMerB from Gordonia otitis showed the highest catalytic biological digestion efficiency. The goMerB site-specific saturation and random mutation libraries were constructed. After two rounds of high-throughput visualization screening, the catalytic activity of the mutant increased 2.5-fold. The distance between the three crucial amino acid sites and methylmercury changed in the mutant, which likely contributed to the enhanced catalytic efficiency. The optimized whole-cell biosensor showed a linear dynamic concentration range of 100 nM to 100 µM (R2 =0.991), satisfactory specificity, and interference resistance. The detection limit of the goMerBt6-MerR-RFP biosensor was 0.015 µM, while the limit of quantitation was 0.049 µM. This study demonstrated the application of synthetic biology for food safety detection and highlighted the future potential of "Lab in a Cell" for hazard analysis.

Synthetic symbiotic bacteria reduces the toxicity of mercury ingested via contaminated food.

Mercury contamination in food poses a significant threat to human health. In this article, we propose a novel approach to solve this problem by enhancing the function of gut microbiota against mercury using a synthetically engineered bacterial strain. An engineered Escherichia coli biosensor MerR with mercury binding function was introduced into the intestines of mice for colonization, whereafter the mice were challenged with oral mercury. Compared with the control mice and mice colonized with unengineered Escherichia coli, the mice with biosensor MerR cells in their gut showed significantly stronger mercury resistance. Furthermore, mercury distribution analysis revealed that biosensor MerR cells promoted the excretion of oral mercury with feces, thereby blocking the entry of mercury into the mice, decreasing the concentration of mercury in the circulatory system and organs, and, thus, attenuating the toxicity of mercury to the liver, kidneys and intestines. Colonization with the biosensor MerR did not result in significant health problems in the mice, nor were genetic circuit mutations or lateral transfers identified during the experiments, thus demonstrating the safety of this approach. This study elucidates the remarkable promise of synthetic biology for modulating gut microbiota function.

Pleurotus Ostreatus Ameliorates Obesity by Modulating the Gut Microbiota in Obese Mice Induced by High-Fat Diet.

Pleurotus ostreatus (PO), a common edible mushroom, contains rich nutritional components with medicinal properties. To explore the effect of PO on ameliorating obesity and modulating the gut microbiota, we administered the mice with a low-fat diet or high-fat diet containing different dosages of PO (mass fraction: 0%, 2.5%, 5% and 10%). The body weight, adipose tissue weight, GTT, ITT, blood lipids, serum biomarkers of liver/kidney function, the gut microbiota and function were measured and analyzed after 6 weeks of PO treatment. The results showed PO prevented obesity, maintained glucose homeostasis and beneficially modulated gut microbiota. PO modified the composition and functions of gut microbiota in obese mice and make them similar to those in lean mice, which contributed to weight loss. PO significantly increased the relative abundance of Oscillospira, Lactobacillus group and Bifidobacterium, while decreased the relative abundance of Bacteroides and Roseburia. The prediction of gut microbiota function showed PO upregulated lipid metabolism, carbohydrate metabolism, bile acid biosynthesis, while it downregulated adipocytokine signaling pathway and steroid hormone biosynthesis. Correlation analysis further suggested the potential relationship among obesity, gut microbiota and the function of gut microbiota. In conclusion, all the results indicated that PO ameliorated obesity at least partly by modulating the gut microbiota.

Serum metalloproteinases MMP-2, MMP-9, and metalloproteinase tissue inhibitors in patients are associated with arteriovenous fistula maturation.

OBJECTIVE: Many vascular surgeons construct arteriovenous fistulas (AVFs) for hemodialysis access as the primary choice access. A significant number of AVFs fail to mature, however, leading to patient frustration and repeated operations. Metalloproteinase (MMP) activity, particularly MMP-2 and MMP-9, may be important for AVF maturation. We therefore sought to identify whether serum MMP levels could serve as a biomarker for predicting future successful AVF maturation. METHODS: Blood was collected from patients with chronic renal insufficiency at the time of surgery for long-term hemodialysis access. Serum was separated from whole blood and ultracentrifuged at 1000g for 10 minutes. Serum aliquots were frozen at -80°C until used for analysis. Enzyme-linked immunosorbent assay was used to assay levels of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase type 2 (TIMP-2), and TIMP type 4 (TIMP-4). Clinical end points were used to divide patients into failed and matured AVF groups. Successful maturation was considered in patients who had specific duplex findings or 1 month of successful two-needle cannulation hemodialysis. MMP/TIMP ratios were calculated as an index of the MMP axis activity because MMP activity parallels alterations in TIMP levels. RESULTS: Of 20 enrolled patients, AVF maturation was successful in 13 and failed in 7. Serum levels of MMP-2/TIMP-2 were significantly higher in patients with matured AVFs vs levels in those that failed (P = .003). Similarly, a trend toward increased serum levels of MMP-9/TIMP-4 was found in patients with successful AVF (P = .06). CONCLUSIONS: MMP-2 and TIMP-2 levels were different among patients whose AVF matured vs those who did not. Further follow-up studies to determine the predictability of AVF maturation using relative patient serum levels of MMP-2 and TIMP-2 should be performed.

Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism.

OBJECTIVES: The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen gamma chain (gammaC) on microvascular endothelial permeability and to examine its molecular mechanism of action. METHODS AND RESULTS: Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that gammaC induced time- and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated gammaC-induced barrier dysfunction. Imaging analyses demonstrated binding of gammaC to endothelial cells; the interaction was inhibited during blockage of the alphavbeta3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to gammaC was attenuated in integrin beta3(-/-) animals. CONCLUSION: Fibrinogen-gamma C terminus directly interacts with the microvascular endothelium causing fluid and protein leak. The endothelial response to gammaC involves an integrin receptor-mediated RhoA-dependent signaling pathway that leads to paracellular hyperpermeability.

Third Generation Whole-Cell Sensing Systems: Synthetic Biology Inside, Nanomaterial Outside.

Whole-cell sensing systems (WCSSs) are highly anticipated in the field of on-site detection. However, due to their low specificity, poor stability, and potential environmental problems, their commercial application is unrealistic. Recently, synthetic biology and nanomaterials have provided potential solutions to these problems, propelling WCSSs into a new generation. Synthetic biology provides a complete solution for the intelligent design and assembly of elements, modules, and genetic circuits. Nanomaterials covering the exterior of the cells provide stable protection, remote control capability, and catalytic ability for the WCSSs, and they can limit the horizontal transfer of genetic elements. These advancements enable personalized customization, intelligent control, and self-destruction in the next generation of cell sensors, promoting their industrialization.

Ultrasensitive magnetic DNAzyme-copper nanoclusters fluorescent biosensor with triple amplification for the visual detection of E. coli O157:H7.

A relatively-simple and high-efficient fluorescent magnetic biosensor based on DNAzyme was established for the detection of E. coli O157:H7. In order to solve the problem of weak signal and low sensitivity in the detection of foodborne pathogenic bacteria, we ingeniously designed a fluorescent sensor based on triple signal amplification of magnetic beads, DNAzyme and photoluminescence. In the detection process, the E. coli-specific RNA-cleaving DNAzyme can specifically identify the target protein in crude intracellular mixture (CIM), which caused its conformation changes and induced rolling circle amplification (RCA) to the generation and luminescence of copper nanoclusters (CuNCs). This cascade amplification design can capture weak signals in the sample. The biosensor also indicated a good linear range from 10 CFU mL-1 to 1000 CFU mL-1 and obtained a limit of detection (LOD) of 1.57 CFU mL-1, which showed a relatively high sensitivity compared with other studies. Furthermore, the biosensor displayed high-efficient detection capability in 1.5 h and good reproducibility (relative standard deviations < 2%). It has been proved that this sensor is feasible to the detection of E. coli O157:H7 in drinking water and apple juice. Moreover, we found that the final detection product can effectively wrap the magnetic beads and can be driven by the magnetic field. And this unexpected discovery will provide ideas for the development of biosensing robots.

A gas reporting whole-cell microbial biosensor system for rapid on-site detection of mercury contamination in soils.

As optical reporting elements, fluorescent proteins are extensively used in whole-cell microbial biosensors. However, the use of these optical reporters is limited in opaque media such as soil. This study described a method utilizing gas as a reporting signal that could be used for the rapid on-site detection of mercury in soil. In this biosensor, the MerR protein could capture mercury ions and then bind the promoter of the efe gene to initiate the synthesis of the ethylene (C2H4)-forming enzyme that produced the gas. The research showed that the mercury ion concentrations could be converted into C2H4 gas signals, which were quantified using a handheld C2H4 sensor. By optimizing the biosensor to improve its anti-interference ability in the system, it could detect mercury ion concentrations in the soil ranging from 0.2 to 20 mg/kg within 45 min, effectively reflecting whether the mercury pollution in the soil exceeded the limit standard. This study provides a simple, inexpensive, and portable method for the on-site detection of soil pollutants.

A test strip platform based on a whole-cell microbial biosensor for simultaneous on-site detection of total inorganic mercury pollutants in cosmetics without the need for predigestion.

Mercury pollutants such as mercuric chloride (HgCl2), mercurous chloride (Hg2Cl2) and mercuric ammonium chloride (Hg(NH2)Cl) are often found in cosmetics. Previous attempts at the on-site detection of mercury were hindered by the complicated and dangerous pretreatment procedure of converting various forms of mercury to Hg (II) ions. In this study, a test strip platform was developed based on a whole-cell microbial biosensor for the simultaneous detection of soluble and insoluble inorganic mercury pollutants in cosmetics without the need for predigestion. The genetic circuits with constitutively expressed MerR as sensor proteins and inducible red fluorescent protein (RFP) as the reporter were introduced into Escherichia coli to construct the mercury detection biosensor. The RFP fluorescence intensity of this biosensor showed a excellent linear relationship (R2 = 0.9848) with the Hg (II) concentration ranging from 50 nM to 10 µM in Luria-Bertani (LB) broth. Further research indicated that this biosensor could respond not only to Hg (II) ions but also to insoluble Hg2Cl2 and Hg2Cl2. The transcriptomic results confirmed the mercury conversion ability of the whole-cell biosensor from a gene expression perspective. This biosensor was embedded on filter paper to form a test strip, which could be used to determine whether the total inorganic mercury pollutants in cosmetics exceeded 1 mg/kg. Therefore, this strip provided a low cost, easy-to-use, and instrument-independent method for the detection of mercury pollution in cosmetics, while this study revealed the unique advantages of microbial biosensors in the automatic bioconversion of targets.

Using the promoters of MerR family proteins as "rheostats" to engineer whole-cell heavy metal biosensors with adjustable sensitivity.

BACKGROUND: Whole cell biosensors provide a simple method for the detection of heavy metals. However, previous designs of them rely primarily on simulation of heavy metal resistance systems of bacteria. RESULTS: This study proposes a strategy for the rational design of metal detection circuits based on sensor proteins of the MerR family. Our results indicate the expression level of sensor protein can be used as a "rheostat" for tuning detection sensitivity with parabola curves to represent the relationships between the detection slopes and the sensor protein levels. This circuits design strategy (named as "Parabola Principle"), is used as a guide for the discovery of optimum metal detection circuits, and the design of biosensors with specific metal detection characteristics. For example, visible qualitative Hg (II) biosensors with a threshold of 0.05 mg/L are successfully constructed. CONCLUSIONS: These results indicate the feasibility of developing a sensor that is much more tunable than what is presented.