Synthetic auxotrophy in which cell viability depends on the presence of an unnatural amino acid (unAA) provides a powerful strategy to restrict unwanted propagation of genetically modified organisms (GMOs) in open environments and potentially prevent industrial espionage. Here, we describe a generic approach for robust biocontainment of budding yeast dependent on unAA. By understanding escape mechanisms, we specifically optimize our strategies by introducing designed \"immunity\" to the generation of amber-suppressor tRNAs and developing the transcriptional- and translational-based biocontainment switch. We further develop a fitness-oriented screening method to easily obtain multiplex safeguard strains that exhibit robust growth and undetectable escape frequency (<~10-9) on solid media for 14 days. Finally, we show that employing our multiplex safeguard system could restrict the proliferation of strains of interest in a real fermentation scenario, highlighting the great potential of our yeast biocontainment strategy to protect the industrial proprietary strains.

This study examines Chinese civil servants\' attitudes toward genetically modified organisms by reviewing a national survey of 3,018 Chinese civil servants. The findings show that Chinese civil servants hold a more positive attitude to GMOs than the wider Chinese \"general public\", with a similar level of genetic scientific literacy and belief in GMOs conspiracy theories and their influence mechanisms. While the Chinese civil servants\' occupational literacy plays an important role in their GMOs attitude. This study provides a new mind-set for studying some specific groups\' attitudes toward GMOs and related food policies.

Genetically modified organisms (GMOs) can be generated to model human genetic disease or plant disease resistance, and they have contributed to the exploration and understanding of gene function, physiology, disease onset and drug target discovery. Here, PertOrg (http://www.inbirg.com/pertorg/) was introduced to provide multilevel alterations in GMOs. Raw data of 58 707 transcriptome profiles and associated information, such as phenotypic alterations, were collected and curated from studies involving in vivo genetic perturbation (e.g. knockdown, knockout and overexpression) in eight model organisms, including mouse, rat and zebrafish. The transcriptome profiles from before and after perturbation were organized into 10 116 comparison datasets, including 122 single-cell RNA-seq datasets. The raw data were checked and analysed using widely accepted and standardized pipelines to identify differentially expressed genes (DEGs) in perturbed organisms. As a result, 8 644 148 DEGs were identified and deposited as signatures of gene perturbations. Downstream functional enrichment analysis, cell type analysis and phenotypic alterations were also provided when available. Multiple search methods and analytical tools were created and implemented. Furthermore, case studies were presented to demonstrate how users can utilize the database. PertOrg 1.0 will be a valuable resource aiding in the exploration of gene functions, biological processes and disease models.

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 ∘C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH4)2SO4. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.

BACKGROUND: With the commercialization of genetically modified organisms (GMOs) in the market, laboratories have undergone a significantly increased workload. A universal analytical approach was designed to achieve cost-efficient and high-throughput GMOs screening with high specificity and accuracy. The approach provides accurate qualification of authorized and unauthorized GMOs.
OBJECTIVE: This article describes the assessment of this analytical approach developed to detect the majority of commercialized GMOs over the world.
METHODS: Seven elements and three events were detected by qPCR in a single laboratory to detect 59 commercialized GMOs. Certificated reference materials and food/feed samples from the Chinese market were also evaluated for the specificity, conformity, and robustness of this approach and were challenged in the interlaboratory study.
RESULTS: The results showed that elements and events selected can best detect GMO presence with good specificity and sensitivity. The results showed a concordance between 97.5 and 99.56% and the variance between 0.65 and 12.88%, which is in line with the minimum requirement of analytical methods of GMO testing.
CONCLUSIONS: The approach validated here can be used to manipulate GMO presence in food and feed and showed the capacity to manipulate GMO trace in the trade and domestic agriculture market in China.
CONCLUSIONS: A universal analytical approach used to track GMO presence was evaluated for its specificity, sensitivity, and robustness.

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.

Bacterial non-coding RNAs (ncRNAs) play important regulatory roles in various physiological metabolic pathways. In this study, a novel ncRNA CsiR (ciprofloxacin stress-induced ncRNA) involved in the regulation of ciprofloxacin resistance in the foodborne multidrug-resistant Proteus vulgaris (P. vulgaris) strain P3M was identified. The survival rate of the CsiR-deficient strain was higher than that of the wild-type strain P3M under the ciprofloxacin treatment condition, indicating that CsiR played a negative regulatory role, and its target gene emrB was identified through further target prediction, quantitative real-time PCR (qRT-PCR), and microscale thermophoresis (MST). Further studies showed that the interaction between CsiR and emrB mRNA affected the stability of the latter at the post-transcriptional level to a large degree, and ultimately affected the ciprofloxacin resistance of P3M. Notably, the base-pairing sites between CsiR and emrB mRNAs were highly conserved in other sequenced P. vulgaris strains, suggesting that this regulatory mechanism may be ubiquitous in this species. To the best of our knowledge, this is the first identification of a novel ncRNA involved in the regulation of ciprofloxacin resistance in P. vulgaris species, which lays a solid foundation for comprehensively expounding the antibiotic resistance mechanism of P. vulgaris.

BACKGROUND: Mosquito-borne diseases affect over half of the human population globally. Multiple studies have shown that chemical insecticides are ineffective because of resistance. Therefore, environmentally safe mosquito population control tools need to be developed. Ribonucleic acid interference (RNAi) is a reverse genetic mechanism recently introduced as a new pest control tool. This technique represents a new class of biorational technology that could combat the increased global incidence of insecticide resistance. The technique has the potential of becoming a critical component of integrated vector control programs.
METHODS: A 3-hydroxykynurenine transaminase (3-HKT) RNAi expression plasmid was constructed, generated and transformed into Chlamydomonas and Chlorella algae. The transgenic algae were then used to feed Ae. aegypti mosquito larvae. The feeding experiments were conducted on a small and large scale with 10 and about 300 larvae, respectively. The mortality rate of the larvae was calculated over 30 days. In addition, histological examination of the insect tissues was performed to examine the extent of tissue damage.
RESULTS: The integumentary system and midguts of larvae fed with transgenic Chlamydomonas were severely damaged. The mortality rate of the larvae fed with transgenic Chlamydomonas ranged from 60 to 100% in small-scale tests. The survival rate of adult mosquitoes was 0.0% in a large-scale feeding experiment when the larvae were fed with transgenic Chlamydomonas. Moreover, when the larvae were fed with transgenic Chlorella, the mortality rate ranged from 6.7% to 43% compared to that fed wild-type Chlorella.
CONCLUSIONS: 3HKT RNAi transgenic algae are in some scales lethal to Ae. aegypti. The findings of this study indicate that technology based on microalgae RNAi may provide a new way to control mosquito populations.

Herein, a surface-enhanced Raman scattering (SERS)-integrated LFS platform was developed for rapid and simultaneous screening of multiple genetically modified organism (GMO) components (promoter, codon, and terminator) in soybean. Research demonstrated that, on the same test line (T line) of single LFS, three different GMP components can be well distinguished with the help of three SERS nano tags. Good linear correlations between SERS signal and concentration of each GMO component were also obtained for quantitative analysis. Of greater importance, whether these multiple analytes coexisted or not, varied in the same concentration trend or not, these multiple GMP components can be rapidly (15 min) and accurately screened with satisfied sensitivity and specificity by decoding the signals on the same T line. We envision that this decoding platform can further improve the potential of LFS and SERS for practical applications and provide a promising alternative for multiple screening of GMO identification in food.