Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff.

Currently, feed enzymes are primarily obtained through fermentation of fungi, bacteria, and other microorganisms. Although the manufacturing technology for feed enzymes has evolved rapidly, the activities of these enzymes decline during the granulating process and the cost of application has increased over time. An alternative approach is the use of genetically modified plants containing complex feed enzymes for direct utilization in animal feedstuff. We co-expressed three commonly used feed enzymes (phytase, ß-glucanase, and xylanase) in barley seeds using the Agrobacterium-mediated transformation method and generated a new barley germplasm. The results showed that these enzymes were stable and had no effect on the development of the seeds. Supplementation of the basal diet of laying hens with only 8% of enzyme-containing seeds decreased the quantities of indigestible carbohydrates, improved the availability of phosphorus, and reduced the impact of animal production on the environment to an extent similar to directly adding exogenous enzymes to the feed. Feeding enzyme-containing seeds to layers significantly increased the strength of the eggshell and the weight of the eggs by 10.0%-11.3% and 5.6%-7.7% respectively. The intestinal microbiota obtained from layers fed with enzyme-containing seeds was altered compared to controls and was dominated by Alispes and Rikenella. Therefore, the transgenic barley seeds produced in this study can be used as an ideal feedstuff for use in animal feed.

Metabolic engineering of Escherichia coli for 2,4-dinitrotoluene degradation.

2,4-Dinitrotoluene (2,4-DNT) as a common industrial waste has been massively discharged into the environment with industrial wastewater. Due to its refractory degradation, high toxicity, and bioaccumulation, 2,4-DNT pollution has become increasingly serious. Compared with the currently available physical and chemical methods, in situ bioremediation is considered as an economical and environmentally friendly approach to remove toxic compounds from contaminated environment. In this study, we relocated a complete degradation pathway of 2,4-DNT into Escherichia coli to degrade 2,4-DNT completely. Eight genes from Burkholderia sp. strain were re-synthesized by PCR-based two-step DNA synthesis method and introduced into E. coli. Degradation experiments revealed that the transformant was able to degrade 2,4-DNT completely in 12 h when the 2,4-DNT concentration reached 3 mM. The organic acids in the tricarboxylic acid cycle were detected to prove the degradation of 2,4-DNT through the artificial degradation pathway. The results proved that 2,4-DNT could be completely degraded by the engineered bacteria. In this study, the complete degradation pathway of 2,4-DNT was constructed in E. coli for the first time using synthetic biology techniques. This research provides theoretical and experimental bases for the actual treatment of 2,4-DNT, and lays a technical foundation for the bioremediation of organic pollutants.

Construction of complete degradation pathway for nitrobenzene in Escherichia coli.

Nitrobenzene is widely present in industrial wastewater and soil. Biodegradation has become an ideal method to remediate organic pollutants due to its low cost, high efficiency, and absence of secondary pollution. In the present study, 10 exogenous genes that can completely degrade nitrobenzene were introduced into Escherichia coli, and their successful expression in the strain was verified by fluorescence quantitative polymerase chain reaction and proteomic analysis. The results of the degradation experiment showed that the engineered strain could completely degrade 4 mM nitrobenzene within 8 h. The formation of intermediate metabolites was detected, and the final metabolites entered the E. coli tricarboxylic acid cycle smoothly. This process was discovered by isotope tracing method. Results indicated the integrality of the degradation pathway and the complete degradation of nitrobenzene. Finally, further experiments were conducted in soil to verify its degradation ability and showed that the engineered strain could also degrade 1 mM nitrobenzene within 10 h. In this study, engineered bacteria that can completely degrade nitrobenzene have been constructed successfully. The construction of remediation-engineered bacteria by synthetic biology laid the foundation for the industrial application of biological degradation of organic pollutants.

Enhanced phytoremediation of TNT and cobalt co-contaminated soil by AfSSB transformed plant.

2,4,6-trinitrotoluene (TNT) and cobalt (Co) contaminants have posed a severe environmental problem in many countries. Phytoremediation is an environmentally friendly technology for the remediation of these contaminants. However, the toxicity of TNT and cobalt limit the efficacy of phytoremediation application. The present research showed that expressing the Acidithiobacillus ferrooxidans single-strand DNA-binding protein gene (AfSSB) can improve the tolerance of Arabidopsis and tall fescue to TNT and cobalt. Compared to control plants, the AfSSB transformed Arabidopsis and tall fescue exhibited enhanced phytoremediation of TNT and cobalt separately contaminated soil and co-contaminated soil. The comet analysis revealed that the AfSSB transformed Arabidopsis suffer reduced DNA damage than control plants under TNT or cobalt exposure. In addition, the proteomic analysis revealed that AfSSB improves TNT and cobalt tolerance by strengthening the reactive superoxide (ROS) scavenging system and the detoxification system. Results presented here serve as strong theoretical support for the phytoremediation potential of organic and metal pollutants mediated by single-strand DNA-binding protein genes. SUMMARIZES: This is the first report that AfSSB enhances phytoremediation of 2,4,6-trinitrotoluene and cobalt separately contaminated and co-contaminated soil.

Metabolic engineering of rice endosperm for betanin biosynthesis.

Betanin has been widely used as an additive for many centuries, and its use has increased because of its market application as an additive, high free radical scavenging activity, and safety, health-promoting properties. The main source of betanin is red beet, but many factors notably affect the yield of betanin from red beets. Betanin is not produced in cereal grains. Thus, developing biofortified crops with betanin is another alternative to health-promoting food additives. Here, rice endosperm was bioengineered for betanin biosynthesis by introducing three synthetic genes (meloS, BvDODA1S, and BvCYP76AD1S). The overexpression of these genes driven by rice endosperm-specific promoter established the betanin biosynthetic pathways in the endosperm, resulting in new types of germplasm - 'Betanin Rice' (BR). The BR grains were enriched with betanin and had relatively high antioxidant activity. Our results proved that betanin can be biosynthesized de novo in rice endosperm by introducing three genes in the committed betanin biosynthetic pathway. The betanin-fortified rice in this study can be used as a functional grain to promote health and as a raw material to process dietary supplements.

Enhancement of naphthalene tolerance in transgenic Arabidopsis plants overexpressing the ferredoxin-like protein (ADI1) from rice.

KEY MESSAGE: The ADI1 Arabidopsis plants enhanced tolerance and degradation efficiency to naphthalene and had great potential for phytoremediation of naphthalene in the plant material before composting or harvesting and removal. Naphthalene is a global environmental concern, because this substance is assumed to contribute considerably to human cancer risk. Cleaning up naphthalene contamination in the environment is crucial. Phytoremediation is an efficient technology to clean up contaminants. However, no gene that can efficiently degrade exogenous recalcitrant naphthalene in plants has yet been discovered. Ferredoxin (Fd) is a key player of biological electron transfer reaction in the PAH degradation process. The biochemical pathway for bacterial degradation of naphthalene has been well investigated. In this study, a rice gene, ADI1, which codes for a putative photosynthetic-type Fd, has been transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants enhanced tolerance and degradation efficiency of naphthalene. Compared with wild-type plants, transgenic plants assimilated naphthalene from the culture media faster and removed more of this substance. When taken together, our findings suggest that breeding plants with overexpressed ADI1 gene is an effective strategy to degrade naphthalene in the environment.

Phytoremediation of phenanthrene by transgenic plants transformed with a naphthalene dioxygenase system from Pseudomonas.

Genes from microbes for degrading polycyclic aromatic hydrocarbons (PAHs) are seldom used to improve the ability of plants to remediate the pollution because the initiation of the microbial degradation of PAHs is catalyzed by a multienzyme system. In this study, for the first time, we have successfully transferred the complex naphthalene dioxygenase system of Pseudomonas into Arabidopsis and rice, the model dicot and monocot plant. As in bacteria, all four genes of the naphthalene dioxygenase system can be simultaneously expressed and assembled to an active enzyme in transgenic plants. The naphthalene dioxygenase system can develop the capacity of plants to tolerate a high concentration of phenanthrene and metabolize phenanthrene in vivo. As a result, transgenic plants showed improved uptake of phenanthrene from the environment over wild-type plants. In addition, phenanthrene concentrations in shoots and roots of transgenic plants were generally lower than that of wild type plants. Transgenic plants with a naphthalene dioxygenase system bring the promise of an efficient and environmental-friendly technology for cleaning up PAHs contaminated soil and water.

Gene expression profiles of Arabidopsis under the stress of methyl viologen: a microarray analysis.

Methyl viologen (MV) is the main ingredient of Paraquat. It is little known about how plants respond to this compound. To understand the mode of MV action and molecular mechanism of plant response, we performed experiments of microarray on Arabidopsis. In MV treated seedling, approximately 6% genes were altered at mRNA levels, including 818 genes increased, whereas 1,440 genes decreased. Studies of these genes expression patterns provided some new information on the reaction process of plant after the treatment with MV. These included signaling molecules for MV response and reactive oxygen species formation, enzymes required for secondary metabolism and, cell wall maintenance and strategy of photostasis balance. The expression kinetics of the genes induced by MV will provides useful information for the abiotic stress defense mechanism in plants.

Expression, purification, and characterization of recombinant mangrove glutamine synthetase.

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg(2+)-dependent biosynthetic activity was strongly inhibited by Ni(2+), Zn(2+), and Al(3+), whereas was enhanced by Co(2+). The apparent K m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH4 (+) respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.

Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.

A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.

Complete biodegradation of the oldest organic herbicide 2,4-Dichlorophenoxyacetic acid by engineering Escherichia coli.

After nearly 80 years of extensive application, the oldest organic herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has caused many problems of environmental pollution and ecological deterioration. Bioremediation is an ideal method for pollutant treatment. However, difficult screening and preparation of efficient degradation bacteria have largely hindered its application in 2,4-D remediation. We have created a novel engineering Escherichia coli with a reconstructed complete degradation pathway of 2,4-D to solve the problem of screening highly efficient degradation bacteria in this study. The results of fluorescence quantitative PCR demonstrated that all nine genes in the degradation pathway were successfully expressed in the engineered strain. The engineered strains can quickly and completely degrade 0.5 mM 2, 4-D within 6 h. Inspiring, the engineered strains grew with 2,4-D as the sole carbon source. By using the isotope tracing method, the metabolites of 2,4-D were found incorporated into the tricarboxylic acid cycle in the engineering strain. Scanning electron microscopy showed that 2,4-D had less damage on the engineered bacteria than the wild-type strain. Engineered strain can also rapidly and completely remedy 2,4-D pollution in natural water and soil. Assembling the metabolic pathways of pollutants through synthetic biology was an effective method to create pollutant-degrading bacteria for bioremediation.

Creation of Environmentally Friendly Super "Dinitrotoluene Scavenger" Plants.

Pervasive environmental contamination due to the uncontrolled dispersal of 2,4-dinitrotoluene (2,4-DNT) represents a substantial global health risk, demanding urgent intervention for the removal of this detrimental compound from affected sites and the promotion of ecological restoration. Conventional methodologies, however, are energy-intensive, susceptible to secondary pollution, and may inadvertently increase carbon emissions. In this study, a 2,4-DNT degradation module is designed, assembled, and validated in rice plants. Consequently, the modified rice plants acquire the ability to counteract the phytotoxicity of 2,4-DNT. The most significant finding of this study is that these modified rice plants can completely degrade 2,4-DNT into innocuous substances and subsequently introduce them into the tricarboxylic acid cycle. Further, research reveals that the modified rice plants enable the rapid phytoremediation of 2,4-DNT-contaminated soil. This innovative, eco-friendly phytoremediation approach for dinitrotoluene-contaminated soil and water demonstrates significant potential across diverse regions, substantially contributing to carbon neutrality and sustainable development objectives by repurposing carbon and energy from organic contaminants.

Stress responses to phenol in Arabidopsis and transcriptional changes revealed by microarray analysis.

Phenols are toxic, environmentally persistent products of the chemical industry that are capable of bioaccumulation and biomagnifications in the food chain. Little is known of how plants respond to this compound. To understand the transcriptional changes under phenol, microarray experiments on Arabidopsis thaliana were performed. Microarray results revealed numerous perturbations in signaling and metabolic pathways. The results indicated that the phenol response was related to reactive oxygen species (ROS) accumulation and oxidative conditions, including ROS generated for pathogen defense.

Phytoremediation of trichlorophenol by Phase II metabolism in transgenic Arabidopsis overexpressing a Populus glucosyltransferase.

Trichlorophenol (TCP) and its derivatives are introduced into the environment through numerous sources, including wood preservatives and biocides. Environmental contamination by TCPs is associated with human health risks, necessitating the development of cost-effective remediation techniques. Efficient phytoremediation of TCP is potentially feasible because it contains a hydroxyl group and is suitable for direct phase II metabolism. In this study, we present a system for TCP phytoremediation based on sugar conjugation by overexpressing a Populus putative UDP-glc-dependent glycosyltransferase (UGT). The enzyme PtUGT72B1 displayed the highest TCP-conjugating activity among all reported UGTs. Transgenic Arabidopsis demonstrated significantly enhanced tolerances to 2,4,5-TCP and 2,4,6-TCP. Transgenic plants also exhibited a strikingly higher capacity to remove TCP from their media. This work indicates that Populus UGT overexpression in Arabidopsis may be an efficient method for phytoremoval and degradation of TCP. Our findings have the potential to provide a suitable remediation strategy for sites contaminated by TCP.

Phytoremediation and phytosensing of chemical contaminant, toluene: identification of the required target genes.

As an industrial chemical produced worldwide in high volumes, toluene is commonly detected in ambient air and water. It can combine with oxygen and form compounds that are harmful to humans. In recent years, phytoremediation has been increasingly applied to repair the environmental damage caused by pollutants. However, insufficient knowledge is available regarding the response of plants to toluene. To detect the potential genes in plants that are related to the sensing mechanism and metabolism of toluene, a microarray analysis has been conducted on Arabidopsis thaliana seedlings grown on toluene-containing media. Following the validation of data and the application of appropriate selection criteria, the results show a coordinated induction and suppression of 202 and 67 toluene-responsive genes, respectively. Within the functional class "metabolism", the genes encoding detoxification proteins represent the most strongly up-regulated group. These include genes encoding cytochrome P450s, glucosyl transferases, and transporters. Subsequently, the toluene-induced genes of Arabidopsis are analyzed in detail.

Analysis of gene expression profile of Arabidopsis genes under trichloroethylene stresses with the use of a full-length cDNA microarray.

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Plants are able to take up a range of harmful organic compounds, including some of the most abundant environmental pollutants like TCE. In this study, complementary DNA microarrays were constructed to have a better view of transcript expression in Arabidopsis thaliana during TCE-induced stress. The microarray analysis demonstrated the complexity of gene expression patterns resulting from TCE. A total of 1,020 transcripts were differentially up-regulated by TCE. Those genes might specifically contribute to the TCE transformation, conjugation, and compartmentation in plant. This study provides informative preliminary data for more in-depth analyses of TCE tolerance in Arabidopsis thaliana.

Metabolic engineering of Escherichia coli for direct production of vitamin C from D-glucose.

BACKGROUND: Production of vitamin C has been traditionally based on the Reichstein process and the two-step process. However, the two processes share a common disadvantage: vitamin C cannot be directly synthesized from D-glucose. Therefore, significant effort has been made to develop a one-step vitamin C fermentation process. While, 2-KLG, not vitamin C, is synthesized from nearly all current one-step fermentation processes. Vitamin C is naturally synthesized from glucose in Arabidopsis thaliana via a ten-step reaction pathway that is encoded by ten genes. The main objective of this study was to directly produce vitamin C from D-glucose in Escherichia coli by expression of the genes from the A. thaliana vitamin C biosynthetic pathway. RESULTS: Therefore, the ten genes of whole vitamin C synthesis pathway of A. thaliana were chemically synthesized, and an engineered strain harboring these genes was constructed in this study. The direct production of vitamin C from D-glucose based on one-step fermentation was achieved using this engineered strain and at least 1.53 mg/L vitamin C was produced in shaking flasks. CONCLUSIONS: The study demonstrates the feasibility of one-step fermentation for the production of vitamin C from D-glucose. Importantly, the one-step process has significant advantages compared with the currently used fermentation process: it can save multiple physical and chemical steps needed to convert D-glucose to D-sorbitol; it also does not involve the associated down-streaming steps required to convert 2-KLG into vitamin C.

Metabolic engineering of Oryza sativa for complete biodegradation of thiocyanate.

Industrial thiocyanate (SCN-) waste streams from gold mining and coal coking have caused serious environmental pollution worldwide. Phytoremediation is an efficient technology in treating hazardous wastes from the environment. However, the phytoremediation efficiency of thiocyanate is very low due to the fact that plants lack thiocyanate degradation enzymes. In this study, the thiocyanate hydrolase module was assembled correctly in rice seedlings and showed thiocyanate hydrolase activity. Rice seedlings engineered to express thiocyanate degrading activity were able to completely remove thiocyanate from coking wastewater. Our findings suggest that transforming the thiocyanate hydrolase module into plants is an efficient strategy for rapid phytoremediation of thiocyanate in the environment. Moreover, the rice seedlings expressing apoplastic or cytoplasmic targeted thiocyanate hydrolase module were constructed to compare the phytoremediation efficiency of secretory/intracellular recombinant thiocyanate hydrolase. The most obvious finding from this study is that the apoplastic expression system is more efficient than the cytoplasm expression system in the phytoremediation of thiocyanate. At last, this research also shows that the secreted thiocyanate hydrolase from engineered rice plants does not influence rhizosphere bacterial community composition.

Screening and predicting progression from high-risk mild cognitive impairment to Alzheimer's disease.

Individuals with mild cognitive impairment (MCI) are clinically heterogeneous, with different risks of progression to Alzheimer's disease. Regular follow-up and examination may be time-consuming and costly, especially for MRI and PET. Therefore, it is necessary to identify a more precise MRI population. In this study, a two-stage screening frame was proposed for evaluating the predictive utility of additional MRI measurements among high-risk MCI subjects. In the first stage, the K-means cluster was performed for trajectory-template based on two clinical assessments. In the second stage, high-risk individuals were filtered out and imputed into prognosis models with varying strategies. As a result, the ADAS-13 was more sensitive for filtering out high-risk individuals among patients with MCI. The optimal model included a change rate of clinical assessments and three neuroimaging measurements and was significantly associated with a net reclassification improvement (NRI) of 0.246 (95% CI 0.021, 0.848) and integrated discrimination improvement (IDI) of 0.090 (95% CI - 0.062, 0.170). The ADAS-13 longitudinal models had the best discrimination performance (Optimism-corrected concordance index = 0.830), as validated by the bootstrap method. Considering the limited medical and financial resources, our findings recommend follow-up MRI examination 1 year after identification for high-risk individuals, while regular clinical assessments for low-risk individuals.