Ectopic expression of an Old Yellow Enzyme (OYE3) gene from Saccharomyces cerevisiae increases the tolerance and phytoremediation of 2-nitroaniline in rice.

2-nitroaniline (2-NA) is an environmental pollutant and has been extensively used as intermediates in organic synthesis. The presence of 2-NA in the environment is not only harmful for aquatic life but also mutagenic for human beings. In this study, we constructed transgenic rice expressing an Old Yellow Enzyme gene, ScOYE3, from Saccharomyces cerevisiae. The ScOYE3 transgenic plants were comprehensively investigated for their biochemical responses to 2-NA treatment and their 2-NA phytoremediation capabilities. Our results showed that the rice seedlings exposed to 2-NA stress, showed growth inhibition and biomass reduction. However, the transgenic plants exhibited strong tolerance to 2-NA stress compared to wild-type plants. Ectopic expression of ScOYE3 could effectively protect transgenic plants against 2-NA damage, which resulted in less reactive oxygen species accumulation in transgenic plants than that in wild-type plants. Our phytoremediation assay revealed that transgenic plants could eliminate more 2-NA from the medium than wild-type plants. Moreover, omics analysis was performed in order to get a deeper insight into the mechanism of ScOYE3-mediated 2-NA transformation in rice. Altogether, the function of ScOYE3 during 2-NA detoxification was characterized for the first time, which serves as strong theoretical support for the phytoremediation potential of 2-NA by Old Yellow Enzyme genes.

Biosynthesis of melatonin from L-tryptophan by an engineered microbial cell factory.

BACKGROUND: The demand for melatonin is increasing due to its health-promoting bioactivities such as antioxidant and sleep benefits. Although melatonin is present in various organisms, its low content and high extraction cost make it unsustainable. Biosynthesis is a promising alternative method for melatonin production. However, the ectopic production of melatonin in microorganisms is very difficult due to the low or insoluble expression of melatonin synthesis genes. Hence, we aim to explore the biosynthesis of melatonin using Escherichia coli as a cell factory and ways to simultaneously coordinated express genes from different melatonin synthesis pathways. RESULTS: In this study, the mXcP4H gene from Xanthomonas campestris, as well as the HsAADC, HsAANAT and HIOMT genes from human melatonin synthesis pathway were optimized and introduced into E. coli via a multi-monocistronic vector. The obtained strain BL7992 successfully synthesized 1.13 mg/L melatonin by utilizing L-tryptophan (L-Trp) as a substrate in a shake flask. It was determined that the rate-limiting enzyme for melatonin synthesis is the arylalkylamine N-acetyltransferase, which is encoded by the HsAANAT gene. Targeted metabolomics analysis of L-Trp revealed that the majority of L-Trp flowed to the indole pathway in BL7992, and knockout of the tnaA gene may be beneficial for increasing melatonin production. CONCLUSIONS: A metabolic engineering approach was adopted and melatonin was successfully synthesized from low-cost L-Trp in E. coli. This study provides a rapid and economical strategy for the synthesis of melatonin.

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.

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.

Metabolic engineering for the biosynthesis of bis-indolylquinone terrequinone A in Escherichia coli from L-tryptophan and prenol.

BACKGROUND: Terrequinone A is a bis-indolylquinone natural product with antitumor activity. Due to its unique asymmetric quinone core structure and multiple functional groups, biosynthesis is more efficient and environmentally friendly than traditional chemical synthesis. Currently, most bis-indolylquinones are obtained by direct extraction from fungi or by chemical synthesis. By focusing on the biosynthesis of terrequinone A, we hope to explore the way to synthesize bis-indolylquinones de novo using Escherichia coli as a cell factory. RESULTS: In this study, a terrequinone A synthesis pathway containing the tdiA-tdiE genes was constructed into Escherichia coli and activated by a phosphopantetheinyl transferase gene sfp, enabling the strain to synthesize 1.54 mg/L of terrequinone A. Subsequently, a two-step isopentenol utilization pathway was introduced to enhance the supply of endogenous dimethylallyl diphosphate (DMAPP) in E. coli, increasing the level of terrequinone A to 20.1 mg/L. By adjusting the L-tryptophan (L-Trp)/prenol ratio, the major product could be changed from ochrindole D to terrequinone A, and the content of terrequinone A reached the highest 106.3 mg/L under the optimized culture conditions. Metabolic analysis of L-Trp indicated that the conversion of large amounts of L-Trp to indole was an important factor preventing the further improvement of terrequinone A yield. CONCLUSIONS: A comprehensive approach was adopted and terrequinone A was successfully synthesized from low-cost L-Trp and prenol in E. coli. This study provides a metabolic engineering strategy for the efficient synthesis of terrequinone A and other similar bis-indolylquinones with asymmetric quinone cores. In addition, this is the first report on the de novo biosyhthesis of terrequinone A in an engineered strain.

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.

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.

ElNFS1, a nitroreductase gene from Enterobacter ludwigii, confers enhanced detoxification and phytoremediation of 4-nitrobenzaldehyde in rice.

4-nitrobenzaldehyde (4-NBA) is a widely used chemical intermediate for industrial application and an important photodegradation product of chloramphenicol. This compound represents a substantial threat to human health and ecosystem due to its genotoxic and mutagenic effect. In this study, the 4-NBA detoxification by transgenic rice overexpressing a bacterial nitroreductase gene, ElNFS1, from Enterobacter ludwigii were investigated. The cytosol-targeted ElNFS1 transgenic plants were selected to comprehensively examine their physio-biochemical responses and phytoremediation potential to 4-NBA. Our results showed that the transgenic plants exhibited strong tolerance to 4-NBA. Overexpression of ElNFS1 could significantly alleviate 4-NBA-induced damages of photosynthetic apparatus and reactive oxygen species overproduction in transgenic plants. The phytoremediation assay revealed that transgenic plants could remove more 4-NBA from the medium than wild-type plants. HPLC and LC-MS assays showed that 4-aminobenzaldehyde was found in the reductive products of 4-NBA. Altogether, the function of ElNFS1 during 4-NBA detoxification was characterized for the first time, which provides a strong theoretical support for the application potential of ElNFS1 transgenic plants on the phytoremediation of 4-NBA.

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.

Rice carotenoid biofortification and yield improvement conferred by endosperm-specific overexpression of OsGLK1.

Carotenoids, indispensable isoprenoid phytonutrients, are synthesized in plastids and are known to be deficient in rice endosperm. Many studies, involving transgenic manipulations of carotenoid biosynthetic genes, have been performed to obtain carotenoid-enriched rice grains. Nuclear-encoded GOLDEN2-LIKE (GLK) transcription factors play important roles in the regulation of plastid and thylakoid grana development. Here, we show that endosperm-specific overexpression of rice GLK1 gene (OsGLK1) leads to enhanced carotenoid production, increased grain yield, but deteriorated grain quality in rice. Subsequently, we performed the bioengineering of carotenoids biosynthesis in rice endosperm by introducing other three carotenogenic genes, tHMG1, ZmPSY1, and PaCrtI, which encode the enzymes truncated 3-hydroxy-3-methylglutaryl-CoA reductase, phytoene synthase, and phytoene desaturase, respectively. Transgenic overexpression of all four genes (OsGLK1, tHMG1, ZmPSY1, and PaCrtI) driven by rice endosperm-specific promoter GluB-1 established a mini carotenoid biosynthetic pathway in the endosperm and exerted a roughly multiplicative effect on the carotenoid accumulation as compared with the overexpression of only three genes (tHMG1, ZmPSY1, and PaCrtI). In addition, the yield enhancement and quality reduction traits were also present in the transgenic rice overexpressing the selected four genes. Our results revealed that OsGLK1 confers favorable characters in rice endosperm and could help to refine strategies for the carotenoid and other plastid-synthesized micronutrient fortification in bioengineered plants.

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.

Metabolic engineering of Escherichia coli for efficient degradation of 4-fluorophenol.

As a kind of refractory organic pollutant, 4-fluorophenol (4-FP) can be degraded by only a few microorganisms with low efficiency because of the great electron-withdrawing ability of fluorine atoms. So it is necessary to artificially construct engineered strain to improve the degradation efficiency and meet the requirements of pollutant degradation. In this study, four genes (fpdA2, fpdB, fpdC, and fpdD) for 4-FP degradation from Arthrobacter sp. strain IF1 were optimized and synthesized and then reconstructed into Escherichia coli by a multi-monocistronic vector to obtain recombinant BL-fpd that could degrade 4-FP efficiently. Under optimized induction conditions (inducing the strain by 2 g/L L-arabinose and 1 mM IPTG at 37 ℃), BL-fpd could completely degrade 2 mM 4-FP, 4-chlorophenol, 4-bromophenol, and 4-nitrophenol into ß-ketoadipate, which could be further metabolized by the bacteria. FpdA2 showed the highest activity towards 4-bromophenol. The strain could completely degrade 1 mM 4-FP in industrial wastewater within 3 h. This study provided a promising strain for the degradation of 4-FP and some other 4-substituted phenols. The construction technologies of multi-monocistronic expression vector may also be used to construct other organic pollutants degrading bacteria.

Enhanced phytoremediation of selenium using genetically engineered rice plants.

Selenium (Se) is a micronutrient essential for human and animal health. However, Se is toxic at high levels because the nonspecific substitution of cysteine by selenocysteine could lead to protein malfunction. In an attempt to prevent nonspecific selenocysteine incorporation into proteins, we simultaneously overexpressed the gene encoding selenocysteine lyase from Homo sapiens (HsSL), which specifically catalyzes the decomposition of selenocysteine into elemental Se0 and alanine, and the gene encoding selenocysteine methyltransferase from Astragalus bisulcatus (AbSMT), which methylates selenocysteine into methylselenocysteine in rice. The transgenic plants showed normal growth under standard conditions. Se treatment resulted in higher levels of alanine and methylselenocysteine in transgenic plants than in wild-type plants, which indicated that this approach might have successfully redirected Se flow in the plant. Overexpression of HsSL and AbSMT in rice also endows transgenic plants with hyposensitivity to Se stress at the seed germination stage. The transgenic plants showed enhanced selenate and selenite tolerance, which was simultaneously supported by fresh weight values. Moreover, our phytoremediation assay revealed that the transgenic plants exhibited greatly improved Se elimination capabilities and accumulated about 38.5% and 128.6% more Se than wild-type plants when treated with selenate and selenite, respectively. This study offers hope that genetically modified plants could play a role in the restoration of Se-contaminated environment.

Metabolic Engineering of Escherichia coli for Methyl Parathion Degradation.

Organophosphate compounds are widely used in pesticides to control weeds, crop diseases, and insect pests. Unfortunately, these synthetic compounds are hazardous and toxic to all types of living organisms. In the present work, Escherichia coli was bioengineered to achieve methyl parathion (MP) degradation via the introduction of six synthetic genes, namely, opdS, pnpAS, pnpBS, pnpCS, pnpDS, and pnpES, to obtain a new transformant, BL-MP. MP and its subsequent decomposition intermediates were completely degraded by this transformant to enter the metabolites of multiple anabolic pathways. The MP-degraded strain created in this study may be a promising candidate for the bioremediation of MP and potential toxic intermediates.

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.

Biodegradation of p-nitrophenol by engineered strain.

p-Nitrophenol (PNP) is an important environmental pollutant and can causes significant environmental and health risks. Compared with the traditional methods, biodegradation is a useful one to completely remove the harmful pollutants from the environment. Here, an engineered strain was first constructed by introducing PNP biodegradation pathway via the hydroquinone (HQ) pathway into Escherichia coli. In the engineered strain BL-PNP, PNP was completely degraded to ß-ketoadipate and subsequently enter the metabolites of multiple anabolic pathways. The high tolerance and rapid degradation ability to PNP enable the engineered strain to have the potential to degrade toxic substances. The engineered strain created in this study can be used as a functional strain for bioremediation of PNP and potential toxic intermediates, and the method of assembling aromatic hydrocarbons metabolic pathway can be used to eradicate nitroaromatic pollutants in the environment.

Optimization and reconstruction of two new complete degradation pathways for 3-chlorocatechol and 4-chlorocatechol in Escherichia coli.

Chlorinated aromatic compounds are a serious environmental concern because of their widespread occurrence throughout the environment. Although several microorganisms have evolved to gain the ability to degrade chlorinated aromatic compounds and use them as carbon sources, they still cannot meet the diverse needs of pollution remediation. In this study, the degradation pathways for 3-chlorocatechol (3CC) and 4-chlorocatechol (4CC) were successfully reconstructed by the optimization, synthesis, and assembly of functional genes from different strains. The addition of a 13C-labeled substrate and functional analysis of different metabolic modules confirmed that the genetically engineered strains can metabolize chlorocatechol similar to naturally degrading strains. The strain containing either of these artificial pathways can degrade catechol, 3CC, and 4CC completely, although differences in the degradation efficiency may be noted. Proteomic analysis and scanning electron microscopy observation showed that 3CC and 4CC have toxic effects on Escherichia coli, but the engineered bacteria can significantly eliminate these inhibitory effects. As core metabolic pathways for the degradation of chloroaromatics, the two chlorocatechol degradation pathways constructed in this study can be used to construct pollution remediation-engineered bacteria, and the related technologies may be applied to construct complete degradation pathways for complex organic hazardous materials.

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.