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PubMed2004

Metabolite profiling as an aid to metabolic engineering in plants.

The past decade has seen some impressive successes in the metabolic engineering of biotechnologically important plant pathways. However, plant metabolic engineering currently proceeds more by trial and error than by intelligent system design. A change in philosophy away from studying pathways in isolation and towards studying metabolism as a network is necessary. To support this development, improvements in technologies for metabolic analysis, a wider adoption of metabolite-profiling approaches and significant innovations in data analysis methodologies are required.

Biotechnology↗
PubMed2005

Bioprospecting in plants for engineered proteins.

For more than two decades, bioengineered plants have produced protein therapeutics for human and animal use. Almost all proteins produced by other existing systems, including antibodies, vaccines and plasma proteins, have now been manufactured in plants. Considering the limitations of microbial and mammalian reactor-based protein-production technologies and the impending bottleneck in manufacturing capacity, plants are now emerging as an attractive alternative system with which to supply the growing need for protein-based therapeutics. However, full realization of the promise of plant-derived engineered proteins requires that we confront the dual challenges of bioequivalence and product consistency, challenges that are largely related to post-translational protein modifications (PTMs) that are crucial to the structure and function of most eukaryotic proteins. Among the protein PTMs, the foremost challenge for bioactivity and acceptance by the pharmaceutical and biotechnology industries and regulatory agencies is glycosylation. Advances made in recent years that 'humanize' plant glycosylation pathways combined with the discovery of terminal sialic acids (SAs) in plants now make feasible the bioengineering in plants of glycoproteins that have mammalian-like glycosylation.

Animals↗
PubMed1996

The healthy worker survivor effect and mortality at two automotive engine manufacturing plants.

Attributes such as time since hire or length of followup may be important in occupational mortality due to the "healthy worker survivor effect." In a regression analysis of mortality odds ratios at two automotive engine plants, strong effects of overall employment duration (latency weighted) were observed in addition to effects for (similarly weighted) cumulative exposures. The duration effects were negative for several cancer and noncancer outcomes, and confounded exposure effects. The lung cancer odds ratio declined to 0.68 (95% CI = 0.51, 0.90) at the mean duration of employment. With control for employment duration, adjusted lung cancer odds ratios for work as millwrights increased from 3.0 to 3.8, and for work in cylinder head production, from 3.3 to 3.9. Several causes of death with strong duration effects were smoking-related, suggesting diminished smoking risk factors with increasing employment duration. Similarly, trends for cirrhosis of the liver mortality suggested the alcohol risk factor is smaller in long-duration workers. If personal risk factors are an important component of the healthy worker survivor effect, they could be powerful negative confounders of exposure-response for related outcomes. Including a term for employment duration in regression models appears to partially correct for healthy worker survivor bias.

Aged↗
PubMed2005

Gene regulation in planta by plant-derived engineered zinc finger protein transcription factors.

The ability to modify plant traits is of great commercial potential in agricultural biotechnology. To this end we have engineered plant-based zinc finger protein transcription factors (ZFP TFs) that minimize the use of non-plant DNA sequences. This novel architecture supports the use of tandem arrays of zinc-finger DNA recognition domains such that the ZFP TF binds a contiguous DNA target site - thus emulating the design of ZFP TFs described previously for mammalian gene regulation. We show that this plant-based ZFP TF architecture supports high affinity DNA binding while allowing the specificity of the DNA-protein interaction to be determined by the amino acid sequences of the recognition helices. This plant-based backbone thus supports the use of previously characterized DNA recognition helices originally identified in a mammalian ZFP context without using mammalian DNA sequences. Moreover, we show that plant-based ZFP TFs employing this new architecture can up-regulate endogenous ADH activity by > 20-fold in transgenic Arabidopsis. Thus plant-based ZFP TFs are shown to be potent regulators of gene expression in vivo.

Alcohol Dehydrogenase↗
PubMed1996

Transgene inheritance in plants genetically engineered by microprojectile bombardment.

Microprojectile bombardment to deliver DNA into plant cells represents a major breakthrough in the development of plant transformation technologies and accordingly has resulted in transformation of numerous species considered recalcitrant to Agrobacterium- or protoplast-mediated transformation methods. This article attempts to review the current understanding of the molecular and genetic behavior of transgenes introduced by microprojectile bombardment. The characteristic features of the transgene integration pattern resulting from DNA delivery via microprojectile bombardment include integration of the full length transgene as well as rearranged copies of the introduced DNA. Copy number of both the transgene and rearranged fragments is often highly variable. Most frequently the multiple transgene copies and rearranged fragments are inherited as a single locus. However, a variable proportion of transgenic events produced by microprojectile bombardment exhibit Mendelian ratios for monogenic and digenic segregation vs events exhibiting segregation distortion. The potential mechanisms underlying these observations are discussed.

Animals↗
PubMed2005

Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC.

Neohesperidin dihydrochalcone (NHDC) is a seminatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-glucoside-2-O-rhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.

Cells, Cultured↗
PubMed1999

Genetic engineering of plant lipids.

Vegetable oils are a major component of human diets, comprising as much as 25% of average caloric intake. Until recently, it was not possible to exert significant control over the chemical composition of vegetable oils derived from different plants. However, the advent of genetic engineering has provided novel opportunities to tailor the composition of plant-derived lipids so that they are optimized with respect to food functionality and human dietary needs. In order to exploit this new capability, it is essential for food scientists and nutritionists to define the lipid compositions that would be most desirable for various purposes.

Dietary Fats, Unsaturated↗
PubMed2002

Nuclear magnetic resonance and plant metabolic engineering.

Nuclear magnetic resonance (NMR) can be used to measure metabolite levels and metabolic fluxes, to probe the intracellular environment, and to follow transport and energetics nondestructively. NMR methods are therefore powerful aids to understanding plant metabolism and physiology. Both spectroscopy and imaging can help overcome the unique challenges that plants present to the metabolic engineer by detecting, identifying, quantifying, and localizing novel metabolites in vivo and in extracts; revealing the composition and physical state of cell wall and other polymers; allowing the identification of active pathways; providing quantitative measures of metabolic flux; and testing hypotheses about the effects of engineered traits on plant physiological function. The aim of this review is to highlight recent studies in which NMR has contributed to metabolic engineering of plants and to illustrate the unique characteristics of NMR measurements that give it the potential to make greater contributions in the future.

Biopolymers↗
PubMed2000

Effect of crop rotation and soil cover on alteration of the soil microflora generated by the culture of transgenic plants producing opines.

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.

Arginine↗
PubMed2006

[Specific promoters used in plant gene engineering].

The choice of specific promoters used within a transgene construct is a vital strategy to achieve the transgene regulation in the temporal, spatial and measurable manner. The strategy has been widely used in diverse aspects of plant gene engineering, such as quality improvement, resistance breeding and bioreactor. In this paper, we describe the structure feature, classification and research method of the specific promoter and its application progresses in plant gene engineering.

Animals↗
PubMed2024

Genetic modification of the shikimate pathway to reduce lignin content in switchgrass (Panicum virgatum L.) significantly impacts plant microbiomes.

UNLABELLED: Switchgrass (Panicum virgatum L.) is considered a sustainable biofuel feedstock, given its fast-impact growth, low input requirements, and high biomass yields. Improvements in bioenergy conversion efficiency of switchgrass could be made by reducing its lignin content. Engineered switchgrass that expresses a bacterial 3-dehydroshikimate dehydratase (QsuB) has reduced lignin content and improved biomass saccharification due to the rerouting of the shikimate pathway towards the simple aromatic protocatechuate at the expense of lignin biosynthesis. However, the impacts of this QsuB trait on switchgrass microbiome structure and function remain unclear. To address this, wild-type and QsuB-engineered switchgrass were grown in switchgrass field soils, and samples were collected from inflorescences, leaves, roots, rhizospheres, and bulk soils for microbiome analysis. We investigated how QsuB expression influenced switchgrass-associated fungal and bacterial communities using high-throughput Illumina MiSeq amplicon sequencing of ITS and 16S rDNA. Compared to wild-type, QsuB-engineered switchgrass hosted different microbial communities in roots, rhizosphere, and leaves. Specifically, QsuB-engineered plants had a lower relative abundance of arbuscular mycorrhizal fungi (AMF). Additionally, QsuB-engineered plants had fewer Actinobacteriota in root and rhizosphere samples. These findings may indicate that changes in the plant metabolism impact both AMF and Actinobacteriota similarly or potential interactions between AMF and the bacterial community. This study enhances understanding of plant-microbiome interactions by providing baseline microbial data for developing beneficial bioengineering strategies and by assessing nontarget impacts of engineered plant traits on the plant microbiome. IMPORTANCE: Bioenergy crops provide an important strategy for mitigating climate change. Reducing the lignin in bioenergy crops could improve fermentable sugar yields for more efficient conversion into bioenergy and bioproducts. In this study, we assessed how switchgrass engineered for low lignin impacted aboveground and belowground switchgrass microbiome. Our results show unexpected reductions in mycorrhizas and actinobacteria in belowground tissues, raising questions on the resilience and function of genetically engineered plants in agricultural systems.

Panicum↗
PubMed2004

Prospects of genetic engineering of plants for phytoremediation of toxic metals.

Bioremediation is gaining a lot of importance in recent times as an alternate technology for removal of elemental pollutants in soil and water, which require effective methods of decontamination. Phytoremediation--the use of green plants to remove, contain or render harmless environmental pollutants--may offer an effective, environmentally nondestructive and cheap remediation method. The use of genetic engineering to modify plants for metal uptake, transport and sequestration may open up new avenues for enhancing efficiency of phytoremediation. Metal chelator, metal transporter, metallothionein (MT), and phytochelatin (PC) genes have been transferred to plants for improved metal uptake and sequestration. Transgenic plants, which detoxify/accumulate cadmium, lead, mercury, arsenic and selenium have been developed. A better understanding of the mechanisms of rhizosphere interaction, uptake, transport and sequestration of metals in hyperaccumulator plants will lead to designing novel transgenic plants with improved remediation traits. As more genes related to metal metabolism are discovered, facilitated by the genome sequencing projects, new vistas will be opened up for development of efficient transgenic plants for phytoremediation.

Biodegradation, Environmental↗
PubMed2004

Transcription factors as tools for metabolic engineering in plants.

The functions of an increasing number of plant transcription factors are being elucidated, and many of these factors have been found to impact flux through metabolic pathways. Because transcription factors, as opposed to most structural genes, tend to control multiple pathway steps, they have emerged as powerful tools for the manipulation of complex metabolic pathways in plants. The review describes the highlights of recent experiments that have targeted transcription factors that control plant metabolic pathways, and discusses their potential as tools for metabolic engineering.

Enzymes↗
PubMed1995

Genetic engineering in plants.

Until now most research, and its funding, has been focused on animal and human health care as well as simple microbiological model systems such as Escherichia coli and yeast. Molecular plant studies have generally lagged behind, often simply adapting discoveries from the animal field to plants. Clearly, good health and the efficient tackling of diseases is crucial for the well-being of humans, and good remedies have a high economic value for the pharmaceutical industry. However, one should not forget that plants are an essential component of the large ecosystem that is our planet. They are not only the basic food producers but they are also necessary for a balanced atmosphere (oxygen production) and stable and viable climates. Especially in this period of demographic explosion and growing environmental deterioration, there is a need to rebuild our agricultural systems. Plants also have a wide variety of 'non-food' uses, for instance as energy sources, construction materials, or cosmetics. Last, but not least, they produce a lot of chemicals that can be used as pharmaceuticals. The growing awareness of the importance of plants has coincided with the development of plant molecular biology. Specific features make them ideally suited for gene engineering and genetic studies in general.

Biotechnology↗