There is also evidence that CRISPR-Cas9 modifications to upstream open reading frames can impact metabolite regulation across species, as vitamin C concentrations increased by 150% in Arabidopsis and lettuce by removing the regulatory effects of the associated uORF . While altering the levels of endogenously produced chemicals is effective, this approach is limited in the diversity of nutrients it can deliver. However, plant synthetic biology offers a means to bypass this limitation through the introduction of heterologous biosynthetic pathways. One of the first successes in this came with the production of golden rice, a ß-carotene enhanced rice grain that was generated through the addition of three genes . Since then, replacement of the daffodil phytoene synthase with a maize phytoene synthase has increased the titers of ß- carotene in golden rice 2 , providing greater nutritional benefits . Furthermore, the authors were able to not only add ß-carotene to rice grains but also increase the levels of iron and zinc with a single genetic insertion. This shows the potential of a biofortified staple crop to have an enhanced nutritive capacity suitable to meet multiple minimal daily requirements. Additional work has shown that the added ß- carotene in the rice grain can be further modified into astaxanthin and canthaxanthin, two antioxidants of interest, with one or two additional genes . Iron deficiency is one of the world’s leading micronutrient deficiencies and is responsible for around one million deaths annually. As such, hydroponic nft channel iron has been a target of biofortification efforts over the last few decades.
Early work showed that the overexpression of a root zinc transporter from Arabidopsis could not only increase the zinc concentration in grains, another significant deficiency, but it also increased the iron content for Hordeumvulgare . The addition of genes increasing nicotianamine, the nicotianamine-iron transporter and the iron storage protein ferritin was shown to have a concerted effect on rice seed iron concentration of more than fourfold . Perhaps, with the combination of nutrient transport and sequestration mechanisms in the grain or fruit, levels of many micronutrients could be enhanced. Other vitamin deficiencies also pose major concerns to human health and plant synthetic biology efforts can alleviate some of these nutrient shortcomings. Inadequate folate consumption is linked to cardiovascular disease, Alzheimer’s disease, and cancer, but it is especially important for pregnant women, as deficiencies can cause neural tube defects or megaloblastic anemia . Folate content in rice has been improved by 150-fold through the expression of a folate binding protein and a folylpolyglutamate synthase , offering a means to increase folate consumption without dietary supplementation. This work also showed that the binding protein provided stability to folate so that it was maintained at a similar level even after months of storage, which is a concern for any crop that does not get eaten immediately after harvest.
Vitamin B6 deficiency has been shown to be linked to cardiovascular disease, diabetes, neurological diseases, as well as nodding syndrome . Efforts to biofortify cassava, a staple vegetable crop in many parts of Africa, have been successful with the expression of the Arabidopsis thaliana genes PDX1.1 and PDX2, which are involved in de novo production of vitamin B6. Root-specific expression of PDX1.1 and PDX2 increased vitamin B6 and B6-glycoside concentrations by 4- and 14-fold, respectively, even after cooking . While biofortification of crops has taken great strides forward, there remain several considerations for effective implementation of this synthetic biology strategy. As more compounds are engineered into crops, it will be important that metabolic flux and regulation are not drastically altered, as this can have detrimental pleiotropic effects on the plant’s health. For example, the overproduction of γ-aminobutyric acid in tomatoes causes gross developmental defects . Moreover, there will be clear challenges in transferring metabolic engineering strategies from one crop to another. There has been moderate success in this, such as with enhancing ß-carotene in golden bananas , but each crop is unique and requires experimentation to optimize production. As we continue to understand the intricacies of plant metabolism, these issues will be more easily addressed.Nutraceuticals are defined as consumed bio-active compounds that are not essential for nutrition but offer other health benefits. These molecules are distinct from pharmacological compounds—though in some cases they do overlap—and do not require extraction and purification or semi-synthetic modifications before final dosage and distribution, as the source of nutraceuticals are the foods themselves.
While these compounds are not essential for growth and development and many of their health benefits are still under investigation, their presence in our diet has the potential to improve human health. One nutraceutical compound that has been of great interest is resveratrol. This naturally occurring compound, found in wine and chocolate among other plants, has been studied since the 1990s for its health benefits due to its association with the “French paradox,” the quandary of a low incidence of heart disease in a population that eats a high-fat diet. Many health benefits, including prevention of cardiovascular disease and treatment of diabetes, have been attributed to resveratrol and validated clinically, but its low concentration in native plants poses a challenge to extraction for medicinal use . Additionally, consumption of purified resveratrol is mostly metabolized, greatly reducing its beneficial effect, but ingestion alongside other food-based phenolic compounds increases resveratrol levels found in blood serum. Therefore, the addition of resveratrol to foods could provide a more efficient means for delivery than purified supplements. Since all plants contain the precursor molecules for resveratrol, 4-coumaroyl-CoA and malonyl-CoA, only a single gene, stilbene synthase, is required to produce minor amounts of the metabolite in any plant. Optimization of precursor pathways would also be necessary to enhance nutraceutical levels in any given food . Glucosinolates, and their hydrolysis products, isothiocyanates, are another family of compounds with great health potential that have been studied thoroughly . A recent review examined the vast diversity of these compounds, totaling 156 unique entries, emphasizing their identification and synthesis in plants . This diversity can be broken down into three types of glucosinolates based on the amino acid precursor: aliphatic, indolic, or benzenic, with the vast diversity derived from secondary modifications to these basal categories. Over decades of study, biosynthetic pathways for each type have been elucidated . Glucoraphanin is a glucosinolate found in high concentration in broccoli and has been a subject of intensive study due to the health benefits of it and its isothiocyanate, sulforaphane . Various studies have displayed the promise of sulforaphane in treating diseases such as neurological disorders, diabetes, cancer, and cardiovascular disease with variable efficacy observed over a vast range of dosages and sources of the metabolite; however, further clinical validation is needed to verify these health benefits . Medicinal interest in glucoraphanin led to its successful production in Nicotiana benthamiana through transient expression, though only small amounts were produced . This was one of the first examples of a complex pathway being heterologously expressed in plants, and with some optimization, nft growing system it could be stably introduced into a crop species for consumption. The use of breeding practices that select plants for favorable agricultural traits can remove important nutrients from cultivated crops. This can also reduce some of the important nutraceutical compounds found in the foods that we eat. For example, there is a complete lack of iridoids, a class of monoterpenes suggested to benefit human health, in most cultivated varieties of blueberry on the market, while every wild species tested was abundant with them . While this was an inadvertent consequence of breeding, it illustrates the need to investigate wild relatives of food crops to identify nutraceuticals lost during domestication.
Using the tools of plant synthetic biology, we should be able to quickly and easily reintegrate the pathways lost into commercial cultivars without the need for the lengthy breeding process. As we move forward in our efforts to produce advanced nutritive foods, it is important to examine the past uses of nutraceuticals in the prevention and treatment of disease. Traditional medicinal plants have seen renewed interest as sources for undiscovered bio-active compounds . This highlights the need for additional compound and pathway discovery efforts. There are many other reviews that discuss the different types of specialized metabolites found in medicinal plant species . Historically, medicinal plant extracts have been used as food additives, but introduction of their biosynthetic pathways into common food crops offers a means of increasing the availability of nutraceuticals to a broader market. In some cases, the biosynthetic pathways are known and efforts are being made to sequence more medicinal plant genomes in order to accelerate enzyme discovery in candidate organisms . Nevertheless, the major limiting step hampering efforts to engineer plant nutraceuticals is still the discovery of the enzymes involved in the biosynthesis of target plant natural products. Thus, new tools to streamline such research is of great interest to the larger plant synthetic biology field.Plants have many attributes that make them an ideal platform for the production of plant natural products. They are autotrophic, can be grown at a large scale, contain various intracellular compartments and tissue types, are used as a nutrient delivery system, and retain similar cellular features to the productproducing plant that may be required for efficient biosynthesis of compounds. Together, this enables countless strategies for the production and administration of plant natural products . However, the state of plant biotechnology has imposed limitations on the utilization of plants. In this section, we focus on the strengths of plants as a production platform and the technologies needed to improve their efficacy.Perhaps one of the greatest strengths of plants as a synthetic biology platform is their scalability. Large-scale plant production has been a focus of human society and technology since the first crops were cultivated. In addition to a long history of scalable plant cultivation for consumption, agriculture also offers a unique platform for the production and extraction of valuable therapeutic plant natural products. Opioids from Papaver somniferum, taxol from Taxus brevifolia, and QS-21 adjuvants from Quillaja saponaria, are several notable examples of therapeutic molecules that are produced and extracted from their native plant host . While many therapeutic chemicals can be extracted from their native producer, many species are difficult to cultivate or suffer from limited yields . Past efforts have focused on the production of nutrients and phytochemicals in microbes or with the use of chemical synthesis, but plants are an ideal platform for the production of small molecules at a large scale, as they are autotrophic and can be grown in non-axenic conditions like an open field. There are two main approaches for the heterologous expression of a specialized metabolic pathway in planta: transient expression or expression in stable plant lines. Transient expression typically utilizes Agrobacterium tumefaciens to insert genes of interest into a host plant. This process allows for the expression and characterization of genes in 3–6 days as opposed to the months required to make stable plant lines . Additionally, multiple strains of Agrobacterium harboring different genes can be co-infiltrated into host tissue, limiting the need for creating multi-gene constructs . These attributes have made it a valuable tool for in planta pathway discovery and protein production. Transient expression is usually conducted in a species of the genus Nicotiana but is typically only applied at small scales, limiting its viability as a production platform. Recent improvements by Reed and Osbourn have demonstrated the scalability and viability of transient expression for the production of phytochemicals. Through the use of full plant transient expression via vacuum infiltration, gram-scale quantities of various triterpenes were able to be produced and purified from Nicotiana benthamiana . As well as being a production platform, transient expression permits the testing of gene construct efficacy prior to stable line generation; however, the efficiency of Agrobacterium-mediated transient expression is variable in different hosts . This poses a limitation of Agrobacterium as a tool to screen genes in host plants. Additionally, transiently expressed pathways in Nicotiana benthamiana do not always function as expected in the desired host, limiting its use for screening constructs before generating stable lines . One method to circumvent these issues is the use of protoplasts, calli, and cell suspension cultures. These cell types are suitable for the use of direct transformation methods such as electroporation, gene gun bombardment, and microinjection. However, generation of these cell lines is often inefficient depending on the species used, produces fragile cells, and may not behave as an intact plant would .