With an increasing global population, new tools are needed to provide highly nutritive foods and low-cost therapeutics to a larger populace. Synthetic biology is a proven approach to reconstitute metabolic pathways for the production of valuable chemicals; so far, microbes have been the production platform of choice. While an extremely robust synthetic biology platform, microbes are unicellular organisms, hindering the reconstitution of certain complex plant metabolic pathways that normally take place in multiple cellular compartments and tissue types . Additionally, many plant enzymes do not function properly in microbial systems . Microbial production also requires the use of expensive, large-scale fermenters and the extraction of metabolites produced, incurring significant costs. Plants are ideally suited for the production of plant natural products as they natively host many of the biosynthetic pathways and produce essential precursor molecules required using light and water. Additionally, plants have a variety of tissues and organelles, allowing for compartmentalization of pathways and toxic intermediates. As humanity’s main source of food, plants are a unique yet practical vehicle for the production and delivery of essential nutrients and nutraceuticals . While plants present an ideal platform, there are still technological bottlenecks hampering our ability to optimize the production and accumulation of specific nutrients and phytochemicals. This review will examine the past successes and future challenges of plant synthetic biology in producing valuable therapeutics and enhancing the nutritive capacity of food crops. Additionally, strawberry gutter system the strengths and weaknesses of plants as a production platform will be addressed.
Plants and the compounds they produce are some of the most important natural resources we have. From the food we eat to many of the medicines we take, plants provide us with a diverse selection of bio-active compounds capable of improving human health. A major goal of plant synthetic biology is to identify biosynthetic pathways for bio-active compounds and transfer them to other plant species to provide alternative sources of these compounds. In this section, we focus on the progress of pharmaceutical compound biosynthetic pathway discovery, efforts for enhancing the nutritive value of plants, and exploration of nutraceuticals for better health and wellness.Plant remedies have provided insight into functional molecules that have drastically improved human healthcare, such as the revolutionization in pain management following the use of opiates from opium poppy and the discovery of the active ingredient, salicin, in willow bark which enabled the synthesis of aspirin by the acetylation of plant derived salicylic acid . However, as was the case with aspirin, it can be difficult to pinpoint the specific metabolite, or combination of metabolites, that result in observed beneficial effects from the entirety of a plant’s metabolome. With the major advances in modern analytical chemistry tools, it has become easier to identify and characterize specific metabolites from any given organism . From this collection of metabolites, specific compounds can be isolated and tested for bioactivity and efficacy against many chronic and deadly diseases. However, knowing the identity of the compound is only the start, as many of these active compounds are found in uncultivated plant varieties or in low abundance therein, impeding the large-scale production necessary for pharmaceutical use . Plant synthetic biology seeks to circumvent some of these problems through heterologous expression of bio-active compounds for large-scale production and extraction.
The first step needed to achieve this goal is to fully characterize the biosynthetic pathway for the compound of interest. Understanding the reactions between pathway intermediates and determining the required parts in a pathway based on standard classes of enzymes can help to fill in the gaps of many incomplete biosynthetic pathways . Additionally, modern sequencing technologies have advanced to the point where whole genome sequencing and transcriptomics in conjunction with co-expression analysis, phylogenomics, gene clustering, and genome-wide association studies of non-model species have become accessible . These techniques enable a route to the rapid discovery of all the parts in complex biosynthetic pathways, which enables heterologous expression of pathways for compound production. A notable example of this is the discovery of the pathway for the anti-inflammatory drug, colchicine, from Gloriosa superba . In this work, the authors used previously generated RNA-seq data from multiple species, generated a new RNA-seq dataset, and used metabolomics to determine eight essential genes in the pathway as well as reconstitute a 16-gene pathway with transient expression in Nicotiana benthamiana to produce the colchicine precursor, N-formyldemecolcine. With these tools in hand, it is time for plant synthetic biology to develop a library of parts and pathways to not only produce natural bio-active compounds for better human health but also new-to-nature compounds which could revolutionize the way we think about wellness. Other examples of heterologous expression of phytochemical pathways have shown it is possible to develop plants into pharmaceutical production platforms.
Crocosmia spp. is an ornamental plant that was found to produce Montbretin A , a potent inhibitor of human pancreatic amylase, which is a promising treatment for type II diabetes awaiting clinical validation . However, MbA is only produced in the corms of Croscosmia spp. in low amounts. Corms are the small vegetative reproductive tissue of the plant, making the propagation and extraction of MbA difficult. Thus, the limitations of Crocosimia spp. make a heterologous host a better suited system for MbA production. The full MbA pathway was recently discovered and its heterologous expression in leaf tissue of N. benthamiana resulted in a measurable but low yield, displaying the need for pathway and potentially host optimization for largescale production. Potential mechanisms for effective pathway optimization include enhancing precursor molecule levels or relaxation of bottleneck steps within the pathway. For example, etoposide is a chemotherapy drug whose precursor previously could only be isolated from the endangered mayapple plant, Sinopodophyllum hexandrum. Optimization of the production of the etoposide precursor, -deoxypodophyllotoxin, was improved by two orders of magnitude through transient expression of eight genes required to produce coniferyl alcohol, allowing for purification of milligram levels . Traditional medicinal plants are another source of pharmacologically important compounds, with several compounds having fully elucidated biosynthetic pathways. Salidroside, isolated from the Rhodiola genus, has a long history of use due to its potential, albeit understudied, benefits for mood stabilization, fatigue, and prevention of cardiovascular disease and cancer . Unfortunately, because of its suggested benefits, some species of Rhodiola are being harvested to near extinction from their native habitats. The recent discovery of its three-enzyme biosynthetic pathway using transcriptomics and metabolomics has enabled the production of salidroside by heterologous expression in N. benthamiana, providing a scalable alternative to extraction from Rhodiola . From another medicinal plant, Catharanthus roseus, the final steps of the complicated, thirtyone enzyme biosynthetic pathway of vinblastine, an important drug in the treatment of leukemia and lymphoma, have been discovered using RNA-seq to identify the genes and transient expression in N. benthamiana to validate vinblastine production . The heterologous expression of this pathway could provide an alternative production platform for the synthesis of this important pharmaceutical. Even with these discoveries, there are many biosynthetic pathways of important medicinal compounds that are not fully understood, such as tentative antivirals found in traditional Chinese medicine . Currently, some of these traditional medicines are undergoing FDA clinical trials to be developed into direct treatment or combinatorial supplements with existing approved drug treatments . With continued progress, grow strawberry in containers there will be a more comprehensive library of pharmacologically relevant phytochemical pathways to draw upon for the treatment of any type of disease. With a greater understanding of the biosynthetic principles behind natural product synthesis, synthetic biologists are producing new-to-nature molecules by utilizing well characterized precursors in combination with modifying enzymes from multiple plant sources. Terpenoids are ubiquitous across the plant kingdom and this diverse class of molecules serves many functions for plants, including but not limited to defense and stress response.
Researchers have demonstrated that heterologous expression of the triterpene, ß-amyrin, with combinations of CYP450 enzymes can produce more than a dozen natural saponins as well as new-to-nature products on a gram scale . These compounds were tested for anti-proliferative and anti-inflammatory effects and demonstrated some effectiveness, illustrating the potential pharmacological efficacy of new-to-nature compounds being produced in plants at a large scale. Other evidence shows that the addition of plant and bacterial enzymes into a plant-specific pathway can also enhance the ability of plants to generate new to-nature compounds. A recent example of this is the production of novel crucifalexins and halogenated derivatives of brassinin by transiently expressing halogenases and various CYP79 genes with different specificities . The authors also demonstrated that the anti-fungal nature of these new compounds was enhanced compared to commercially available products, showing an alternative approach to developing a suite of new compounds using the parts and pathways that are readily available.One of the primary ways plant synthetic biology can aid in health and wellness is by alleviating malnutrition which is still prevalent across the entire world. Beyond the macromolecular necessities of food, an even greater portion of the world experiences micronutrient deficiencies—termed “hidden hunger”– especially in regard to vitamin A, iodine, zinc, and iron . A recent review has an in depth analysis of the types of deficiencies as well as some strategies that have been used to biofortify crops. The production of palatable crops that provide adequate amounts of macroand micronutrients that can grow in diverse biomes is one of the cheapest and most practical ways to ameliorate malnutrition . Plant metabolic engineering has the potential to address many of these nutrient shortcomings through both the manipulation of existing pathways and the introduction of additional plant pathways. Traditionally the enhancement of crop species has occurred through breeding practices which involves a series of genetic crosses and backcrosses to take a desirable trait from one variety of plant into a commercial cultivar. This process requires multiple plant generations and can introduce undesirable effects due to tight gene linkage. These practices have been in use for thousands of years and have improved almost every crop that we eat today, yet the arduous nature of plant breeding has limited the varieties of crops that are actively cultivated for consumption. With the advent of gene-editing technologies, scientists can directly modify specific DNA sequences to produce desired changes in the plant genome within one generation. An example of this is in the improvement of ground cherry, a species closely related to tomato . In this work, the authors use the clustered regularly interspaced short palindromic repeats –CRISPR-associated protein-9 nuclease system to generate null mutations in Ppr-SP5G and SlCLV3 promoter region to impart desirable traits into this crop, such as flowering and fruit size. Additional examples of de novo domestication from wild tomatoes have shown the potential of nutritive enhancement along with cultivation improvement, retaining the disease and abiotic stress resistance of the wild varieties . With the use of new tools to perform genetic modifications, such as CRISPRCas9, species of plants that are difficult to cultivate can be modified for use as crops, thereby diversifying available food options. This could in turn enhance the availability of nutrients that are limited throughout the world. Additionally, the advent of a CRISPR-Cas9 with less restriction on the genetic site of protein binding will allow for greater flexibility in the use of this tool . Gene editing further allows for the alteration and enhancement of specific endogenous biosynthetic pathways to produce biofortified crop species. Work has shown that silencing of lycopene ε-cyclase in the carotenoid pathway can enhance the natural levels of ß-carotene while reducing the α-carotene levels . Other examples show that the manipulation of plastid identity—such as the conversion of chloroplast to chromoplast—can enhance the overall carotenoid levels. One study converted chloroplasts to chromoplasts in tomato during early fruit development through the ectopic expression of the Arabidopsis thaliana ORANGE gene containing a SNP . Another study converted chloroplasts to chromoplasts throughout the plant through the expression of plastid-targeted microbial phytoene synthase in multiple plant species; however, the transgenic plants suffered from decreased photosynthetic efficiency in leaf tissue . This highlights the importance of finding the balance between high titers and stable growth. Additionally, the levels of zeaxanthin, a carotenoid with potential benefits in eye health, have been increased by the elimination of zeaxanthin deepoxidase in the algal species Chlamydomonas reinhardtii . The enhancement of desirable carotenoids could easily be translated into higher plant species and much effort is being made for their use in non-model organisms .