A recent study utilized an industrially scalable delivery of self-replicating viral vectors to transform a variety of crop species, such as tomato, potato, and spinach, with successful alterations to reproduction, stature, and drought tolerance . This method could befurther developed for the introduction of biosynthetic pathways for bio-active phytochemicals into single generations of crop species. Advancements in the host range and efficacy of transient expression will make plants a better platform for the production of plant natural products and the characterization of pathways in hosts before the generation of stable plant lines. Transgenic plants can be grown in open fields, allowing plant natural products to be purified at large scales. However, the generation of stable plant lines is arduous, typically requiring the use of low-efficiency transgene delivery methods with Agrobacterium or particle bombardment, the generation of calli, the growth of the new, transgenic plant, vertical hydroponic nft system and the confirmation that the transgene is properly inserted. This results in long regeneration times, a requirement for numerous generations of backcrossing, and limits the size and location of transgene insertions.
A series of studies have begun to ameliorate this by creating transformation methods in maize that have increased the number of genotypes amenable to transformation, removed the need to generate calli, and utilized systems to remove selectable markers . Additionally, improvements over the size of transgenes that can be inserted have been made. Dong et al. utilized CRISPR-Cas9 in rice to perform targeted insertion of the 5.2 kb carotenoid biosynthesis pathway in a genomic safe harbor to limit adverse insertion effects. This method also limits the segregation of transgenes in subsequent generations, potentially reducing the number of crosses needed. With simplification of breeding efforts, the production of nutraceuticals and phytochemicals in transgenic plants will be ready for market more rapidly by requiring less screening of transgenic plant lines for insertional effects. Another example utilized a gene stacking method called TransGene Stacking II to generate and insert a 31 kb cassette encoding an anthocyanin pathway into rice grain . The utilization of gene stacking and targeted integration will enable long, complex pathways to be inserted with minimal adverse insertion effects . Together, these techniques have improved the feasibility of making stable plant lines expressing complex metabolic pathways for nutraceuticals and phytochemicals.
Many transformation techniques that generate stable transgenics require lengthy and expensive approval processes before becoming marketable . The use of gene editing tools with high target specificity that can be removed from future generations is able to circumvent these approval processes. One example is the modification of soybeans through TALEN site-directed mutagenesis, wherein natural soybean oil contains high levels of linoleic acid, an undesirable compound increasing the risk of heart attacks . In this work, FAD2-1 was disrupted, reducing the content of undesirable linoleic acid while simultaneously increasing its desirable precursor, oleic acid. With the discovery of DNA-free CRISPR-Cas9 genome editing in plants, these same types of modifications could be made to other nutritive regulator genes in a much shorter timescale . While these systems are subject to less regulation, they are unable to incorporate genes in heterologous pathways, limiting their use in plant synthetic biology. In addition to regulatory concerns, criticism of genetically modified crops by the public has slowed the advancement of crop engineering. Further engagement between scientists and the public will be needed to improve the overall opinion of transgenic crops .While supplements generated through microbial production or extraction from a native plant host can often be used as a source for the consumption of nutrients, plants serve as both a production platform and a vector for the delivery of nutritional and therapeutic small molecules. In addition to removing the need for costly purification processes, the delivery of small molecules through edible plants provides a low-cost solution to delivering critical nutrients and medicines.
The expression of pathways to improve the nutrient profile of staple crops is thus a promising method to improve access to the health benefits of a diverse diet. Anthocyanins are commonly consumed flavonoids with antioxidant activity that are believed to play a preventive role in many diseases, including cancer . With the expression of two transcription factors from A. majus by a fruit-specific promoter, tomatoes, normally devoid of anthocyanins, accumulated high concentrations of 2.83 ± 0.46 mg/g fresh weight and displayed purple coloring. Subsequent feeding experiments in cancer susceptible Trp53−/− mice with purple, anthocyanin-producing tomatoes significantly improve life span compared to control groups fed a normal diet or diet supplemented with red tomatoes . If made available to the public, purple tomatoes could serve as a broadly available source of health promoting anthocyanins. Additionally, anthocyanin-producing purple rice was developed through the expression of eight transgenes driven by endosperm-specific promoters, resulting in anthocyanin accumulation of 1 mg/g dry weight . By generating these crops, nutraceuticals can be delivered through the foods we eat, rather than with the use of a supplement. This has the potential to improve the accessibility of nutraceuticals. Artemisinin is a sesquiterpene lactone used in the treatment of malaria, whose biosynthesis has received a large amount of attention. One study optimized the production of artemisinin through the compartmentalization of the biosynthesic pathway to the cytosol, mitochondria, and chloroplast in N. tabacum . Crushed tobacco leaves synthesizing artemisinin, wildtype tobacco leaves, and pure artemisinin were then fed to mice infected with Plasmodium berghei, and parasitemia was monitored. Mice fed with tobacco leaves containing artemisinin showed reduced parasitemia and delayed onset of malaria symptoms compared to mice fed wild type tissue and pure artemisinin . While tobacco is not considered an edible crop, this approach shows the potential for engineered foods to serve as a delivery vehicle for important therapeutics. As plant synthetic biology advances and the yields of therapeutic small molecules in plants are improved, developing methods to control the dose of the target molecule will be needed.As organisms with multiple cellular compartments and tissue types, plants are ideally suited for the expression of complex metabolic pathways that require compartmentalization . The precise control over localization is particularly important when pathway intermediates are toxic. Strictosidine is an important intermediate in the production of the chemotherapy drug, vincristine. The strictosidine aglycone is a toxic intermediate in the strictosidine biosynthetic pathway that is normally sequestered to the vacuole before glycosylation. Viral induced gene silencing of a suspected strictosidine-glycosyl transporter, CfNPF2.9, resulted in blackening of leaf tissue in C. roseus, displaying the necessary role of transporters in plant natural product biosynthesis . As many metabolic pathways are compartmentalized, the use of transporters permits the movement of key intermediates synthesized in one compartment to the location of the enzymes required for the final steps of a biosynthetic pathway. This is further emphasized in the biosynthesis of aliphatic and indolic glucosinolates, which takes place in the cytosol and chloroplast. One study utilized a bile acid transporter to improve the heterologous yields of a precursor to the chemopreventive glucosinolate, glucoraphanin. Bile acid transporter 5 was demonstrated to be a plastidic transporter of the methioninederived α-keto acid and increased the production of the glucoraphanin precursor, dihomomethionine, by 10-fold . Many metabolites are synthesized in one tissue and transported to another as a means of storage or defense. Intercellular glucoraphanin transporters have been identified, which are responsible for the transport of glucoraphanin to seeds in A. thaliana .
The identification of these transporters allows for the transport of chemopreventive glucoraphanin to seeds of crops species to improve dietary consumption. Additionally, nft hydroponic system the use of transporters could allow for the concentration of metabolites into a specific tissue type, which would simplify harvest and extraction. Thus, there is a need to increase our understanding of transporters to enable the utilization of compartmentalization in the heterologous expression of metabolic pathways. However, most current methods are low throughput relying on the generation of complex yeast strains or plants with mutant transporters . One method utilizing Xenopus laevis oocytes expressing putative transporters has been demonstrated to improve the throughput of transporter screening by multiplexing transporters . Moreover, there is also a severe lack of structural information on plant specialized metabolite transporters, with less than a handful of known structures . An increase in the number of solved metabolite transporter structures could alleviate the need for enzymatic characterization through the use of prediction software, such as TransportTP . The compartmentalization of plant natural products into different tissue types provides a unique means for the production and storage of nutraceuticals and phytochemicals. Tissue-specific promoters enable the expression of metabolic pathways in a select tissue type. In one study, a bidirectional, embryo-specific promoter was used to drive the expression of anthocyanin genes in maize . The bidirectional promoter was then engineered with additional cis-elements to generate a construct expressing anthocyanin genes in both the endosperm and embryo . Additionally, specific plant tissues, such as trichomes, naturally accumulate large amounts of valuable plant natural products, thus making them promising targets for metabolic engineering. In one study, two genes required for methylketone synthesis, ShMKS1 and ShMKS2, were expressed constitutively alone or together in N. tabacum, A. thaliana, and cultivated tomato . While this resulted in methylketone production, it also caused severe lesions. However, by expressing ShMKS2 using a trichomespecific promoter, a significant increase in methylketone levels was observed with no other morphological defects . In another study, the trichomes of G. hitsutum L. were engineered to produce melanin through the expression of two genes driven by a cotton fiber-specific promoter. This enabled the production of brown-colored cotton fibers containing melanin, which could serve as a UV protectant and a natural pigment . These studies show the efficacy of tissue-specific expression as a means to produce useful small molecules. Future research should aim to identify and develop tissue-specific promoters that allow for targeted expression of pathways. Plant compartmentalization also enables organelles to produce and accumulate high amounts of a specific molecule of interest . Much work has been done to improve the availability of plant natural product precursors by targeting enzymes to specific organelles. Amorpha-4,11-diene is an important precursor to artemisinin. By targeting two enzymes involved in amorpha-4,11- diene synthesis to the chloroplast, amorpha-4,11-diene concentrations were improved by 40,000-fold compared to plants targeting the two enzymes to the cytosol . Additionally, the localization of enzymes can alter its stability or activity. One study found that targeting tryptophan decarboxylase to the chloroplast improved accumulation and stability of the enzyme compared to its cytosolic counterpart . Targeting enzymes to different cellular compartments can substantially improve the function of enzymes and the production of plant natural products.The expression of heterologous pathways can introduce a tremendous metabolic burden on the host organism. The use of large amounts of cellular resources, such as carbon sources, ATP, and NADH, can lead to undesirable physiologicalchanges that reduce host fitness and product titers . Mitigating the metabolic burden of heterologous pathways can be accomplished through host engineering. Additionally, the use of engineered hosts can improve the yields of target metabolites. Microbial synthetic biologists have used host engineering to improve the production of various important small molecules, which can serve as an example for plant synthetic biology. Phenylalanine is an important precursor for the synthesis of many plant natural products, such as phenylpropanoids and aromatic glucosinolates as well as many industrial applications, such as aspartame production . The E. coli strain, ATCC31884, has been engineered to overproduce phenylalanine. One study utilized ATCC31884 alongside changes to phenylalanine, tyrosine, and shikimate biosynthesis to improve the production of chorismate. The high chorismate producing strain was then used to produce muconic acid, which resulted in yields of 1.5 g/L after 48 h of growth . ATCC31884 has been further altered to improve the diversity of chemicals it can be engineered to make. Huang et al. knocked down two genes required for phenylalanine expression in ATCC31884 while expressing a feedback-insensitive mutant of tyrA to improve the levels of tyrosine, which enabled the strain to make 15- fold greater caffeic acid than the previous highest yield . These examples of microbial host engineering should serve as inspiration for plant synthetic biologists to engineer plant production platforms for optimized production. Plants like N. benthamiana could be altered in a similar fashion to E. coli strain ATCC31884 as a plant background designed for the production of phenylalanine-derived plant natural products.