These high value products split into two major classes: high value small molecules and proteins. The anti-malarial drug Artemisinin is a high value small molecule naturally produced in non-model plants. Fast-growing tobacco plants have recently been engineered to produce the drug, potentially expanding production capacity. Plants have been the primary production platform for high value small molecules for millennia – medicines, spices, and drugs have mostly been sourced from plant hosts. In modern times, the large-scale production of cannabis and opium poppies attest to the scalability and cost-efficacy of in planta small molecule production. Despite advances in microbial engineering and synthetic chemistry, plants remain the production platforms of choice for these high value small molecules, demonstrating the low cost and high scale that can be achieved with plant systems. With an appropriate ideotype compromising between fermentable sugars and high-value co-products, engineered lignocellulosic biomass crops may be economically viable. Rather than a single ideotype for all biomass crops, different crops may be more amenable hosts to particular applications. Complex metabolic pathways to produce high value small molecules have been successfully implemented in model plant species, and some biomass crops seem particularly amenable to metabolic engineering for high value small molecule production as well as overall modifications to the total carbon allocation . Ultimately, engineered feedstock crops that produce co-products may help offset costs associated with a future plant based bio-economy that will have to compete with petrochemicals.Dedicated crops have been used in first-generation food-to-ethanol production for over 100 years,cannabis vertical farming and in the United States annual production has increased 10-fold since 199022.

Ethanol accounts for over 90% of all bio-fuel produced in the United States, nearly all of which is derived from dedicated fields of corn, consuming 38% of corn production. The production of bio-fuel products from food crops causes competition between food and fuel, raising the price of staple foodstuffs. Lignocellulosic ‘second generation’ bio-fuels substantially reduce this problem by either growing on marginal land where food crops are not viable, or by production from agricultural residues rather than diverting a food crop into the bio-fuel pathway. Bio-fuels have sometimes been presented as an environmentally friendly and low-carbon alternative to fossil fuels, but current implementations have failed to deliver substantial GHG emission reductions. Bio-fuels grown from established agricultural fields generally achieve GHG emission reductions of 20–80% compared to fossil fuels95. However, land use change associated with the conversion of natural land to bio-fuel production leads to a ‘carbon debt’ that takes decades to centuries to pay back, negating any GHG savings96. Furthermore, conversion of natural land to bio-fuel production is a major driver of rainforest loss97. Growing bio-fuel feedstock on marginal lands and producing bio-fuel as one of multiple products are the two main strategies to reduce this trade-off. Here we consider re-designing bio-fuel feedstock crops to reduce cell wall recalcitrance, increase biomass per acre, and generate additional products not only add value but also improve resource use efficiency. Modern biotechnology has expanded the possibilities of crop ideotypes by allowing for plant phenotypes not attainable through classical breeding. Petrochemical fuels have been instrumental for global industrialization, and their use remains indispensable at present. However, climate considerations as well as the practical limitations inherent in using a finite resource call for the development of alternative sources of liquid fuel and materials. Plant biomass is the most viable means of production sufficiently scalable to take the place of petrochemicals in the economy of the future, and ideotype breeding serves as a useful paradigm for the design and improvement of biomass feedstock crops. No matter the goal of a plant engineering project, gene expression and the regulation of its expression is crucial for synthetic biology applications.

At its core, the regulation of when, where, and how strong a gene is expressed underpins the phenomenon of multicellular life. Intricate networks of transcriptional elements harness complex arrangements of “logic gates” to conduct the development of a multi-system organism from a single embryonic cell. The development of synthetic regulatory systems that provide control of when a gene is expressed , where it is expressed , and the level of expression without disrupting endogenous processes would allow for the engineering of complex, multilayered, synthetic gene circuits in future endeavors. In the work presented here, we sought to develop novel tools for the modulation of transgene expression in plants by developing synthetic orthogonal regulatory elements. These elements allow for the refinement of the engineering scheme by providing the means to tune the desired synthetic genetic circuit with a higher level of precision. We also developed a series of synthetic genetic circuits building upon our preliminary work with synthetic transcriptional regulators for deployment in sorghum. Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their utility in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli. Plants offer a unique platform to address many imminent challenges that face society, as future engineering efforts hold promise in promoting sustainable agriculture, renewable energy, and green technologies. However, the tools to effectively modify and engineer plants are still in their infancy.

One major hurdle has been the development of genetic parts that enable precise control of transgene expression in plants. Many genetic and metabolic engineering strategies require robust and accurate control of multiple genes to optimize synthetic pathways, regulate flux, and introduce new traits. The ability to modulate gene expression provides a direct approach to address these tasks. However, the majority of plant engineering efforts are limited to a small number of characterized constitutive promoters, which may result in unintended pleiotropic effects or toxicity issues and are limited in their range of expression strength. This work was inspired by previous studies that have successfully engineered orthogonal gene expression tools employing a reductionist and modular approach to parts design. Our unique strategy builds upon this approach by blending elements from both yeast and plants and could theoretically be applied to any TF type. Additionally, many of these previous systems were constructed using repetitive cis-element DNA sequences in the promoter design, while an important aspect of our approach is maximizing sequence diversity while tuning promoter strength. Specifically, we sought to build an expansive and diverse library of synthetic transcriptional regulators for plant engineering with components from various yeast transcription factor systems in conjunction with plant-specific regulatory DNA sequences. With this approach we developed a method that can be expanded beyond the well-characterized Gal4- based synthetic systems that have been used in the past . In general, parts design was approached in three ways: altering TF DNA-binding dynamics at the regulatory promoter by introducing DNA cis-element variation with randomly concatenated cis-elements,cannabis drying rack modulating RNA polymerase II recruitment by testing various plant minimal promoters, and directly modifying TFs from disparate families through truncation and fusion of activation or repression domains to generate new synthetic TFs, or trans-elements . A major finding of our work is that plant minimal promoter sequences can be leveraged for the design of chimeric promoters. By utilizing cis-elements from yeast in combination with a plant minimal promoter, we can generate functional synthetic promoters that interact with both the orthologous yeast TF as well as the endogenous transcriptional machinery of the plant. The minimal promoter, also known as a core promoter, lies directly upstream of the transcription start site and is where the transcription pre-initiation complex including RNA-polymerase II binds. Thus, the minimal promoter region facilitates the assembly and stabilization of the pre-initiation complex leading to variable levels of basal and activated transcription determined by this interaction. The binding of additional TFs, specifically orthogonal trans-elements in this work, at the regulatory promoter upstream of the minimal promoter can stimulate or repress this basal level of transcription providing multiple layers of regulation131. Using this strategy, we characterized a diverse set of synthetic promoters and synthetic trans-elements composed of discrete genetic parts from various TF families. Promoters were constructed with a collection of TF-binding cis-elements appended upstream of a plant minimal promoter.We tuned promoter strength by modifying basal transcription through minimal promoter variation and modulating the binding dynamics of the trans-element with concatenated cis-element variation. The initial library of synthetic promoters was developed for the yeast TF, Gal4, by appending random combinations of five well-characterized Gal4 upstream activation sequences , to varied plant minimal promoters. Through our approach, we explored how these CCEs and minimal promoters contribute to the overall strength of a synthetic promoter.

We then expanded upon this design strategy to TFs from disparate protein families and assembled a full suite of trans-elements and synthetic promoters for each. By expanding our library to contain both cis- and trans-element variation, we generated and tested more than 500 unique promoter/TF pairs resulting in a wide range of transcriptional output potential. Our library of TF binding cis-elements, plant minimal promoters, and TF fusion proteins demonstrates a novel method for the design, construction, and characterization of new tools for the controlled modulation of gene expression for various plant synthetic biology applications. As a proof of concept for our promoter design strategy, we utilized the well-characterized TF Gal4 fused to the VP16 activation domain as the activating trans-element. The heterologous nature of yeast TFs like Gal4 provides an opportunity to leverage a purely orthogonal system in plants, decoupling the transcriptional regulation of transgenes from those endogenous to the genome. To test the abundance of parts generated in this study, we used a high throughput transient expression assay in Nicotiana benthamiana. This system allows for the rapid screening of parts using a combinatorial approach, with each element cloned into individual binary vectors. A diversity of known Gal4 cis-elements were manually curated from native promoters in the yeast Gal regulon, each with a distinct nucleotide sequence and assumed to exhibit a diversity of dissociation constants with Gal4. We hypothesized that these deviations would lead to variation in the transcriptional output of each synthetic promoter, depending on the combination and position of cis-elements used in the CCE design. Minimal promoters from a collection of plant promoter sequences were appended to the CCEs generated with these Gal4 cis-elements to produce complete synthetic promoters . This strategy also presents an opportunity to investigate the effect the minimal promoter has on gene expression outside the context of its endogenous sequence. Additionally, introducing nucleotide diversity to our promoter constructs may limit the potential for transcriptional silencing often observed when identical sequences are used multiple times in gene stacking efforts. We screened these randomized combinations to measure the strength of the promoter in the presence of our synthetic trans element and the basal expression of our promoters in the absence of the TF. A measurement of Green Fluorescent Protein fluorescence was used as the proxy for transcriptional output, while the constitutive expression of a Red Fluorescent Protein was used as a normalization metric, with the ratio of GFP over RFP providing normalized values for the output of each construct. As expected, we observed a distribution of expression strengths while avoiding the usage of identical sequences, demonstrating a strategy for tuning gene expression . In many cases, it is not necessary to constitutively express all transgenes, and thus state-specific regulation of gene expression provides an additional dimension of control over a genetic circuit. For example, the constitutive over expression of a given protein may act as a sink on cellular resources resulting in overall detrimental effects. Similarly, various agricultural traits often result in fitness costs, and thus targeted expression of these genes may curtail unintended consequences. To further evaluate the efficacy of our parts in planta, we generated stable lines in A. thaliana.