Nature truly inspires beautiful forms, from simple multicellular life to complex organisms like plants and humans, each with numerous distinct tissues and systems layered to generate the whole. A sophisticated mechanism that allows for a massive collection of cells, all containing the same genetic code, to differentiate into complex living forms is the cell-specific modulation of gene expression. A main focus of our group, as plant synthetic biologists, is the development of tools and strategies for the engineering of plant-based green technologies. To achieve these goals, it is imperative to expand the tool set available to plant engineers. We have traditionally been constrained by a dearth of characterized constitutive promoters for use in planta, thus limiting our capacity to modulate the expression strength of introduced transgenes. These tools will be crucial for the future development of more elegant engineering regimes in the future, as they will require the incorporation of numerous signals and outputs. One day we hope to have the capacity to integrate complex tunable synthetic genetic circuits with native systems for the engineering of modified plant lines, whether for the production of optimal biofuel feedstocks or introducing new desirable traits for nutraceutical or pharmaceutical purposes.
Microbes have been engineered with complex genetic circuits and metabolic pathways for a wide variety of applications [2,3,4]. Yet, plants have long remained in the background of this synthetic biology/metabolic engineering boom. While they are amazing organisms, plants are much more challenging to work with. As sessile creatures, plants have developed a multitude of defense mechanisms to fend off would be attackers, whether it be fungus, insects, or pesky transposons. In particular, the natural phenomenon of gene-silencing in plants, a major defense mechanism for limiting the transposon load, has often thwarted plant engineering efforts [5,6]. It has been observed that stacking identical promoters for the expression of transgenes often leads to the silencing of these genes within a few generations, not a good thing for long-term sustainable solutions. These mechanisms largely function by targeting homologous regions of DNA/RNA deemed to be a threat to the genetic fidelity of the plant, mainly transposons in nature, or in our case, engineers attempting to slam synthetic segments of DNA into their genome.
So, we have two main problems: 1) how can we modulate the expression strength of introduced transgenes, and 2) how can we keep the plant from silencing these genes? We have aimed to tackle both issues simultaneously by building chimeric transcriptional regulators based on yeast transcription factors and their associated binding sites along with minimal core promoters from plants to build a synthetic system that should operate in an orthogonal fashion within the plant [7,8,9]. Our strategy was to increase the genetic sequence diversity of our chimeric synthetic promoters to both limit transcriptional silencing while also engendering promoters of varying expression strength. In general, our system consists of three components: the trans-element (modified yeast transcription factors), cis-elements (modified yeast DNA sequences that allow for the binding of the trans-element at the proximal promoter), and a minimal or core promoter (the region where the transcription pre-initiation complex assembles allowing for active gene transcription, these were manual curated from native plant promoters from numerous plant species) [Figure 1].
By altering the sequence and the position of each cis-element in concert with a varying minimal plant promoter, we were successful in demonstrating the design and functionality of numerous unique chimeric promoters in planta.
The next phase was to explore the orthogonality of our system in stably transformed plant lines. As an initial proof of concept, we chose to show that our system could enable the expression of three reporter genes in a tissue-specific or environmentally responsive manner. By expressing our trans-element under a native promoter, we were able to integrate our simple genetic circuit with the endogenous signaling system of the plant [Figure 2]. We observed the expression of reporter genes only in the desired state, demonstrating a functionality of our parts as well as their orthogonality (no phenotypic anomalies were observed in the engineered lines).
These results demonstrate that out designed DNA parts can be expanded beyond our simple proof-of-principle to more ambitious plant engineering endeavors. Additionally, we were successful in creating transcriptional repressors that interact with the same promoters. These elements allow for a degree of repressor logic to be built into synthetic circuits, expanding the potential for the development of multi-gated logic principles in future designs.
We continued to expand our tool set by generating and characterizing synthetic promoters and synthetic transcription factors from four additional transcription factor families. In doing so, we also explored the prospect of designing minimal and modular trans-elements with varying truncations and functional reconstruction through the fusion of alternate regulatory domains. As expected, not all modifications result in the desired outcome, but by testing numerous iterations of each we demonstrated the viability of our approach while generating functional synthetic transcription factors. As we look towards expanding these design principles in future work, we envision developing layered synthetic circuits that incorporate numerous endogenous signals with multi-input logic gates that require the use of both repressors and activators in tandem. Additionally, our chimeric promoter design has the potential to be expanded to other eukaryotic organisms, as long as you include a minimal promoter from your native host, you can design complete synthetic promoters that interact with the orthogonal transcription factor as well as the endogenous transcriptional machinery of the host. This is an exciting time for the nascent plant synthetic biology community, and we hope our developed tools and resources will prove useful for future advances in plant engineering.
Michael Belcher & Patrick Shih
Joint BioEnergy Institute and University of California, Davis
Design of orthogonal regulatory systems for modulating gene expression in plants: https://www.nature.com/articles/s41589-020-0547-4
1: Shih, P. M. (2018). Towards a sustainable bio-based economy: Redirecting primary metabolism to new products with plant synthetic biology. Plant Science, 273, 84-91.
2: Belcher, M. S., Mahinthakumar, J., & Keasling, J. D. (2020). New frontiers: harnessing pivotal advances in microbial engineering for the biosynthesis of plant-derived terpenoids. Current Opinion in Biotechnology, 65, 88-93.
3: Ro, Dae-Kyun, et al. "Production of the antimalarial drug precursor artemisinic acid in engineered yeast." Nature 440.7086 (2006): 940-943.
4: Elowitz, Michael B., and Stanislas Leibler. "A synthetic oscillatory network of transcriptional regulators." Nature 403.6767 (2000): 335-338.
5: Okamoto, H. & Hirochika, H. Silencing of transposable elements in plants. Trends Plant Sci. 6, 527–534 (2001).
6: Morel, J. B., Mourrain, P., Béclin, C. & Vaucheret, H. DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis. Curr. Biol. 10, 1591–1594 (2000).
7: Baker, C. R., Booth, L. N., Sorrells, T. R. & Johnson, A. D. Protein modularity, cooperative binding, and hybrid regulatory states underlie transcriptional network diversification. Cell 151, 80–95 (2012).
8: Joshi, C. P. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res 15,6643–6653 (1987).
9: Fordyce, P. M. et al. De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis. Nat. Biotechnol. 28, 970–975 (2010).