Additionally, it is important to note that many classically used promoters have been incorrectly labeled as ‘constitutive,’ as they are not expressed in all tissues, nor do they express at similar levels across various tissue types. Thus, this broad stroked approach and traditional reliance on ‘constitutive’ promoters represents a major barrier that separates the level of engineering complexity that can be deployed in model microbial versus plant systems. A key hurdle has been how to artificially design promoter elements that replicate elements of spatial and temporal control over gene expression. In comparison to microbial efforts, these challenges are unique and specific to plants and have not yet been thoroughly addressed. The majority of previous studies that have required tissue specific expression of transgenes have utilized endogenous promoters; however, these promoters cannot modulate expression strength, thus demonstrating the one-dimensional limitation of being restricted to these endogenous sequences. The distinct output of each combination of synthetic promoter and trans-element enables the expression of numerous transgenes at varying strengths,plant grow trays providing an additional dimension of control over synthetic genetic circuits. Eventually, these techniques can be expanded to design and build complex multi-layered gene expression systems with synthetic elements A rapidly growing global population has led to an increase in the motivation and enthusiasm for the engineering of plants to tackle impending societal challenges.
Many of these efforts have focused on agronomy, with the goal of increasing crop output per hectare by optimizing plant traits associated with abiotic/biotic stress, disease resistance, bio-fortification, and sustainability. Additionally, there is a growing interest in using plants for ‘molecular farming’, with engineered lines grown for the harvest of high-value compounds and proteins. In order to deliver on such agricultural biotechnology solutions, tools will need to be developed that address basic challenges in controlling transgene expression strength, enabling tissue-specific expression, and stacking multiple genes without risks of gene silencing. With these concerns in mind, we have developed a strategy for designing and testing synthetic transcriptional regulators and have demonstrated their utility in planta. Expanding the capacity for synthetic plant biologists to perform targeted and precise engineering will be indispensable for future complex multi-gene engineering efforts.Promoters are thought to be unidirectional in driving gene expression, often denoted as a single headed arrow, though they inherently show bidirectional behavior in most cases. Generally, the product of upstream transcription is short lived uaRNAs , and while currently unknown, may themselves have a function much like siRNAs and miRNAs. While we are not interested in the uaRNAs specifically, this phenomenon demonstrates the capacity for transcription at both the 5’ and 3’ end of the promoter. This leads us to believe there is potential to engineer a single promoter that drives productive transcription of both the sense and antisense strands of the flanking DNA.
This could be quite beneficial when designing synthetic gene circuits with numerous gene-coding sequences by limiting the number of elements required for desired functionality. Especially, when generating GMO plants that require years of work between generations when producing modified lines; and are limited in the amount of transgenic material that can be introduced with each cycle. We theorize that the system we developed for engineering synthetic promoters can be applied for the development of synthetic bidirectional promoters that generate independent mRNA sequences at both ends. In tandem with bidirectional promoter activity, it would be possible to link multiple CDSs with 2A peptide regions to develop polycistronic expression systems for use in plants . This would be especially promising, allowing for the expression of six genes with a single promoter and two terminators. This has the possibility to revise the current approach to plant engineering, which requires a promoter/terminator pair for each CDS. Decreasing the “genetic real estate” used for non-coding sequences like promoters and terminators would increase the capacity for the inclusion of functional CDSs in a single transformation event, accommodating more complex, layered, and stacked engineering schemes. Even though these first designs did not produce robust levels of output, we were successful in demonstrating bidirectional expression highlighting the potential for engineering bidirectional promoters. We have targeted the core promoters as the likely source for optimization of these primary designs, as additional work done with select Jores et al. core promoters found them to be under performing in our tobacco system .
Further comparison of methods also highlighted a key incongruity that is often overlooked. Their measurements were all taken at the mRNA level using barcoded sequences to measure transcription, a sound method for the examination of gene transcription. In our tobacco system, we analyze protein accumulation as a measurement of gene transcription. While both methods are correctly employed, they are measuring different aspects of the central dogma. Inconsistencies are often observed when correlating mRNA abundance and the resulting protein titers. In a theoretical scenario, mRNA with a short half-life and high translation efficiency could produce similar amounts of protein as mRNA with a long half-life and low translation efficiency. If one were to measure the protein abundance, it could be assumed the transcriptional/translational dynamics of both mRNAs were similar, due to similar titers. When measuring the abundance of each mRNA, one would find drastic differences due to varying transcript dynamics and could incorrectly assume this pattern would be observed at the protein level. When selecting core promoters based on mRNA measurements, we inappropriately assumed this would correlate with high levels of protein production, which is the goal for our promoter designs. The second iteration of our bidirectional promoters will shift back to using core promoter sequences mined from dicots, or tobacco specifically, that correlate with high levels of protein accumulation in the native system. These core promoters can then be substituted into our scheme for the optimization of bidirectional promoter design. Additionally, the Zm00001d041672 5’UTR would be substituted with varied 5’UTRs from the tobacco HSP family to determine the ideal elements for our application. The interest in plant natural products spans the entirety of human civilization, with references of plant-based medical remedies being documented throughout history. Plants have diversified over time to create an array of biosynthetic pathways that produce specialized metabolites with an immense range of chemical structure and function184. This can be attributed to their sedentary nature, which drives the evolution of numerous traits that chemically modulate interactions with an ever-changing environment. Advances in genome sequencing/mining has led to the characterization of numerous biosynthetic pathways responsible for the synthesis of alkaloids, terpenoids, phenolics, polyketides, and many more. Unfortunately, the large-scale production of many high-value molecules is hindered in plants. This is mainly due to the considerable energy and time input required to amass a purified product, which is often synthesized at very low concentrations in the native plant host. Synthetic biology offers a remedy to the challenge of mass production via microbial engineering. Introducing the genes of a biosynthetic pathway for a target molecule into a microbial host delivers a system for the mass production of plant natural products via fermentation in bioreactors. Saccharomyces cerevisiae has emerged as an appealing host for the bio-production of plant natural products due to its well-characterized genome, the understanding of native biosynthetic pathways,custom grow rooms the plethora of genetic tools available, and the presence of an endoplasmic reticulum as a eukaryotic microorganism. The latter is of great importance when dealing with plant natural products specifically, as many of the plant-derived enzymes required for biosynthesis are ER bound or interact directly with components of the ER. By optimizing biosynthetic pathway functionality in yeast, it is possible to achieve production titers of high-value plant natural products at far greater a concentration then the native plant host without the need for vast plots of land or fertilizer input. Terpenoids are a vast and diverse class of molecules with industrial and medicinal importance. Microorganisms, mainly Escherichia coli and Saccharomyces cerevisiae, have become choice platforms for the biosynthesis of terpenoids due to recent advances in synthetic biology and metabolic engineering.
New techniques for gene discovery have expanded our search space for novel terpene synthesis pathways and unlocked unrealized potential for the microbial production of more complex derivatives. Additionally, numerous advances in host and pathway engineering have allowed for the production of terpenoids requiring oxidation and glycosylation, effectively expanding the potential target space. These advances will lay the foundation for the microbial biosynthesis of a seemingly infinite domain of terpenoids with varying applications. Over 50,000 known terpenoids are naturally produced across all kingdoms of life and constitute the largest class of natural products. Though, the majority are the result of specialized metabolism in plants. Terpenoids are of major interest due to their applications in the food, pharmaceutical, and cosmetic industries as well as their potential as liquid fuels. Microbial biosynthesis is an optimal platform for the isolation and purification of individual terpenoid compounds. Additionally, this limits the need to grow, harvest and extract plant material, which generally contains low concentrations of the desired product along with complex mixtures of similar compounds and prevents the over harvesting of ecologically sensitive species. Terpenes are synthesized from C5 isoprene building blocks derived from the mevalonate or 1- deoxy-D-xylulose 5-phosphate pathway. These units are then condensed to produce larger and more complex molecules. The major terpene families are the hemiterpenes , monoterpenes , sesquiterpenes , diterpenes , triterpenes , and tetraterpenes . This space is further expanded by the oxidation, via cytochrome P450 oxygenases , and glycosylation, via glycosyltransferases , of the base terpene skeleton/scaffold . Furthermore, prenyltransferases add terpene units to other molecules to generate complex terpene composites, or meroterpenoids, which includes the cannabinoids. Within the last two years, large strides have been made in the microbial production of terpenoids from cannabis and hops, with a focus on prenylated chalcones, flavanones, bitter acids, and cannabinoids due to their distinct bio-active properties. All of these products result from the concatenation of a common isoprenoid precursor with a second fatty acid-derived precursor by a prenyltransferase. Prenyltransferases in cannabis and hops are severely under characterized yet are responsible for the large diversity within the cannabis & hop terpenomes. A combination of gene network and phylogenetic analyses has been used to identify candidate genes for many of these molecules. Microbial engineering is currently limited to the biosynthesis of known and well characterized terpenoid products. Computational biology, genomics, and transcriptomics are powerful tools for the identification of novel terpenoid synthesis pathways, ushering in a new era of synthetic biology. Yeast has proven to be a desirable host for the production of these complex terpenoids due to its endogenous MEV pathway, the presence of an endoplasmic reticulum to anchor membrane-associated plant enzymes, and the plethora of genetic tools for the metabolic engineering of this chassis. Bacterial engineering for terpene production has also seen recent success, but there is limited capacity for the expression of CYPs in these hosts without extensive modification of the native enzymes. Here, we focus on recent advances in the discovery of terpenoid synthesis pathways and their heterologous expression in microorganisms, as well as the expansion of the terpenoid target space through host engineering and the utilization of plant-derived CYPs and GTs. Detailed transcriptomics performed on extractions from glandular trichomes as well as female floral tissues of Cannabis sativa at varying developmental stages indicate that candidate genes involved in terpene and cannabinoid synthesis have abundant expression in trichomes. A recent study acquired high quality glandular trichome transcriptomes from nine different commercial cannabis strains in an attempt to characterize terpene synthases. A phylogenetic analysis of the whole genome contig database of C. sativa also revealed 24 o-methyltransferases and eight putative aromatic prenyltransferases, one of which was characterized as the penultimate synthase for cannaflavins A and B . Additionally, a total of more than 22,000 expressed sequence tags from several hop trichome-specific cDNA libraries have been deposited in the TrichOME database . This comprehensive transcriptome is a significant resource for further research into natural product pathway discovery and has massively advanced the potential for microbial biosynthesis of multiple terpenoids. Additionally, a recently unveiled metadata platform MaveDB aims to distribute and interpret data from multiplexed assays of variant effect and may be a valuable tool to inform rational engineering. There have been several key advancements in the production of terpenoids from both hops and cannabis in recent years. In 2018, an industrial brewing yeast was engineered to produce primary flavor determinants in hopped beer, allowing for a hoppy tasting beer in the absence of additional hops.