Abstract:Programmable transcriptional factors based on the CRISPR
architecture
are becoming commonly used in plants for endogenous gene regulation.
In plants, a potent CRISPR tool for gene induction is the so-called
dCasEV2.1 activation system, which has shown remarkable genome-wide
specificity combined with a strong activation capacity. To explore
the ability of dCasEV2.1 to act as a transactivator for orthogonal
synthetic promoters, a collection of DNA parts was created (GB_SynP)
for combinatorial synthetic promoter … Show more
“…To investigate if this copper‐sensing dCasEV2.1 system would enable control over pheromone biosynthesis, we assembled the coding sequence of each pathway enzyme with a synthetic promoter activated by the dCasEV2.1 system. To reduce the amount of sequence repeated within each transcriptional unit, and therefore minimize the potential for gene silencing, pathway genes were each assembled with synthetic promoters that had minimal sequence similarity (Moreno‐Giménez et al ., 2022 ). Each promoter consisted of three parts: a distal region consisting of random sequence unique to each promoter and lacking any known transcription factor binding sites (parts GB2815, GB3269 and GB3270); a proximal region containing three copies of the gRNA recognition site flanked by random sequence (parts GB3275, GB3276 and GB3277) and a constant minimal DFR core region (part GB2566).…”
Summary
Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species‐specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low‐cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9‐based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.
“…To investigate if this copper‐sensing dCasEV2.1 system would enable control over pheromone biosynthesis, we assembled the coding sequence of each pathway enzyme with a synthetic promoter activated by the dCasEV2.1 system. To reduce the amount of sequence repeated within each transcriptional unit, and therefore minimize the potential for gene silencing, pathway genes were each assembled with synthetic promoters that had minimal sequence similarity (Moreno‐Giménez et al ., 2022 ). Each promoter consisted of three parts: a distal region consisting of random sequence unique to each promoter and lacking any known transcription factor binding sites (parts GB2815, GB3269 and GB3270); a proximal region containing three copies of the gRNA recognition site flanked by random sequence (parts GB3275, GB3276 and GB3277) and a constant minimal DFR core region (part GB2566).…”
Summary
Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species‐specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low‐cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9‐based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.
“…We reasoned that the observed signal saturation could be due to the choice of Luz as the connection point for the transcriptional output, as we earlier determined that the HispS gene and to a lesser extend the H3H gene, are the transcriptionally limiting steps in the pathway 9 (see Supplementary Fig. 1).…”
Engineered autonomous bioluminescence (EAB) offers many potential applications in Plant Synthetic Biology, notably as in vivo reporter system. Current EAB reporter configurations are limited for quantitative applications due to low dynamic range. We reconfigured the Neonothopanus nambi fungal bioluminescence (NeoLuc) pathway to serve as a high-throughput and inexpensive reporter for quantitative analysis of gene expression. We showed that by configuring the first committed step in the pathway (HispS) as the transcriptional entry point instead of the fungal luciferase, the dynamic range of the output increased dramatically, equaling that of the FLuc/RLuc reporter, and outperforming it in high throughput capacity. Furthermore, the inclusion of an enhanced GFP as normalizer allowed transient ratiometric measurements in N. benthamiana. Fast and rich datasets generated by the NeoLuc/eGFP system enabled us to undertake the optimization of new challenging synthetic gene circuits, including a complex agrochemical/optogenetic dual input switch for tight control of engineered metabolic pathways.
“…In contrast, the discovery of enzymes catalyzing light-emitting caffeic acid cycle in the fungus Neonothopanus nambi (Fig. 1a ) quickly translated into the development of multicellular organisms with autonomous luminescence 6 – 8 and reporter tools for transient expression assays in planta 9 – 11 . Fig.…”
The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
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