Supercoiling-mediated feedback rapidly couples and tunes transcription

Johnstone, Christopher P.; Galloway, Kate E.

doi:10.1016/j.celrep.2022.111492

Cited by 50 publications

(34 citation statements)

References 65 publications

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“…3-fold decrease for SGD, 4-fold decrease for GS, 1-fold decrease for GO) (Figure D). The repetitive use of the strong promoter may have caused homology-mediated epigenetic silencing or disturbed the transcriptional dynamics, when using the 4TU_SGD_GS_GO_Redox1 vector. , This is the likely cause of the decrease in PCA yield, as the levels of Redox2 and SAT coinfiltrated on separate vectors remained high under all conditions (Supplementary Figure S3). Notably, in the 2TU_SGD_GS + 2TU_GO_Redox1 coinfiltration experiments, the relative gene expression of SGD, GS, and GO was significantly higher compared to levels of these same genes in not only the 4 TU construct 4TU SGD_GS_GO_Redox1, but also with the positive control in which all genes were expressed from separate plasmids (All SlUbq10 coinfiltration) (Figure D).…”

Section: Resultsmentioning

confidence: 99%

“…The repetitive use of the strong promoter may have caused homology-mediated epigenetic silencing or disturbed the transcriptional dynamics, when using the 4TU_SGD_GS_GO_Redox1 vector. 40,41 This is the likely cause of the decrease in PCA yield, as the levels of Redox2 and SAT coinfiltrated on separate vectors remained high under all conditions (Supplementary Figure S3). Notably, The statistically significant difference between PCA yields are marked with an asterisk.…”

Section: Acs Synthetic Biologymentioning

confidence: 98%

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Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

et al. 2022

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Vinblastine is a chemotherapy agent produced by the plant Catharanthus roseus in small quantities. Currently, vinblastine is sourced by isolation or semisynthesis. Nicotiana benthamiana is a plant heterologous host that can be used for reconstitution of biosynthetic pathways as an alternative natural product sourcing strategy. Recently, the biosynthesis of the latestage vinblastine precursors precondylocarpine acetate, catharanthine, and tabersonine have been fully elucidated. However, the large number of enzymes involved in the pathway and the unstable nature of intermediates make the reconstitution of late-stage vinblastine precursor biosynthesis challenging. We used the N. benthamiana chassis and a state-of-art modular vector assembly to optimize the six biosynthetic steps leading to production of precondylocarpine acetate from the central intermediate strictosidine (∼2.7 mg per 1 g frozen tissue). After selecting the optimal regulatory element combination, we constructed four transcriptional unit assemblies and tested their efficiency. Finally, we successfully reconstituted the biosynthetic steps leading to production of catharanthine and tabersonine.

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Section: Resultsmentioning

confidence: 99%

Section: Acs Synthetic Biologymentioning

confidence: 98%

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

et al. 2022

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“…Furthermore, 44.9% of all TUs overlap with either GapR or TopoI peaks, and 73.1% of these overlapping regions are contributed by only GapR peaks. Previous studies have shown that DNA supercoils generated by ongoing transcription activities could modulate the cooperativity of adjacent RNA polymerase molecules on the same gene and also impact the transcription of adjacent genes 20 21 . Therefore, the observation that more GapR than TopoI peaks reside in adjacent genes suggest that positive supercoiling also plays a more pervasive role than negative supercoiling in regulating E. coli transcription.…”

Section: Resultsmentioning

confidence: 99%

Differential roles of positive and negative supercoiling in organizing theE. coligenome

Guo

Zhou

et al. 2022

Preprint

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This study aims to explore whether and how positive and negative supercoiling contribute to the three-dimensional (3D) spatial organization of the bacterial genome. We used recently published GapR ChIP-seq and Topo-seq data, which marks positive and negative supercoiling sites, respectively, to study how GapR and TopoI binding sites correlate with the corresponding contact frequencies obtained from the Hi-C data of the E. coli chromosome. We found that chromosomal loci enriched with GapR or TopoI binding sites have overall higher Hi-C contact frequencies than sites that do not contain the corresponding binding sites, with GapR peaks corresponding to higher spatial contacts than TopoI peaks. Additionally, we found that Hi-C contact frequencies alone could identify GapR, but not TopoI, binding sites with high accuracy. Finally, we found that majority of positive and negative supercoils coincides with highly active transcription units, with a minor group likely associated with replication. Our results suggest that both positive and negative supercoiling enhance chromosomal interactions, but the positive supercoils contribute significantly more than the negative supercoils to bring distant chromosomal loci closer in space.

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“…Using ratiometric reporter assays, we found that the position in which genes are located within multigene constructs differentially affects their expression ( Figure 3 ). In other systems, the dominance of the upstream genes has been proposed to be caused by positive supercoiling accumulates downstream of constitutively active genes, altering RNA polymerase binding and initiation (Johnstone and Galloway, 2022). In our work, we found the extent to which genes were affected by co-assembly to be sequence-dependent ( Figure 3 ).…”

Section: Discussionmentioning

confidence: 99%

Tunable control of insect pheromone biosynthesis inNicotiana benthamiana

Kallam

Moreno-Giménez

Mateos-Fernández

et al. 2022

Preprint

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Synthetic regulatory elements that provide control over the timing and levels of gene expression are useful for maximizing yields from heterologous biosynthetic pathways. Previous work has demonstrated that plants and microbes can be engineered to produce insect sex pheromones, providing a route for low-cost production of these compounds, which are valued for species-specific control of agricultural pests. Strong constitutive expression of pathway genes can lead to toxicity and metabolic loads, preventing normal growth and development and thus limiting biomass and affecting overall yields. In this study we demonstrate the ability to inducibly control the accumulation of Lepidopteran sex pheromones in leaves of Nicotiana benthamiana. Further, we show how construct architecture influences expression and product yields in multigene constructs, applying this to control the relative expression of genes within the pathway, thereby tuning the accumulation of pheromone components. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

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Supercoiling-mediated feedback rapidly couples and tunes transcription

Cited by 50 publications

References 65 publications

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine in Nicotiana benthamiana

Differential roles of positive and negative supercoiling in organizing theE. coligenome

Tunable control of insect pheromone biosynthesis inNicotiana benthamiana

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