A library of synthetic transcription activator‐like effector‐activated promoters for coordinated orthogonal gene expression in plants

Abstract

A library of synthetic promoters containing the binding site of a single designer transcription activator-like effector (dTALE) was constructed. The promoters contain a constant sequence, consisting of an 18-base long dTALE-binding site and a TATA box, flanked by degenerate sequences of 49 bases downstream and 19 bases upstream. Forty-three of these promoters were sequenced and tested in transient assays in Nicotiana benthamiana using a GUS reporter gene. The strength of expression of the promoters ranged from around 5% to almost 100% of the viral 35S promoter activity. We then demonstrated the utility of these promoters for metabolic engineering by transiently expressing three genes for the production of a plant diterpenoid in N. benthamiana. The simplicity of the promoter structure shows great promise for the development of genetic circuits, with wide potential applications in plant synthetic biology and metabolic engineering.

On the road to C4 rice: advances and perspectives

“…Synthetic biology has also provided a potential solution to this paucity of promoters in rice. Brückner et al () described a system compatible with the Golden Gate cloning which utilises multiple promoters (Synthetic TALE Activated Promoters or STAPs) designed to be orthogonal to the genome of the plant to be transformed, which can be activated by a single Transcription Activator‐Like Effector (TALE). This approach provides the opportunity to build multiple transcriptional units driven by different promoters on the same gene construct, trans ‐activated by a single transcription factor.…”

Synthetic Botany

“…For instance, Nicotiana benthamiana leaves can be infiltrated with Agrobacterium at multiple regions of a single leaf and screened for gene expression a few days after infection. This allows relatively fast characterization of libraries of genetic components in a common genetic background (Sparkes et al 2006;Engler et al 2014;Brückner et al 2015). Transient leaf agroinfiltration in Nicotiana has been adopted as an efficient method for the optimization of metabolic pathways, such as artemisinin (van Herpen et al 2010) and triterpene biosynthesis pathways (reviewed in Thimmappa et al 2014), and the bioproduction of vaccines (D'Aoust et al 2008, Mardanova et al 2015.…”

Rational Design of Minimal Synthetic Promoters for Plants

“…While all core histones and the linker histone, H1, have been shown to associate with transiently delivered exogenous DNA in mammalian cells (albeit with aberrant stoichiometry) (66, 67), this has not been investigated in plant cells. Several synthetic promoters and cognate orthogonal TFs for plants, including those that can be induced by chemical signals, have been engineered for plant systems (24)(25)(26). In this study we aimed to expand on those efforts, creating regulatory elements of different strengths for use in the construction of larger genetic circuits, particularly biosynthetic pathways, in which it is desirable to control the relative expression levels of different proteins.…”

“…To identify a functional basic design for minimal synthetic plant promoters (MinSyn-P), we first built and tested synthetic promoters with a range of expression levels that respond to orthogonal TFs. The initial design was based on previously reported synthetic Transcriptional Activator Like Effector (TALE)-responsive synthetic elements to which single binding sites for TALES were added (26). These synthetic promoters consist of 19 bps of random sequence, followed by a second region of random sequence of flexible length (to which CREs are added), a TATA box sequence (TATATAA) and a 43 bp minimal core including transcriptional start site (TSS) (Figure 2A).…”

“…Progress has been made by engineering variations of natural promoters for tissue-specific expression (23). In addition, a number of synthetic regulatory elements comprised of binding sites for orthogonal TFs fused to a minimal core promoter have been used to enable inducible constitutive expression (24)(25)(26). However, to initiate the flow of transcription, these require a non-orthogonal promoter to drive expression of the orthogonal TF, for which natural are typically used (27).…”