TAL Effectors As Artificial Transcription Factors For Synthetic Biology Applications

Synthetic biology promises to deliver advances in the production of commodity chemicals and fuels as well as novel genetic circuits and biosensors. In order to accomplish these feats, artificial transcription factors that could be engineered to target any DNA sequence of interest would be highly beneficial. Previous efforts have either used zinc finger technology, antisense RNA, or site-specific DNA binding proteins (native or heterologous) to accomplish design goals, but these strategies require screening or the use of a particular DNA binding site. Transcription activator-like effectors (TALEs), which may be designed to bind any sequence of interest, have recently emerged as an alternative for the regulation of gene expression at the transcriptional level. These plant virulence factors from Xanthomonas species have already been used for genome editing and as transcription factors in eukaryotes, including in human cell lines, and hold the potential to adjust the expression levels of genes involved in pathways of interest in prokaryotic organisms.

The potential clinical applications of TALEs, which have been the major focus to date, do not necessitate a quantitative understanding of TALE DNA binding activity. On the other hand, metabolic engineering applications, such as the repression of genes in central metabolism to increase the yield of a desired product, would benefit greatly from a quantitative understanding of the relationship between TALE primary structure and activity in order to finely tune the expression of genes and associated metabolite concentrations. To assess the validity of this approach, a TALE was designed to target the lacO1 operator. This designer transcription factor  was able to repress expression of a red fluorescent reporter gene downstream of an IPTG-inducible promoter in Escherichia coli.¹ Further, the expression of this TALE was shown to alter the ratio of expression of two genes in an artificial operon harboring the lacO1 operator in the intergenic space.¹ While these proof-of-concept experiments illustrate that TALEs retain their functionality in prokaryotes, efforts are currently underway to gain a quantitative understanding of how TALE structure affects activity in vivo. In addition, this technology will be applied to improve the yields of free fatty acids in an overproducing strain of E. coli.

1. Politz, M. C.; Copeland, M. F.; Pfleger, B. F., Chemical Communications 2013.