Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Abstract

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions.

Modular optimization of multi-gene pathways for fatty acids production in E. coli

“…Engineering mutant and hybrid promoters with modified RNA polymerase, activator or repressor-binding activities has been widely used in metabolic engineering for transcriptional finetuning and pathway optimization 17,27,28 . However, the genetic recalcitrance of the bacteriophage T7 promoter has restricted our ability to manipulate the consensus promoter core sequence ( À 35 and À 10 region) and the lacI repressor binding region (lacO) 29 .…”

Precision genetic modifications: a new era in molecular biology and crop improvement

“…Transcription activator-like effectors fused to known repressor domains can also repress transcription, as shown in mammalian and yeast cells (Blount et al 2012;cong et al 2012), bacteria (Politz et al 2013) and Drosophila (crocker and stern 2013). In plants, an AvrBs3-like effector protein (hax3 from X. camprestris pv.…”

“…however, the possibility to use TAlEs for other traits remains to be explored, although their application in other fields, like synthetic biology, is increasing steadily (Blount et al 2012).…”

Synthetic Biology: opportunities for Chilean bioindustry and education

“…Most biotechnological developments rely on the modifi cation of existing natural components such as the recent generation of genome editing tools based on mechanisms of Xanthomona's infection of plants (Garg et al, 2012;Boch et al, 2009;Moscou et al, 2009;Blount et al, 2012). Chile has a unique opportunity to contribute to expanding this repertoire of biological components, processes and chassis by tapping into its genetic biodiversity.…”