Engineering Trans-Acting Regulators to Dynamically Control Gene Expression in Bacteria

Simple and predictable trans-acting regulatory tools are needed in the fields of metabolic engineering and synthetic biology and to build complex regulatory circuits and optimize the levels of native and heterologous gene products (Copeland et al., Curr Opin Biotechnol, 2014). Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. Previously, we demonstrated that a TALE effector repurposed to bind the lac operator is able to inhibit transcription initiation and elongation events in E. coli (Politz et al., ChemComm, 2013). In this presentation, I will summarize the state of TALEs as bacterial transcription factors and describe our recent efforts to expand their versatility to create an inducible TALE system (Copeland, Politz et al., Nature Chemical Biology, 2016). To do so, the TALE backbone was engineered to contain tobacco-etch virus (TEV) protease recognition sites. The resulting engineered TALEs maintain repression of their target genes, but are degraded following induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to LacI-IPTG but with the added flexibility of being operator agnostic. In this talk, I will describe our efforts to apply the TALE technology in metabolic engineering efforts in E. coli to produce oleochemicals. I will also compare the TALE technology with CRISPRi technology developed by many groups, but here with constructs we developed for use in the fast growing cyanobacterium Synechococcus sp. strain PCC7002. I will describe how we are using these technologies to increase the photosynthetic production of chemicals in cyanobacteria.