Trans-acting repressors as tools for metabolic engineering in Escherichia coli

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Trans-regulators, such as TALEs, CRISPR/Cas, and sRNAs, can be easily designed to bind target sequences due to the simplicity of the rules governing their interactions with nucleic acids. These trans-regulators enable us to repress the expression of genes at the transcriptional or post-transcriptional levels, enabling us to down-regulate gene expression without prior manipulation of cis-acting and/or chromosomal target DNA sequences. We are now harnessing these trans-repressors to target genes encoding enzymes involved in central metabolism in an engineered strain of E. coli in to improve the yield and titer of free fatty acids and fatty alcohols. We are particularly interested in knocking down, but not knocking out, genes encoding TCA cycle enzymes, such as sucABCD or gltA, in order to both reduce acetyl-CoA flux into the cycle and the loss of carbon as CO₂. These strategies demonstrate the utility of using trans-acting repressors to tune down gene expression and as tools for rapidly screening candidates for future gene knockouts.