Design of a tunable bacterial gene expression system using engineered σ factors.

Unlabelled: Extracytoplasmic function (ECF) σ factors selectively upregulate expression of specific genes in bacteria. These σ factors, belonging to the σ family, are much smaller than the primary, housekeeping σ factor with two helical domains that interact with the Pribnow box and the -35 element of the promoter DNA. Structural studies reveal that promoter specificity in a σ factor is determined by the interactions between a loop (L3) and the Pribnow box element. Similarly, the efficiency of transcription initiation is governed by the polypeptide linker between the two promoter-binding domains. Both these polypeptide segments are dynamic and poorly conserved among ECF σ factor homologs. This feature hitherto limited insights from protein-DNA interactions to be correlated with transcription initiation efficiency. Here, we describe an approach to characterize these features that govern the dynamic range of gene expression using chimeric σ. The L3 loop and linker polypeptides in these σ chimeras were replaced by the corresponding segments from 10 annotated and functional ECF σ's. and measurements to determine the effect of these polypeptide replacements provided an experimentally validated σ chimera- gene expression level data set. We illustrate the utility of this chimeric σ library in improving the efficiency of a biosynthetic pathway in . In a two-enzyme step, unaffected by feedback inhibition and substrate concentration, we show an increase in desired product levels by altering the relative intracellular levels of the target enzymes using this library of σ factors. The chimeric σ library thus demonstrates the feasibility of engineering σ factors to achieve bespoke expression levels of target genes for diverse applications in synthetic microbiology.

Importance: The synthesis of organic compounds involves the action of multiple enzymes in a biosynthetic pathway. Incorporating such biosynthetic pathways into microbes often leads to substantial cellular and metabolic stress resulting in low titers of the target compound. This limitation can be offset, in part, by optimizing enzyme efficiency and cellular enzyme concentration. The former involves significant efforts to achieve improvements in catalytic efficiency with the caveat that the metabolic load on a microbial cell imposed by the overexpression of the exogenous enzyme could result in reduced cell fitness. Here, we demonstrate the feasibility of engineered σ factors to modulate gene expression levels without significant genetic engineering. We note that changing the sequence of two flexible polypeptide loops without any changes to the structural scaffold of the transcription initiation factor σ could modulate the expression levels of the target genes. This ability provides a route to improve the efficiency of a biosynthetic pathway without altering the overall genomic makeup. The σ chimera library thus provides an avenue for pre-determined conditional gene expression of specific genes in .