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Deciphering the regulatory genome of Escherichia coli, one hundred promoters at a time.

ABSTRACT: Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ?65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than a E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.

SUBMITTER: Ireland WT

PROVIDER: S-EPMC7567609 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Deciphering the regulatory genome of <i>Escherichia coli</i>, one hundred promoters at a time.

Ireland William T WT Beeler Suzannah M SM Flores-Bautista Emanuel E McCarty Nicholas S NS Röschinger Tom T Belliveau Nathan M NM Sweredoski Michael J MJ Moradian Annie A Kinney Justin B JB Phillips Rob R

eLife 20200921

Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium <i>Escherichia coli</i>, for ≈65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than a <i ...[more]

PMID: 32955440

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Project description:BACKGROUND:The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two ?70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. RESULTS:We showed that, in genome-integrated E. coli expression systems that used ?70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. CONCLUSIONS:Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Dataset Information

Deciphering the regulatory genome of Escherichia coli, one hundred promoters at a time.

Publications

Deciphering the regulatory genome of <i>Escherichia coli</i>, one hundred promoters at a time.

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