Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance.

One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1-MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1-MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h(-1) while that of wild type was 0.20 h(-1). When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h(-1)) became twice that of the wild type (0.09 h(-1)). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.

A fluorescence-based high-throughput assay for the discovery of exchange protein directly activated by cyclic AMP (EPAC) antagonists.

BACKGROUND: The discovery, more than ten years ago, of exchange proteins directly activated by cAMP (EPAC) as a new family of intracellular cAMP receptors revolutionized the cAMP signaling research field. Extensive studies have revealed that the cAMP signaling network is much more complex and dynamic as many cAMP-related cellular processes, previously thought to be controlled by protein kinase A, are found to be also mediated by EPAC proteins. Although there have been many important discoveries in the roles of EPACs greater understanding of their physiological function in cAMP-mediated signaling is impeded by the absence of EPAC-specific antagonist. METHODOLOGY/PRINCIPAL FINDINGS: To overcome this deficit, we have developed a fluorescence-based high throughput assay for screening EPAC specific antagonists. Our assay is highly reproducible and simple to perform using the "mix and measure" format. A pilot screening using the NCI-DTP diversity set library led to the identification of small chemical compounds capable of specifically inhibiting cAMP-induced EPAC activation while not affecting PKA activity. CONCLUSIONS/SIGNIFICANCE: Our study establishes a robust high throughput screening assay that can be effectively applied for the discovery of EPAC-specific antagonists, which may provide valuable pharmacological tools for elucidating the biological functions of EPAC and for promoting an understanding of disease mechanisms related to EPAC/cAMP signaling.

cAMP-CRP activator complex and the CytR repressor protein bind co-operatively to the cytRP promoter in Escherichia coli and CytR antagonizes the cAMP-CRP-induced DNA bend.

Initiation of transcription from the cytRP promoter in Escherichia coli is activated by the cAMP-CRP complex and negatively regulated by the CytR repressor protein. By combining gel retardation and footprinting assays, we show that cAMP-CRP binds to a single site centered at position -64 and induces a considerable bend in the DNA. CytR binds to a region immediately downstream from, and partially overlapping, the CRP site, and induces a modest bend into the DNA. In combination, cAMP-CRP and CytR bind co-operatively to cytRP forming a nucleoprotein complex in which the proteins directly interact with each other and bind to the same face of the DNA helix. CytR binding concomitantly antagonizes the cAMP-CRP-induced bend. This study indicates that the minimal DNA region required to obtain CytR regulation consists of a single binding site for each of cAMP-CRP and CytR. The case described here, in which a protein-induced DNA bend is modulated by a second protein, may illustrate a mechanism that applies to other regulatory systems.