Enhancing E. coli isobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP)

Abstract

The limited isobutanol tolerance of Escherichia coli is a major drawback during fermentative isobutanol production. Different from classical strain engineering approaches, this work was initiated to improve E. coli isobutanol tolerance from its transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP). Random mutagenesis libraries were generated by error-prone PCR of crp, and the libraries were subjected to isobutanol stress for selection. Variant IB2 (S179P, H199R) was isolated and exhibited much better growth (0.18 h(-1) ) than the control (0.05 h(-1) ) in 1.2% (v/v) isobutanol (9.6 g/L). Genome-wide DNA microarray analysis revealed that 58 and 308 genes in IB2 had differential expression (>2-fold, p < 0.05) in the absence and presence of 1% (v/v) isobutanol, respectively. When challenged with isobutanol, genes related to acid resistance (gadABCE, hdeABD), nitrate reduction (narUZYWV), flagella and fimbrial activity (lfhA, yehB, ycgR, fimCDF), and sulfate reduction and transportation (cysIJH, cysC, cysN) were the major functional groups that were up-regulated, whereas most of the down-regulated genes were enzyme (tnaA) and transporters (proVWX, manXYZ). As demonstrated by single-gene knockout experiments, gadX, nirB, rhaS, hdeB, and ybaS were found associated with strain isobutanol resistance. The intracellular reactive oxygen species (ROS) level in IB2 was only half of that of the control when facing stress, indicating that IB2 can withstand toxic isobutanol much better than the control.

Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7

“…In recent years, engineering components of global transcription machinery has been explored to fulfill the requirement of fine-tuning or reprogramming microbial cellular transcription profile. In prokaryotic microbes, a few key regulators have been successfully engineered to alter Escherichia coli ( E. coli ) and Zymomonas mobilis phenotypes, including sigma factor σ 70 [2, 45], alpha subunit of RpoA [27], exogenous regulator IrrE [9], global regulator Hha & H-NS [21, 22], cAMP receptor protein (CRP) [11, 53]. In eukaryotic microbes, the transcriptional machinery is more complex, with a large set of general and specific transcription factors involved [15].…”

Engineering of global regulator cAMP receptor protein (CRP) in Escherichia coli for improved lycopene production

“…This offers a unique opportunity to induce a simultaneous global transcription-level alteration that could potentially impact cellular properties. The engineering of CRP has been applied to improve various endurance-related phenotypes of E. coli, such as the tolerance to biofuel toxicity (Chong et al, 2014(Chong et al, , 2013aZhang et al, 2012b), osmotic pressure (Zhang et al, 2012a), acetate or low pH (Chong et al, 2013b;Basak et al, 2014), organic solvent toxicity and oxidative stress . However, beyond the tolerance phenotypes, the reframing of the entire E. coli transcriptional machinery through CRP engineering to improve the metabolite overproduction has not been reported.…”

Succinate production positively correlates with the affinity of the global transcription factor Cra for its effector FBP in Escherichia coli

“…Global regulation has attracted increasing attention because of its effective regulation of the expression of multiple pathway-related genes [4] and required cell phenotypes [5]. Furthermore, transcriptional engineering approaches have been regarded as important [6, 7]. In prokaryotes, gene expression is controlled by transcription factors (TFs) via their binding to specific DNA sequences [8].…”