Abstract
Producing high concentrations of biobutanol is challenging, primarily because of the toxicity of butanol toward cells. In our previous study, several butanol tolerance-promoting genes were identified from butanol-tolerant Escherichia coli mutants and inactivation of the transcriptional regulator factor Rob was shown to improve butanol tolerance. Here, the butanol tolerance characteristics and mechanism regulated by inactivated Rob are investigated. Comparative transcriptome analysis of strain DTrob, with a truncated rob in the genome, and the control BW25113 revealed 285 differentially expressed genes (DEGs) to be associated with butanol tolerance and categorized as having transport, localization, and oxidoreductase activities. Expression of 25 DEGs representing different functional categories was analyzed by quantitative reverse transcription PCR (qRT-PCR) to assess the reliability of the RNA-Seq data, and 92% of the genes showed the same expression trend. Based on functional complementation experiments of key DEGs, deletions of glgS and yibT increased the butanol tolerance of E. coli, whereas overexpression of fadB resulted in increased cell density and a slight increase in butanol tolerance. A metabolic network analysis of these DEGs revealed that six genes (fadA, fadB, fadD, fadL, poxB, and acs) associated with acetyl-CoA production were significantly upregulated in DTrob, suggesting that Rob inactivation might enhance butanol tolerance by increasing acetyl-CoA. Interestingly, DTrob produced more acetate in response to butanol stress than the wild-type strain, resulting in the upregulation expression of some genes involved in acetate metabolism. Altogether, the results of this study reveal the mechanism underlying increased butanol tolerance in E. coli regulated by Rob inactivation.
Highlights
Butanol has received increased attention as a high-energydensity fuel and bulk chemical feedstock (Gu et al, 2011)
Strain DTrob showed a similar growth trend to control BW25113 in absence of butanol (Figure 1A), and the maximum cell density of the DTrob exhibit 94, 49, and 18% higher than that of BW25113 under 0.75, 1, and 1.25% (v/v) butanol stress, respectively, indicating that the DTrob strain was able to tolerate 1–1.25% and that the improvement in relative growth decreased with increasing butanol concentrations
The per unit intracellular butanol concentration (PIC) of the DTrob strain was 76, 56, and 78% of that of the control BW25113 under 0.75, 1, and 1.25% (v/v) butanol stress at 4 h, respectively (Figures 1B–D), which shows that inactivation of Rob can result in an enhanced ability to efflux butanol out of the cell to improve the tolerance, and the highest efflux capacity was shown in the presence of 1% butanol
Summary
Butanol has received increased attention as a high-energydensity fuel and bulk chemical feedstock (Gu et al, 2011). Traditional biobutanol is produced by Clostridium species and can reach a titer of approximately 20 g/L (Qureshi and Blaschek, 2000). The butanol titers produced by engineered Escherichia coli strains can reach approximately 20 g/L in batch fermentation without any antibiotics or inducers and up to 30 g/L with the continuous removal of butanol (Shen et al, 2011; Jang and Lee, 2015; Dong et al, 2017), indicating that E. coli is a potential butanol-producing bacterium. The current production of butanol is limited to approximately 20 g/L due to the toxicity of butanol toward bacteria; this toxicity is a bottleneck in butanol production (Qureshi and Blaschek, 2000). It is important to improve the butanol tolerance of chassis strains and to explore tolerance-associated mechanisms to promote the highly efficient production of butanol
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