Abstract
A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1– E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h−1 in 62 g/L ethanol, higher than that of the control at 0.06 h−1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.
Highlights
The use of bioethanol as alternative fuel has drawn greater attention than ever due to recent energy crisis and environmental concerns [1], and production of ethanol from microbial fermentation is of practical value in replacing fossil fuel utilization
Recombinant plasmids with mutated crp inserts were transformed into competent E. coli JW5702 Dkan and the total error-prone library size was greater than 106
The mutated crp inserts of these ‘‘winners’’ were digested, re-ligated to freshly prepared plasmid pKSC, and the resulting recombinant plasmids were re-transformed into E. coli JW5702 Dkan background to eliminate false positives or chromosomal mutations
Summary
The use of bioethanol as alternative fuel has drawn greater attention than ever due to recent energy crisis and environmental concerns [1], and production of ethanol from microbial fermentation is of practical value in replacing fossil fuel utilization. Different microorganisms, including yeast [2,3], Zymomonas mobilis [4,5] and E. coli [6,7] have been engineered for selective production of ethanol. The highest reported ethanol yield attained through E. coli xylose fermentation is around 60 g/l [8]. The yields and titers from the microbial fermentation is usually held back by the accumulation of toxic end-product ethanol [9,10]. There are two conventional approaches to improve strain performance under ethanol stress: i) ‘‘random approach’’ with UV/chemical mutagens [11] and adaptive evolution [8,12] ii) ‘‘rational approach’’ of using metabolic engineering tools [13,14]. As for the ‘‘rational approach’’, the lack of detailed metabolism knowledge for many microorganisms often limits its use [15]
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