Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance

Abstract

The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor protein (CRP) of Escherichia coli can improve cell phenotypes. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. The target phenotype in this study is strain osmotolerance. Amino acid mutations were introduced to CRP by either error-prone PCR or DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1-MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1-MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to the wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9 M NaCl, the growth rate of MT6 is 0.113 h(-1), while that of WT is 0.077 h(-1). MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose, and sucrose. DNA microarray analysis showed that genes involved in colanic acid biosynthesis are up-regulated in the absence of salt stress, whereas carbohydrate metabolic genes are differentially expressed under NaCl stress when comparing MT6 to WT. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth.

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

“…coli DH5α (Invitrogen, San Diego, USA) was used as the host organism and cultured in modified LB medium supplemented with 0.1 % glucose and 10 mM MgSO 4 (LBGMg). Plasmid pKSCP was obtained from our previous studies (Zhang et al 2012). Restriction enzymes were purchased from Fermentas (Burlington, Canada).…”

“…Both error-prone PCR and DNA shuffling libraries were constructed as described previously (Zhang et al 2012). In brief, error-prone PCR was performed using GeneMorph® II Random Mutagenesis Kit according to the manufacturer's instructions.…”

“…After cAMP binding to CRP, the CRP-cAMP complex can act as a dual regulator by inducing sequence-specific DNA recognition within or near the promoters of the genes that CRP regulates (Botsford and Harman 1992). Our group has successfully engineered CRP before to improve cell osmotolerance (Zhang et al 2012), and here in this work, we would like to broaden the application to enhance cell 1-butanol tolerance.…”

Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

“…Selection from genomic overexpression libraries is another common approach that has been employed to develop strains with improved growth phenotypes under stress conditions, including exposure to high levels of acetate, butanol, hydrogen peroxide, or inhibitory compounds present in lignocellulosic hydrolysates (16)(17)(18)(19)(20). Global transcription machinery engineering (gTME), where a global transcriptional regulator is targeted for mutagenesis in order to quickly generate complex phenotypic changes based on transcriptome perturbation (21), is a more targeted method that has also been widely utilized to create new host strains (21,22). Each of these methods has its own benefits and drawbacks relating to the degree of phenotypic variation that can be introduced, the ability to relate a specific set of genetic changes to a phenotypic change, and the effort required to implement the method.…”

Osmotolerance in Escherichia coli Is Improved by Activation of Copper Efflux Genes or Supplementation with Sulfur-Containing Amino Acids

“…KEYWORDS osmotolerance, CusS, copper efflux, Cu(I), methionine, Escherichia coli R esistance to high osmotic pressure is a desirable phenotype for engineered bacterial strains for the efficient production of fuels and chemicals in industrial bioprocesses (1)(2)(3)(4)(5)(6). Batch fermentation with a high initial glucose concentration is also desirable in industrial production, as it eliminates the need for glucose feeding and simplifies the bioprocess.…”