Rewiring global regulator cAMP receptor protein (CRP) to improve E. coli tolerance towards low pH.
Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we tried to improve strain acidic tolerance from its transcription level, i.e. we adopted error-prone PCR method to engineer global regulator cAMP receptor protein (CRP) of Escherichia coli to improve its performance at low pH. The best mutant AcM1 was identified from random mutagenesis libraries based on its growth performance. AcM1 almost doubled (0.113h(-1)) the growth rate of the control (0.062h(-1)) at pH 4.24. It also demonstrated better thermotolerance than the control at 48°C, whose growth was completely inhibited at this temperature. Quantitative real time reverse transcription PCR results revealed a stress response overlap among low pH stress-, oxidative stress- and osmotic stress-related genes. The chief enzyme responsible for cell acid tolerance, glutamate decarboxylase, demonstrated over twofold activity in AcM1 compared to the control. Differential binding properties of AcM1 mutant CRP with Class-I, II, and III CRP-dependent promoters suggested that modifications to native CRP may lead to transcription profile changes. Hence, we believe that transcriptional engineering of global regulator CRP can provide a new strain engineering alternative for E. coli.
School of Chemical & Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
Souvik
S
Basak
School of Chemical & Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore.
Hefang
H
Geng
School of Chemical & Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore. Electronic address: rrjiang@ntu.edu.sg.
Rongrong
R
Jiang
D019342:Acetic Acid / Q000378:metabolism*; D000222:Adaptation, Physiological / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002373:Cyclic AMP Receptor Protein / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D019802:Succinic Acid / Q000378:metabolism*; D013696:Temperature
D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; C481662:crp protein, E coli; D019802:Succinic Acid; D005968:Glutamate Decarboxylase; D019342:Acetic Acid
Random mutagenesis; Strain engineering; Transcriptional engineering; cAMP receptor protein