Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli
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ORIGINAL PAPER
Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli Dayanidhi Sarkar Æ Khandaker Al Zaid Siddiquee Æ Marcos J. Arau´zo-Bravo Æ Takahiro Oba Æ Kazuyuki Shimizu
Received: 23 December 2007 / Revised: 9 May 2008 / Accepted: 30 June 2008 / Published online: 22 July 2008 Ó Springer-Verlag 2008
Abstract To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from footprinting data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-
Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally. Communicated by Erko Stackebrandt. D. Sarkar K. A. Z. Siddiquee M. J. Arau´zo-Bravo K. Shimizu (&) Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, 680-4, Kawazu, Iizuka, Fukuoka 820-8502, Japan e-mail: [email protected]; [email protected] T. Oba Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Aikawa-Machi 1465-5, Kurume, Fukuoka 839-0861, Japan K. Shimizu Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan
regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Keywords Escherichia coli cra mutant cra.edd mutant cra.edd.iclR mutant DNA microarray
Introduction The central metabolic pathways of Escherichia coli are controlled by a number of global regulators depending on the carbon sources available and the culture condition. Among them, the catabolite repressor/activator protein (Cra) initially characterized as the fructose repressor, FruR plays an important role in the control of carbon flow in E. coli (Moat et al. 2002; Saier and Ramseier 1996; Saier et al. 1997). The genes such as ptsHI, pfkA, pykF, acnB, edd–eda, fruBKA, mtlADR, gapB are reported to be negatively controlled, while ppsA, fbp, pckA, acnA, icd, aceA, aceB, and cydA,B are positively controlled by the Cra protein (Saier and Ram
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