Metabolic impact of transient genetic perturbations in Escherichia coli and Pseudomonas putida

• Grant number: 13/24087-8
• Support type: Scholarships in Brazil - Post-Doctorate
• Effective date (Start): August 01, 2014
• Effective date (End): July 31, 2016
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: José Gregório Cabrera Gomez
• Grantee: Karel Olavarria Gamez
• Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Despite the huge amount of available -omics data, relevant characteristics of the living beings such as self-construction, robustness and reorientation of the metabolic fluxes are poorly understood. We propose a novel experimental approach to tackle this challenge: we will provoke transitory perturbations upon the addition of a non-hydrolysable inducer. Our case studies will be 1) the temporary substitution of the glyceraldehyde-3-phosphate dehydrogenase from Escherichia coli (NAD-G3PDH) by the non-phosphorylating NADP-preferring glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutants (NADP-G3PDH); and 2) the temporary expression of the phosphofructokinase (PFK) from E. coli in Pseudomonas putida. To accomplish the temporal replacement of the NAD-G3PDH by the NADP-G3PDH in E. coli, the gene encoding for the NAD-G3PDH will be replaced by a modified version, encoding for a tagged NAD-G3PDH, degradable by an inducible protease. The genes encoding for this protease and the NADP-G3PDH (both under the control of the Lac promoter) will be expressed upon the addition of isopropyl-²-D-thiogalactoside (IPTG). In P. putida, the pfkA gene (under the control of the Lac promoter) will be expressed using a suitable plasmid. Using continuous cultures it will be possible to control the addition and outflow of the IPTG and, concomitantly, observe the distributions of the metabolic fluxes, the concentrations of the main metabolites and the transcriptional profile before, during and after the perturbations. The gathered data will be the input to model the physiological responses upon this kind of perturbations.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

OLAVARRIA, KAREL; MARONE, MARINA PUPKE; OLIVEIRA, HENRIQUE DA COSTA; RONCALLO, JUAN CAMILO; DA COSTA VASCONCELOS, FERNANDA NOGALES; DA SILVA, LUIZIANA FERREIRA; CABRERA GOMEZ, JOSE GREGRIO. Quantifying NAD(P)H production in the upper Entner-Doudoroff pathway from Pseudomonas putida KT2440. FEBS OPEN BIO, v. 5, p. 908-915, 2015. Web of Science Citations: 1.