title: Metabolic profiling of recombinant Escherichia coli cultivations based on high-throughput FT-MIR spectroscopic analysis.
abstract: Escherichia coli is one of the most used host microorganism for the production of recombinant products, such as heterologous proteins and plasmids. However, genetic, physiological and environmental factors influence the plasmid replication and cloned gene expression in a highly complex way. To control and optimize the recombinant expression system performance, it is very important to understand this complexity. Therefore, the development of rapid, highly sensitive and economic analytical methodologies, which enable the simultaneous characterization of the heterologous product synthesis and physiologic cell behavior under a variety of culture conditions, is highly desirable. For that, the metabolic profile of recombinant E. coli cultures producing the pVAX-lacZ plasmid model was analyzed by rapid, economic and high-throughput Fourier Transform Mid-Infrared (FT-MIR) spectroscopy. The main goal of the present work is to show as the simultaneous multivariate data analysis by principal component analysis (PCA) and direct spectral analysis could represent a very interesting tool to monitor E. coli culture processes and acquire relevant information according to current quality regulatory guidelines. While PCA allowed capturing the energetic metabolic state of the cell, e.g. by identifying different C-sources consumption phases, direct FT-MIR spectral analysis allowed obtaining valuable biochemical and metabolic information along the cell culture, e.g. lipids, RNA, protein synthesis and turnover metabolism. The information achieved by spectral multivariate data and direct spectral analyses complement each other and may contribute to understand the complex interrelationships between the recombinant cell metabolism and the bioprocess environment towards more economic and robust processes design according to Quality by Design framework. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:285-298, 2017.
authors.affiliation: Faculty of Engineering, Catholic University of Portugal, Rio de Mouro, 2635-631, Portugal.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Sales
authors.affiliation: Faculty of Engineering, Catholic University of Portugal, Rio de Mouro, 2635-631, Portugal.
authors.forename: Filipa
authors.initials: F
authors.lastname: Rosa
authors.affiliation: Faculty of Engineering, Catholic University of Portugal, Rio de Mouro, 2635-631, Portugal.
authors.forename: Bernardo R
authors.initials: BR
authors.lastname: Cunha
authors.affiliation: Faculty of Engineering, Catholic University of Portugal, Rio de Mouro, 2635-631, Portugal.|Faculty of Engineering, Lusophone University of Humanities and Technology, Campo Grande 376, Lisbon, 1749-019, Portugal.
authors.forename: Pedro N
authors.initials: PN
authors.lastname: Sampaio
authors.affiliation: Faculty of Engineering, Catholic University of Portugal, Rio de Mouro, 2635-631, Portugal.|Institute of Telecommunications, Instituto Superior Técnico, University of Lisbon, Av. Rovisco Pais, Lisboa, 1049-001, Portugal.|ISEL-Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, Rua Conselheiro Emídio Navarro 1, Lisboa, 1959-007, Portugal.
authors.forename: Marta B
authors.initials: MB
authors.lastname: Lopes
authors.affiliation: ISEL-Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa, Rua Conselheiro Emídio Navarro 1, Lisboa, 1959-007, Portugal.
authors.forename: Cecília R C
authors.initials: CRC
authors.lastname: Calado
mesh_terms: D000465:Algorithms; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D055442:Metabolome / Q000502:physiology*; D015999:Multivariate Analysis; D025341:Principal Component Analysis; D011995:Recombination, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
keywords: FT-MIR spectroscopy; PCA; bioprocess; metabolic profiling