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.

A Simple, Label-Free, and High-Throughput Method to Evaluate the Epigallocatechin-3-Gallate Impact in Plasma Molecular Profile

“…FTIR spectroscopy on the mid-infrared region of the spectra (between 4000 and 400 cm −1 ) covers molecular fundamental vibrations of most common biomolecules. There are three main spectral regions that mainly reflect bond stretching and bond deformations: between 3600 and 2000 cm −1 , mostly due to vibrations between X-H (where X can be C, O, or N), as present in amide A (≈3200 cm −1 ) of proteins, CH 3 (≈2955 and ≈2870 cm −1 ) and CH 2 (≈2918 and ≈2850 cm −1 ) groups from lipids; 1800-1500 cm −1 , mostly due to double bonds as C=O, C=C, and C=N, as present in amide I (≈1650 cm −1 ) and amide II (≈1550 cm −1 ) of proteins, C=O as from phospholipids esters (≈1740 cm −1 ); and 1500-400 cm −1 , known as the fingerprint region, due to overlapped vibrations from diverse molecules [26,27].…”

“…This vibrational spectroscopic technique is simple to apply, with a sample requiring for most cases a simple pre-processing step such as dehydration. It is economic, as no expensive reagents are needed and it boast a rapid workflow, as one spectra is usually acquired in 1 min [26][27][28]. Furthermore, the technique also presents diverse modes of detection, from transmission, transflectation, and attenuated total reflection.…”

“…Furthermore, the technique also presents diverse modes of detection, from transmission, transflectation, and attenuated total reflection. In the present work, it was selected as a detection mode on the basis of transmission analysis conducted in a high-throughput mode, by using microplates with 96 micro-wells [26][27][28][29].…”

A Chemometric Tool to Monitor and Predict Cell Viability in Filamentous Fungi Bioprocesses Using UV Chromatogram Fingerprints

“…PCA is one of the most commonly used chemometric techniques for compressing high volumes of process data (e.g., spectroscopic sensors [36][37][38]) into few meaningful process features. We used PCA models to identify process trends using UV chromatographic dataset at 260 nm.…”

A phenotypic screening bioassay forEscherichia colistress and antibiotic responses based on Fourier‐transform infrared (FTIR) spectroscopy and multivariate analysis

“…Metabolomics techniques have very high sensitivity for individual metabolites, and protocols are well established (Krishnan et al ), however, in a context of phenotypic screening, focus is put on distinguishing different phenotypes, rather than on characterizing a given phenotype, thus features such as throughput, automation and cost of analysis are highly important (Athamneh et al ). In that regard, Fourier‐transform infrared spectroscopy (FTIRS) acquires phenotypic profiles in a high‐throughput, rapid, label‐free, automatable, considerably inexpensive and reasonably simple mode (Sales et al ). FTIRS may be applied to discriminate and quantify diverse molecules, from proteins, nucleic acids, lipids through to diverse metabolites (Lopes et al ; Rosa et al ; Sampaio et al ), with the advantage of representing the whole omics of a cell (Bellisola and Sorio ).…”