Carboxylic acid consumption and production by Corynebacterium glutamicum_LC-MS/MS
Corynebacterium glutamicum is well-known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Fast growth and robustness against oscillatory oxygen availability, which can occur in large-scale bioreactors, are advantageous properties of this bacterium. However, previous studies of the effect of gradients in scale-down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation by C. glutamicum. This study addresses the impact of carboxylic acids, e.g. acetate and L-lactate, on the cultivation process and their potential role in scale up related performance losses. In order to mimic a discontinuous oxygen supply, a fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied. One focus of this study is to identify relative changes in the proteome due to the differing availability of carboxylic acids under discontinuous oxygen supply.
Sample Processing Protocol
Samples were taken in triplicate from two biological replicates. Two milliliters of cell suspension were centrifuged (10 min, 4 °C, 21,500 x g). The pellet was immediately frozen and stored at −80 °C. Further processing was performed as described in detail by Voges et al., 2012. A mechanical cell disrup-tion was performed, followed by the addition of 15N labeled whole cell internal standard to the cell extract and tryptically digested. Subsequent analysis was performed with a LC-MS/MS using an Infinity 1260 HPLC system (Agilent) hyphenated to a Q-ToF 6600 mass spectrometer (AB Sciex, Darmstadt, Germany) as described by Limberg et al., 2017. Voges R, Noack S. Quantification of proteome dynamics in Corynebacterium glutamicum by 15N-labeling and selected reaction monitoring. J Proteomics. 2012;75:2660-2669. Limberg M, Schulte J, Aryani T, Mahr R. Metabolic profile of 1, 5-diaminopentane producing Corynebacterium glutamicum under scale-down conditions: Blueprint for robustness to bioreactor. Biotechnol. 2017;114:560-575.
Data Processing Protocol
Data analysis and peak integration was performed with the software package PeakView® (AB Sciex). Protein identification and quantification was carried out with the software ProteinPilot™ (AB Sciex). The resulting fold change levels represent the ratio of normalized target protein peak area, which were obtained under stress conditions, to the normalized peak area, obtained under reference conditions. All results were normalized to the 15N labeled internal standard. Corresponding statistical analysis was performed via MarkerView™ (AB Sciex). The analysis was performed at the Research Center Jülich. Proteins with a fold change of between 0.5 and 2 and a p value (student test) below 0.05 were considered.
Conrady M, Lemoine A, Limberg MH, Oldiges M, Neubauer P, Junne S. Carboxylic Acid Consumption and Production by Corynebacterium glutamicum. Biotechnol Prog. 2019:e2804 PubMed: 30851150