13C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

Kamalrul Azlan Azizan; Habtom W Ressom; Eduardo R Mendoza; Syarul Nataqain Baharum

doi:10.7717/peerj.3451

¹³C based proteinogenic amino acid (PAA) and metabolic flux ratio analysis of Lactococcus lactis reveals changes in pentose phosphate (PP) pathway in response to agitation and temperature related stresses

PeerJ. 2017 Jul 5:5:e3451. doi: 10.7717/peerj.3451. eCollection 2017.

Authors

Kamalrul Azlan Azizan^#¹, Habtom W Ressom^#², Eduardo R Mendoza^#^{3

4}, Syarul Nataqain Baharum^#¹

Affiliations

¹ Metabolomics Research Laboratory, Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor, Malaysia.
² Departments of Oncology, Georgetown Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, D.C., United States of America.
³ Institute of Mathematics, University of the Philippines Diliman, Quezon City, Metro Manila, Philippines.
⁴ Membrane Biochemistry Group, Max Planck Institute of Biochemistry, Planegg, Germany.

^# Contributed equally.

Abstract

Lactococcus lactis subsp. cremoris MG1363 is an important starter culture for dairy fermentation. During industrial fermentations, L. lactis is constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response of L. lactis to several stresses has been described, the adaptation mechanisms at the level of in vivo fluxes have seldom been described. To gain insights into cellular metabolism, ¹³C metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) were used to measure the flux ratios of active pathways in the central metabolism of L. lactis when subjected to three conditions varying in temperature (30°C, 37°C) and agitation (with and without agitation at 150 rpm). Collectively, the concentrations of proteinogenic amino acids (PAAs) and free fatty acids (FAAs) were compared, and Pearson correlation analysis (r) was calculated to measure the pairwise relationship between PAAs. Branched chain and aromatic amino acids, threonine, serine, lysine and histidine were correlated strongly, suggesting changes in flux regulation in glycolysis, the pentose phosphate (PP) pathway, malic enzyme and anaplerotic reaction catalysed by pyruvate carboxylase (pycA). Flux ratio analysis revealed that glucose was mainly converted by glycolysis, highlighting the stability of L. lactis' central carbon metabolism despite different conditions. Higher flux ratios through oxaloacetate (OAA) from pyruvate (PYR) reaction in all conditions suggested the activation of pyruvate carboxylate (pycA) in L. lactis, in response to acid stress during exponential phase. Subsequently, more significant flux ratio differences were seen through the oxidative and non-oxidative pentose phosphate (PP) pathways, malic enzyme, and serine and C1 metabolism, suggesting NADPH requirements in response to environmental stimuli. These reactions could play an important role in optimization strategies for metabolic engineering in L. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of how L. lactis regulates central metabolism under various conditions.

Keywords: 13C metabolic flux ratio analysis; GC-MS; L. lactis MG1363; PAAs.

Grants and funding

This work was supported by the Ministry of Science, Technology and Innovation Malaysia (MOSTI) (02-01-02-SF0987 and DLP-2013-024). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.