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.
Highlights
Lactococcus lactis is a lactic acid bacterium (LAB) that produces lactate as its main catabolic by-product (Gaspar et al, 2013; Zhao et al, 2013)
We used 13C-based metabolic flux analysis and gas chromatography mass spectrometry (GC-MS) to determine the fractional contributions of L. lactis central metabolism in response to 30 ◦C with agitation (30WA), 30 ◦C without agitation (30WOA) and 37 ◦C without agitation (37WOA)
Intracellular fatty acids (FAAs) were higher than extracellular FAAs and proteinogenic amino acids (PAAs)
Summary
Lactococcus lactis is a lactic acid bacterium (LAB) that produces lactate as its main catabolic by-product (Gaspar et al, 2013; Zhao et al, 2013). The flavor formation ability of L. lactis depends on the generation and uptake of amino acids by the bacterium (GarciaCayuela et al, 2012; Tanous et al, 2005; Van Kranenburg et al, 2002). It was demonstrated that L. lactis is auxotrophic for several amino acids that they cannot synthesize from simpler nitrogen sources (Ayad et al, 1999; Trip, Mulder & Lolkema, 2013; Van Kranenburg et al, 2002; Wegmann et al, 2007). Amino acids are important in biochemical reactions and metabolic networks (Cocuron, Tsogtbaatar & Alonso, 2017). They are involved as building blocks for protein synthesis, as signaling molecules and in determining physiological conditions (Tanaka et al, 2013)
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