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

Abstract

Lactococcus lactissubsp.cremorisMG1363 is an important starter culture for dairy fermentation. During industrial fermentations,L. lactisis constantly exposed to stresses that affect the growth and performance of the bacterium. Although the response ofL. lactisto several stresses has been described, the adaptation mechanisms at the level ofin vivofluxes 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 ofL. lactiswhen 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 ofL. 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) inL. 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 inL. lactis. Overall, the integration of systematic analysis of amino acids and flux ratio analysis provides a systems-level understanding of howL. lactisregulates central metabolism under various conditions.