Abstract
Lactobacillus sanfranciscensis LSCE1 was selected as a target organism originating from recurrently refreshed sourdough to study the metabolic rerouting associated with the acid stress exposure during sourdough fermentation. In particular, the acid stress induced a metabolic shift toward overproduction of 3-methylbutanoic and 2-methylbutanoic acids accompanied by reduced sugar consumption and primary carbohydrate metabolite production. The fate of labeled leucine, the role of different nutrients and precursors, and the expression of the genes involved in branched-chain amino acid (BCAA) catabolism were evaluated at pH 3.6 and 5.8. The novel application of the program XCMS to the solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) data allowed accurate separation and quantification of 2-methylbutanoic and 3-methylbutanoic acids, generally reported as a cumulative datum. The metabolites coming from BCAA catabolism increased up to seven times under acid stress. The gene expression analysis confirmed that some genes associated with BCAA catabolism were overexpressed under acid conditions. The experiment with labeled leucine showed that 2-methylbutanoic acid originated also from leucine. While the overproduction of 3-methylbutanoic acid under acid stress can be attributed to the need to maintain redox balance, the rationale for the production of 2-methylbutanoic acid from leucine can be found in a newly proposed biosynthesis pathway leading to 2-methylbutanoic acid and 3 mol of ATP per mol of leucine. Leucine catabolism to 3-methylbutanoic and 2-methylbutanoic acids suggests that the switch from sugar to amino acid catabolism supports growth in L. sanfranciscensis in restricted environments such as sourdough characterized by acid stress and recurrent carbon starvation.