The glycolytic pathway was clearly repressed, supporting previous findings [15, 19]. Among these genes were pfk (0.5-1.1) encoding 6-phosphofructokinase (Pfk), and fba (0.7-1.1) coding for fructose-bisphosphate aldolase, both acting at the initial steps of glycolysis. In addition, gpm3 encoding Ferroptosis assay one of the five phosphoglycerate mutases present in the 23K genome, acting in the lower part of glycolysis, was also down-regulated (0.7-0.9). MF1053 down-regulated pyk (0.7) encoding pyruvate kinase (Pyk)

that competes for PEP with the PTS (Figure 2). Its activity results in the production of pyruvate and ATP, and it is of major importance in glycolysis and energy production in the cell. MF1053 also showed a stronger down-regulation of pfk than the other strains (Table 1). Similar to several other lactobacilli, pfk is transcribed together with pyk [43, 44], and in many microorganisms the glycolytic flux depends on the activity of the two enzymes encoded from this operon [43, 45]. At the protein level, we previously

observed both Pfk and Pyk expressed at a lower level for all the three strains [19], however this was not confirmed at the level of gene expression for 23K and LS 25. We could also not confirm the lower protein expression of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and enolase previously seen in LS 25 [19]. The latter three enzymes are encoded from the central glycolytic operon (cggR-gap-pgk-tpi-eno) together with triose-phosphate isomerase and the putative central glycolytic genes regulator Saracatinib datasheet (CggR) [46]. Besides the cggR gene being down-regulated in MF1053 and LS 25, no change in gene expression was seen of these central glycolytic genes. Thus at the transcription level it is not obvious that the LS

25 strain down-regulate the glycolytic pathway more efficiently than the other strains, Dipeptidyl peptidase as previously suggested [19]. Interestingly, all the strains showed an induction (1.4-2.3) of mgsA encoding methylglyoxal synthase, which catalyzes the conversion of dihydroxyacetone-phosphate to methylglyoxal (Figure 2). The presence of this gene is uncommon among LAB and so far a unique feature among the sequenced lactobacilli. The methylglyoxal pathway represents an energetically unfavourable bypass to the glycolysis. In E. coli, this bypass occurs as a response to phosphate starvation or uncontrolled carbohydrate metabolism, and enhanced ribose uptake was shown to lead to the accumulation of methylglyoxal [47, 48]. As suggested by Chaillou et al. [7], such flexibility in the glycolytic process in L. sakei may reflect the requirement to deal with glucose starvation or to modulate carbon flux during co-metabolism of alternative carbon sources. Breakdown of methylglyoxal is important as it is toxic to the cells [49]. An induction of the lsa1158 gene contiguous with mgsA was seen for 23K and MF1053.