Exopolysaccharide biosynthesis and regulation in Leuconostoc mesenteroides influences water holding capacity of fermented plant-based milks.

Physical instability remains a major challenge in fermented plant-based yoghurts in the absence of added texturizing agents. Cultures can improve water holding capacity (WHC) through production of exopolysaccharide (EPS). This study identified key genes required for sucrose-dependent EPS biosynthesis in Leuconostoc mesenteroides 399 and assessed their contribution to WHC during plant-based milk fermentation. A forward genetic screen for spontaneous mutants with reduced slime formation on sucrose agar identified a loss of function mutation in the predicted dextransucrase gene dsr1. Subsequent screening using the dsr1 mutant yielded isolates with mutations in a levansucrase gene, a major facilitator superfamily (MFS) transporter, or a ScrR family transcriptional repressor, each showing complete loss of EPS. These results indicated that only one dextransucrase and one levansucrase are the enzymes involved in EPS production under the conditions tested. Proteomic and RNA analyses showed that mutations in either the MFS or ScrR genes abolished sucrose-induced expression of these EPS biosynthetic enzymes. The MFS mutant displayed impaired growth on sucrose, consistent with a primary role in sucrose uptake. In fermentation assays, wild type L. mesenteroides 399 combined with an acidifying Lactococcus starter increased WHC in soy and almond milks supplemented with 2% sucrose, whereas no increase was observed in oat or rice milks. EPS defective mutants did not increase WHC in sucrose supplemented soy or almond milks, relative to controls. These findings define a sucrose responsive EPS regulatory pathway in L. mesenteroides 399 and demonstrate that dsr1 is essential for WHC improvement in plant-based milk fermentations.