Strategies for Improving Lactobacillus for Dairy and Biofuel Industrie

Our principal goal is to examine genetic variation among lactobacilli, so to determine strategies to select or engineer strains with industrial utility. This dissertation describes studies concerning two applications of lactobacilli in the cheese and biofuel industries. The first application addresses the functional improvement of Italian-style cheeses that undergo undesirable browning during ripening and storage conditions---a phenomenon likely pertaining to the cheeses' microbiota and its metabolism of a small browning pre-cursor, methylglyoxal (MG). The second application describes a strain engineering strategy for improving alcohol tolerance of lactobacilli biofuel biocatalysts.;The first study evaluates the role of different Lactobacillus toward MG-mediated browning of Parmesan cheese extract (PCE). Our results demonstrate that lactobacilli metabolize MG by way of a thiol-independent reduction to acetol and 1,2-propanediol. Furthermore, the strain L. brevis 367 reduces MG exclusively to 1,2-propanediol, which correlates to both its ability to significantly decrease MG concentrations in PCE, as well as its significantly higher tolerance to MG, in comparison to other lactobacilli screened. Together, these results describe a major metabolic pathway of MG in lactobacilli, while particular strains may have the potential to prevent browning when applied as adjunct cultures during the manufacturing of Parmesan cheese.;The second study determines the impact of MG biosynthesis by Lactobacillus on browning and end-product formation in PCE. Our results demonstrate that L. casei overexpressing MG synthase in PCE leads to its browning and the formation of heterocyclic amines including bioactive beta-carboline derivatives. These findings implicate non-starter lactic acid bacteria may exacerbate the formation of melanoidin by enzymatic conversion of triosphosphates to MG despite atypical conditions supporting Maillard browning.;The third study develops a conditional hypermutation system in Lactobacillus and evaluates its utility during chemostatic adaptation to isobutanol. Our results indicate isobutanol adapted isolates share common mutations associated with genes whose functions relate to redox, membrane-transport, central-carbon metabolism, and cell-division. Additionally, our results demonstrate a single nucleotide polymorphism upstream of the opp operon (oligopeptide transport) causes isobutanol tolerance in L. casei 12A by increasing gene expression of opp genes, oppA and dppB, in the presence of isobutanol. Therefore, these findings suggest oligopeptide transport genes could serve as engineering targets for isobutanol tolerance of potential lactobacilli biofuel biocatalysts. In addition, this study demonstrates the utility of hypermutation in conjunction with adaptive evolution as a valuable strategy in discovering novel targets for engineering industrially important phenotypes of lactobacilli.