Construction of prophage-free and highly-transformable Limosilactobacillus reuteri strains and their use for production of 1,3-propanediol

Abstract

The lactic acid bacterium Limosilactobacillus reuteri (formerly Lactobacillus reuteri) is a desirable host for the production of 1,3-propanediol (1,3-PDO) from glycerol when 1,3-PDO is used in the food or cosmetic industry. However, the production is hindered by strain instability, causing cell lysis, and difficult gene manipulation. This study reveals that the stability of L. reuteri DSM 20016 and its 1,3-PDO production, especially in the alcohol dehydrogenases (ADHs)-deletion mutants, are greatly enhanced after the deletion of two prophages (Phi 3 and Phi 4) present in the L. reuteri's chromosome. The resulting phage-free and ADHs-deletion mutant could produce >825 mM 1,3-PDO in 48 h without cell lysis at the theoretical maximum yield on glucose of similar to 2 mol/mol. Compared to the wild-type strain, the mutant exhibited a 45.2% increase in 1,3-PDO production titer and a 2.1-fold increase in yield. In addition, this study reports that the transformation efficiency of L. reuteri Delta adh2 Delta adh6 bmutant strains were greatly enhanced by >300-fold after the deletion of prophage 03, probably due to the removal of a restriction-modification (RM) system which resides in the phage genome. With improved stability and higher transformation efficiency, recombinant L. reuteri DSM 20016 Delta adh2 Delta adh6 Delta Phi 3 Delta Phi 4 can be a more reliable and amenable host for industrial applications.