O-016. Metabolic Engineering of Lactococcus lactis for the Development of a One-Step Bioconversion of Lactose into Tagatose

J-H. Lee, D. J. O'Sullivan;
Univ. of Minnesota, St. Paul, MN.

Background: Tagatose is a natural hexose sugar with 92% the sweetness of sucrose, but only 39% of the calories. It also has functional effects such as prebiotic properties, tooth-friendly attributes and a low glycemic index. However, tagatose production involves a multi-step enzymatic process that renders it a high cost food ingredient, thus limiting its use as a low calorie sweetener. A bioinformatic analysis of the complete genome sequence of Lactococcus lactis indicates that inactivation of either the lacC and lacD gene would result in the buildup of a phosphorylated tagatose intermediate during growth on lactose. Previous studies have demonstrated that L. lactis can dephosphorylate such intermediates and secrete them outside the cell. It is therefore proposed that metabolic engineering of L. lactis by inactivating either lacC or lacD would result in the build up of tagatose in the medium when growing in lactose, as growth could continue due to the metabolism of the glucose moiety of lactose. Methods: The sequences of the lacC and lacD genes were obtained from the L. lactis complete genome sequence. To specifically knockout these genes, a targeted group II intron procedure was used. The lacC/lacD - retargeting of the group II intron was performed using PCR and cloned into pACD4K-C, an E. coli vector, and then the retargeted intron and reverse transcriptase (RT) region were cloned into pMSP3535, an E. coli - L. lactis shuttle cloning vector that contains the nisin promoter and positive regulator for its expression in L. lactis host cells. After electroporation into L. lactis C2 and knockout of lacC and lacD genes, tagatose production was investigated using HPLC. Results: Inactivation of lacD gene in L. lactis C2 was performed successfully using the TargeTronTM Gene Knockout System and the pMSP3535 L. lactis expression vector. Growth of this lacD deficient L. lactis C2 mutant in a lactose growth medium resulted in the accumulation of tagatose in the medium. Conclusion: This one-step bio-conversion of lactose into tagatose may be a cost effective approach for the production of this low calorie sweetener from a whey based medium.