Use of a Novel Escherichia coli-Leuconostoc Shuttle Vector for Metabolic Engineering of Leuconostoc citreum To Overproduce d -Lactate

Determination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, fromLeuconostoc mesenteroides subsp.mesenteroides KCTC13302 revealed that it contains 20,721 bp, a G+C content of 38.7%, and 18 open reading frames. Comparative sequence and mung been nuclease analyses of pMBLT00 showed that pMBLT00 replicates via the theta replication mechanism. A new, stableEscherichia coli-Leuconostoc shuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced intoLeuconostoc ,Lactococcus lactis , andPediococcus . This shuttle vector was used to engineerLeuconostoc citreum 95 to overproduced -lactate. TheL. citreum 95 strain engineered using plasmid pMBLT02, which overexpressesd -lactate dehydrogenase, exhibited enhanced production of optically pured -lactate (61 g/liter, which is 6 times greater than the amount produced by the control strain) when cultured in a reactor supplemented with 140 g/liter glucose. Therefore, the shuttle vector pMBLT02 can serve as a useful and stable plasmid vector for further development of ad -lactate overproduction system in otherLeuconostoc strains andLactococcus lactis .