title: Use of a novel Escherichia coli-leuconostoc shuttle vector for metabolic engineering of Leuconostoc citreum to overproduce D-lactate.
abstract: Determination of the complete nucleotide sequence of a cryptic plasmid, pMBLT00, from Leuconostoc 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, stable Escherichia coli-Leuconostoc shuttle vector, pMBLT02, which was constructed from a theta-replicating pMBLT00 replicon and an erythromycin resistance gene of pE194, was successfully introduced into Leuconostoc, Lactococcus lactis, and Pediococcus. This shuttle vector was used to engineer Leuconostoc citreum 95 to overproduce d-lactate. The L. citreum 95 strain engineered using plasmid pMBLT02, which overexpresses d-lactate dehydrogenase, exhibited enhanced production of optically pure d-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 a d-lactate overproduction system in other Leuconostoc strains and Lactococcus lactis.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Suwon, South Korea.
authors.forename: Han Seung
authors.initials: HS
authors.lastname: Chae
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Lee
authors.forename: Ju-Hoon
authors.initials: JH
authors.lastname: Lee
authors.forename: Si Jae
authors.initials: SJ
authors.lastname: Park
authors.forename: Pyung Cheon
authors.initials: PC
authors.lastname: Lee
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001482:Base Composition; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004917:Erythromycin / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism*; D013294:Lactococcus lactis / Q000235:genetics; D007934:Leuconostoc / Q000235:genetics*; D060847:Metabolic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010376:Pediococcus / Q000235:genetics; D010957:Plasmids; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D019344:Lactic Acid; D004917:Erythromycin; D007770:L-Lactate Dehydrogenase