RESUMO
A cryptic plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733 was isolated, characterized, and used for the construction of a cloning vector to engineer Leuconostoc species. pMBLR00 is a rolling circle replication plasmid, containing 3,370 base pairs. Sequence analysis revealed that pMBLR00 has 3 open reading frames: Cop (copy number control protein), Rep (replication protein), and Mob (mobilization protein). pMBLR00 replicates by rolling circle replication, which was confirmed by the presence of a conserved double-stranded origin and single-stranded DNA intermediates. An Escherichia coli-Leuconostoc shuttle vector, pMBLR02, was constructed and was able to replicate in Leuconostoc citreum 95. pMBLR02 could be a useful genetic tool for metabolic engineering and the genetic study of Leuconostoc species.
Assuntos
Leuconostoc/genética , Plasmídeos/isolamento & purificação , Proteínas de Bactérias/genética , DNA Helicases/genética , Replicação do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Vetores Genéticos , Genética Microbiana/métodos , Biologia Molecular/métodos , Dados de Sequência Molecular , Fases de Leitura Aberta , Análise de Sequência de DNA , Transativadores/genéticaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/genética , Vetores Genéticos , Ácido Láctico/metabolismo , Leuconostoc/genética , Engenharia Metabólica/métodos , Composição de Bases , Replicação do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Expressão Gênica , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactococcus lactis/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Pediococcus/genética , Plasmídeos , Seleção Genética , Análise de Sequência de DNARESUMO
Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids, also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer for C40 acyclic lycopene, C40 dicyclic ß-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion, we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information can serve as the basis for the subsequent development of microorganisms for carotenoids of interest.
