RESUMEN
Methylotrophic bacteria are widespread microbes which can use one carbon compound as their only carbon and energy sources. Here we report the finished, annotated genome sequence of the methylotrophic bacterium Methylovorus sp. strain MP688, which was isolated from soil for high-level production of pyrroloquinolone quinone (PQQ) in our lab.
Asunto(s)
Genoma Bacteriano , Methylophilaceae/genética , Methylophilaceae/metabolismo , Cofactor PQQ/metabolismo , Methylophilaceae/aislamiento & purificación , Datos de Secuencia Molecular , Microbiología del SueloRESUMEN
Recombineering, a new genetic engineering technology based on high efficiency in vivo homologous recombination, can be used in target DNA knock-in, knock-out and gene cloning. In the process of gene subcloning mediated by Recombineering technique, high-quality target DNA fragments were difficult to obtain using in vitro overlapping PCR,therefore the efficiency of in vivo homologous recombination was severely interrupted. To solve this problem, some technology improvements have been established based on the principle of Red recombinases. The PCR DNA fragments of egfp and kan genes with complementary sequences on the end of each fragment were co-introduced into a pcDNA3.1 vector and Red recombinases containing E. coli DY331 host cells by electroporation. A recombinant plasmid pcDNA3.1-egfp-kan was screened directly by antibiotic marker. The positive rates can reach to 45%. The EGFP gene expression of pcDNA3.1-egfp-kan can be observed by transient transfection of 293 eukaryotic cells.
Asunto(s)
Escherichia coli/genética , Fusión Génica/genética , Proteínas Fluorescentes Verdes/genética , Recombinasas/genética , Recombinación Genética , Bacteriófago lambda/enzimología , Bacteriófago lambda/genética , ADN Recombinante/genética , Electroporación , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/metabolismo , Plásmidos/genética , Recombinasas/metabolismoRESUMEN
Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli, being a candidate for enzymic detection of PQQ, is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21 (DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method, over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain, named MP606, was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC, absorption spectra assay, and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences, overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95% . The highest value is with two strains of Methylovorus, which reached at 99%.
