Premium
Development of an efficient host‐vector system of Ruminiclostridium josui
Author(s) -
Wang Yayun,
Okugawa Kei,
Kunitake Emi,
Sakka Makiko,
Kimura Tetsuya,
Sakka Kazuo
Publication year - 2018
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/jobm.201700620
Subject(s) - isoschizomer , plasmid , restriction enzyme , transformation efficiency , transformation (genetics) , escherichia coli , electroporation , recognition sequence , biology , restriction site , shuttle vector , microbiology and biotechnology , pbr322 , dna , gene , genetics , vector (molecular biology) , recombinant dna , gene expression , dna methylation , agrobacterium
Although Ruminiclostridium josui (formerly Clostridium josui ), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host‐vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui . Here, we partially purified restriction endonuclease Rjo I from R. josui cell extract using column chromatography. Further characterization showed that Rjo I is an isoschizomer of Dpn I, recognizing the sequence 5′‐G met ATC‐3′, where the A nucleotide is Dam‐methylated. Rjo I cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 ( dam − / dcm − ) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 10 3 transformants/μg of DNA was obtained using a square‐wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin‐encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui , a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui .