Bifidobacterium-Escherichia coli Shuttle Vector Series pKO403, with Temperature-Sensitive Replication Origin for Gene Knockout in Bifidobacterium.

A series of Bifidobacterium-Escherichia coli shuttle vectors (pKO403-lacZ'-Cm, pKO403-lacZ'-Sp, pKO403-lacZ'-p15A) were constructed based on the pKO403 backbone, which carries a temperature-sensitive replication origin. These vectors carry the lacZ'α fragment, overhung by two facing type IIS restriction sites, for blue-white selection and seamless gene cloning. These vectors are useful for gene knockout or multigene integration into the chromosome of Bifidobacterium.

Levansucrase from Leuconostoc mesenteroides NTM048 produces a levan exopolysaccharide with immunomodulating activity.

OBJECTIVES: A levansucrase from Leuconostoc mesenteroides NTM048 was cloned and expressed and its enzymatic product was characterized. RESULTS: The fructansucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The recombinant enzyme was purified as a single protein and its properties investigated. The polymer produced by the recombinant enzyme was identified as levan by various means including TLC and NMRs, and the enzyme was identified as a GH68 levansucrase. The enzyme was optimal at pH 5.5-6 and 30 °C, and its activity was stimulated by Ca(2+). The levan produced by this strain induced IgA production in mice. CONCLUSION: Leuconostoc mesenteroides, a probiotic strain, possessed levansucrase which catalyzed the produced levan that had immunomodulating activity.

Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor.

Glycan conversion of glycoprotein via the transglycosylation activity of endo-ß-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate.

The pyrE Gene as a Bidirectional Selection Marker in Bifidobacterium Longum 105-A.

We constructed a deletion mutant of the pyrE gene in Bifidobacterium longum 105-A. A pyrE knockout cassette was cloned into pKKT427, a Bifidobacterium-Escherichia coli shuttle vector, and then introduced into B. longum 105-A by electroporation. The transformants were propagated and spread onto MRS plates containing 5-fluoroorotic acid (5-FOA) and uracil. 5-FOA-resistant mutants were obtained at a frequency of 4.7 × 10(-5) integrations per cell. To perform pyrE gene complementation, the pyrE gene was amplified by PCR and used to construct a complementation plasmid (pKKT427-pyrE (+)). B. longum 105-A ∆pyrE harboring this plasmid could not grow on MRS plates containing 5-FOA, uracil and spectinomycin. We also developed a chemically defined medium (bifidobacterial minimal medium; BMM) containing inorganic salts, glucose, vitamins, isoleucine and tyrosine for positive selection of pyrE transformants. B. longum 105-A ∆pyrE could not grow on BMM agar, but the same strain harboring pKKT427-pyrE (+) could. Thus, pyrE can be used as a counterselection marker in B. longum 105-A and potentially other Bifidobacterium species as well. We demonstrated the effectiveness of this system by constructing a knockout mutant of the xynF gene in B. longum 105-A by using the pyrE gene as a counterselection marker. This pyrE-based selection system will contribute to genetic studies of bifidobacteria.

A targeted gene knockout method using a newly constructed temperature-sensitive plasmid mediated homologous recombination in Bifidobacterium longum.

Bifidobacteria are the main component of the human microflora. We constructed a temperature-sensitive (Ts) plasmid by random mutagenesis of the Bifidobacterium-Escherichia coli shuttle vector pKKT427 using error-prone PCR. Mutant plasmids were introduced into Bifidobacterium longum 105-A and, after screening approximately 3,000 colonies, candidate clones that grew at 30 °C but not at 42 °C were selected. According to DNA sequence analysis of the Ts plasmid, five silent and one missense mutations were found in the repB region. The site-directed mutagenesis showed only the missense mutation to be relevant to the Ts phenotype. We designated this plasmid pKO403. The Ts phenotype was also observed in B. longum NCC2705 and Bifidobacterium adolescentis ATCC15703. Single-crossover homologous-recombination experiments were carried out to determine the relationship between the length of homologous sequences encoded on the plasmid and recombination frequency: fragments greater than 1 kb gave an efficiency of more than 10(3) integrations per cell. We performed gene knockout experiments using this Ts plasmid. We obtained gene knockout mutants of the pyrE region of B. longum 105-A, and determined that double-crossover homologous recombination occurred at an efficiency of 1.8 %. This knockout method also worked for the BL0033 gene in B. longum NCC2705.

Acquired tolerance to oxidative stress in Bifidobacterium longum 105-A via expression of a catalase gene.

For improvement of tolerance to oxidative stress in Bifidobacterium longum 105-A, we introduced the Bacillus subtilis catalase gene (katE) into it. The transformant showed catalase activity (39 U/mg crude protein) in the intracellular fraction, which increased survival by ∼100-fold after a 1-h exposure to 4.4 mM H(2)O(2), decreased de novo H(2)O(2) accumulation, and increased survival in aerated cultures by 10(5)-fold at 24 h. The protection level was better than that conferred by exogenously added catalase.

Determination of the ribosome-binding sequence and spacer length between binding site and initiation codon for efficient protein expression in Bifidobacterium longum 105-A.

The frequency of the ribosome-binding site (RBS) and spacer length between binding site and initiation codon were analyzed using informatics, and compared the activities of various synthetic RBSs and spacers in Bifidobacterium longum 105-A. As the result, AAGGAG and a 5 nt spacer length produced the most efficient protein expression.