The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous endjoining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3 + transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright

The bottom-fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)-type and two S. bayanus (Sb)-type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p-hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G-418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3 + /Scura3Δ::TDH3p-hygro, SbURA3 + /Sbura3Δ::KanMX), which was plated on 5-fluoroorotic acid (5-FOA) plates to generate the desired Ura -homozygous disruptant (Scura3Δ::TDH3p-hygro/Scura3Δ::TDH3p-hygro Sbura3Δ::KanMX/Sbura3Δ:: KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom-fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1-overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3Δ lager strain will be a useful host for constructing strains of relevance to brewing.

Although results of recent studies suggest that fermented foods strongly affect the gut microbiota composition and that they relieve inflammatory bowel disease symptoms, some reports have described that fermented foods increase some inflammation markers based on differences in fermented food materials. This study evaluated the effects of fermented plant extract (FPE) on dextran sulfate sodium (DSS)-induced colitis in mice and the effects on fecal microbiota composition in humans. Mice fed 5% FPE with 3% DSS (FPE group) showed no body weight loss, atrophy of colonic length, or bloody stool, similar to mice fed a basal diet (negative group), whereas mice fed 3% DSS (positive group) exhibited those effects. Concentrations of inflammation markers IL-6 and TNF-α were not significantly different between FPE and negative groups; however, those concentrations became higher in the positive group. 16S ribosomal RNA gene sequencing was used to characterize fecal microbiota in healthy women before and after 3-month FPE supplementation. The FPE supplementation induced increases in Firmicutes phyla and in Clostridiales order, which play a central role in inflammation suppression. These results suggest that FPE enhances Clostridiales growth in the gut and that it has an anti-inflammatory effect.

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