Transcriptional factor Cat8 is involved in regulation of xylose fermentation in engineered Saccharomyces cerevisiae

Aim. The aim of this work is the construction of cat8Δ strain on the base of xylose-fermenting S. cerevisiae strain and evaluation of the xylose fermentation rate. Methods. The CAT8 deletion cassette harboring natNT2 gene flanking with 5’ and 3’ non-coding regions of CAT8 gene has been constructed. After transformation by the cassette the cat8Δ strain was selected on the nourseothricin containing medium. Xylose fermentation experiments of constructed strain was performed in mineral medium supplemented with xylose under oxygen-limited conditions. Results. Xylose-fermenting cat8Δ S. cerevisiae strain has been constructed by homologous recombination of the CAT8 deletion cassette with target sequences in the genome of GS010 strain. The cat8Δ strain possessed increase in ethanol accumulation, ethanol yield, rate of ethanol production and productivity of ethanol synthesis relative to the parental GS010 strain for 9.5, 6, 20 and 12 %, respectively. Conclusions. The mutant of the xylose-fermenting S. cerevisiae strain with knock out of the CAT8 gene coding for transcriptional activator, has been constructed. The cat8Δ mutant showed 9.5 % increase in ethanol production from xylose relative to parental strain. Keywords: alcoholic fermentation, xylose, S. cerevisiae, Cat8.

An efficient gene deletion system for Bacillus thuringiensis

It is not easy to manipulate biosynthetic genes of Bacillus thuringiensis since there is a powerful methyl-specific restriction system in this microorganism. In this study, a PCR-based system was used to delete polyphosphate kinase gene (ppk) of Bacillus thuringiensis israelensis (Bti) by replacing the wild-type gene with a cassette containing the apramycin resistance gene as selectable marker. λ-Red was used to promote recombination in Escherichia coli between a PCR-amplified apramycin resistance cassette (linear deletion cassette selectable in E. coli and Bti) and Bti DNA on a plasmid. The isolated mutant plasmid was transferred to Bti by conjugation. Double cross-over transformants were screened for their antibiotic resistance and the mutation was proven by PCR, southern blot hybridization and RT-PCR. The described method, which uses the advantage of quick plasmid construction in E. coli and simple transformation of linear deletion cassette, is very useful to delete entire gene/genes of Bti without any polar effects on genes transcriptionally downstream.