Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

Improvement of the growth defect in salt- and ethanol-tolerant yeast mutagenized with error-prone DNA polymerization by using backcross cell fusion.

Salt- and ethanol-tolerant mutants of Saccharomyces cerevisiae, isolated from the uracil-requiring mutant derived from Taiken No. 396 by proofreading-deficient DNA polymerization, showed less growth than their parent strain. The fusants, between these tolerant mutants and the lysine-requiring mutant from Taiken No. 396 obtained by the protoplast fusion, indicated improved growth.

Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase delta.

Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae. To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase delta is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase delta, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1, which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.

Characterization of an alpha-ketoglutarate-resistant sake yeast mutant with high organic acid productivity.

A yeast with high organic acid productivity was isolated from alpha-ketoglutarate-resistant mutants obtained by mutagenizing a sake yeast, Kyokai no. 701 (K-701). The new strain, 20G-R39, produces about twice as much malate and succinate as the parental strain. DNA microarray analyses revealed that the transcriptional levels of genes involved in the TCA cycle, oxidative phosphorylation, and respiration were higher in strain 20G-R39 than in strain K-701. Expression of these genes is regulated by the Hap2/3/4/5p complex, and especially by expression of the HAP4 gene. In a Northern blot analysis, the transcriptional level of the HAP4 gene was higher in strain 20G-R39 than in strain K-701. We constructed a plasmid that expresses the HAP4 gene constitutively and introduced it into strain K-701. The HAP4-overexpression-strain produced more malate and succinate than strain K-701 both in YPD medium and in a sake brewing test. These results indicate that strain 20G-R39 produces more organic acids than strain K-701 because strain 20G-R39 has a higher level of expression of the HAP4 gene than strain K-701.