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Department of Cosmetology, Dongkang College, Gwangju 61200, Republic of Korea(Received April 4, 2016; Revised May 9, 2016; Accepted May 10, 2016)ABSTRACT: To improve antioxidant glutathione (GSH) content and saccharification ability in sake yeasts of Saccharomyces cerevisiae, the γ-glutamylcysteine synthetase gene (GSH1 ) from S. cerevisiae, glucoamylase gene (GAM1 ) and α-amylase gene (AMY ) from Debaryomyces occidentalis were co-expressed in sake yeasts for manufacturing a refreshing alcoholic beverage abundant in GSH from rice starch. The extracellular GSH content of the recombinant sake yeasts increased 1.5-fold relative to the parental wide-type strain. The saccharification ability by glucoamylase of the new yeast strain expressing both GAM1 and AMY genes was 2-fold higher than that of the yeast strain expressing only GAM1 gene when grown in the culture medium containing 2% (w/v) rice starch. It generated 11% (v/v) ethanol from 20% (w/v) rice starch and consumed up to 90% of the starch content after 7 days of fermentation.Key words: Saccharomyces cerevisiae, fermentation, glutathione, rice starch, saccharification*For correspondence.

To produce an alcoholic beverage containing anthocyanins that can act as antioxidants and have anticarcinogenic activities and antihypertensive effects.High starch-assimilating sake yeast strain of Saccharomyces cerevisiae co-expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis using the double rDNA-integration system was developed. The new strain grew substantially using 5 % (w/v) purple sweet potato flour as the sole carbon source. Its cell yield reached 14.5 mg ml(-1) after 3 days. This value was 2.4-fold higher than that of the parental wild-type strain. It produced 12 % (v/v) ethanol from 20 % (w/v) purple sweet potato flour and consumed 98 % of the starch content in purple sweet potato flour after 5 days of fermentation.We have produced a health-promoting alcoholic beverage abundant in anthocyanins from purple sweet potato.

To contruct amylolytic industrial strains of Saccharomyces cerevisiae which produce ethanol efficiently from raw starch, the Bacillus amyloliquefaciens α-amylase genes (Amy) or Aspergillus awamori glucoamylase genes (GA1) was separately introduced into the ribosomal DNA loci in the chromosomes of the raw starch fermenting-parental strain (ATCC 9763/YIpδAGSAδ), using double 18S rDNA-integration system. Ethanol production after 3 days of fermentation by the strain that produced ethanol most efficiently from raw starch (ATCC 9763/YIpδAGSAδ/YIpAG2rD) among the transformant strains was 1.5-times higher than that by the parental strain. This new strain generated 9.2% (v/v) ethanol (72 g/L) from 20% (w/v) raw corn starch and consumed 75% of the raw starch content during the same period.

Industrial strains of a polyploid, distiller's Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l(-1)) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.

Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis alpha-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the delta-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and alpha-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.

To estimate the inhibitory effect of Lithospemum erythrorhizon root extract on melanin biosynthesis, we tested its inhibitory effects on tyrosinase promoter in B16 mouse melanoma cells. Lithospermum erthrorhizon root extract had inhibitory effect above on tyrosinase promoter at and exhibited no cytotoxicity under . Also, melanin biosynthesis decreased approximately and at and , respectively. Therefore, Lithospermum erythrorhizon root extract would be considered very effective regulator of tyrosinase promoter and melanin biosynthesis.

The methanol extract of Houttuynia cordata repressed the expression of tyrosinase gene of Bl6 mouse melanoma cell containing tyrosinase promoter. The extract repressed expression of tyrosinase gene by about 13 and 45 at 10g/mL and 100 g/mL, respectively. In the MTT assay, the same extract exhibited very low cytotoxicity at 1g/mL, 10g/mL, and 100 g/mL, respectively. The fractions of ethyl acetate, butyl alcohol, and water did not showed the repressive effect on the expression of tyrosinase gone, but methylene chloride fraction layer repressed highly at 10g/mL and 100g/mL.

The gene encoding Schwanniomyces occidentalis α-amylase (AMY) was introduced into the chromosomal δ sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an δ-integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The α-amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.

The application of Phaffia rhodozyma as an astaxanthin source in the aquaculture industry is limited because of the low carotenoid content of natural isolates. We constructed carotenoid hyperproducing hybrids of P. rhodozyma by protoplast fusion from parental strains that produced approx. 1600 μg carotenoid/g yeast and were auxotrophic for tryptophan, leucine, methionine or arginine. The hybrids were stable and consistently produced > 2000 μg carotenoid/g yeast. Karyogamy was confirmed by the isolation of recombinants after mitotic segregation of parental auxotrophic genetic markers, the increased content of DNA/cell and the presence of a single nucleus per cell. The results of this study indicated that protoplast fusion is promising for strain improvement in P. rhodozyma .

To develop a strain of Saccharomyces cerevisiae that produces ethanol directly from starch, two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis alpha-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. The GA1 and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae using the double delta-integration system. The GAM1 gene was constitutively expressed under the corresponding promoter using the double 18S rDNA-integration system. The recombinant industrial strain secreting biologically active alpha-amylase, glucoamylase and debranching enzyme was able to ferment starch to ethanol in a single step. The new strain produced 8% (v/v) ethanol (62.8 g l(-1)) from 20% (w/v) soluble starch after 2 days, fermentation.

The gene encoding Schwanniomyces occidentalis alpha-amylase (AMY) was introduced into the chromosomal delta sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an delta-integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The alpha-amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.