Breeding of Yeast Strain with Starch Utilizing and Alcohol Fermenting Ability by Protoplast Fusion

전분분해활성과 알코올 발효능을 보유한 효모의 육종

  • Published : 2008.06.28

Abstract

The fusants which contain starch utilizing ability and alcohol fermenting ability were developed by protoplast fusion of Saccharomyces cerevisiae KOY-1 and Saccharomyces diastaticus KCTC 1804. Sacharomyces cerevisiae KH-12 was obtained by haploid induction from Saccharomyces cerevisiae KOY-1. The auxotropic mutants of yeast were obtained by using an ethylmethane sulfonate (EMS). The frequency of protoplast formation in Saccharomyces cerevisiae KOY-1 $(Met^-)$ and Saccharomyces diastaticus KCTC 1804 $(Trp^-)$ were 90.5% and 97.7%, respectively. The frequency of fusant formation was $1.79{\times}10^{-4 }$ for the regenerated protoplast and the 1,000 fusants were obtained. Fusant FA 776 was selected as a potential yeast which contain an alcohol fermenting ability in the starch medium. The genetic stability was 4.64% for 10 passages of generation. Fusant FA 776 produced 13mg/ml of alcohol in 24% starch medium and showed 1.86-fold higher alcohol fermenting ability than Saccharomyces diastaticus KCTC 1804.

Saccharomyces diastaticus KCTC 1804 균주와 Saccharomyces cerevisiae KOY-1 균주를 이용하여 전분분해활성과 알코올 발효능을 동시에 가지는 효모융합체를 개발하였다. Saccharomyces cerevisiae KOY-1 균주의 단수체 유도를 통하여 Saccharomyces cerevisiae KH-12 균주를 획득하였다. EMS를 사용한 돌연변이 유발을 통하여 Saccharomyces cerevisiae KH-12 균주의 Met 영양요구성 변이주를 획득하였다. Lyticase 처리로 Saccharomyces cerevisiae KOY-1 $(Met^-)$, Saccharomyces diastaticus KCTC 1804$(Trp^-)$ 균주의 원형질체 형성률은 각각 90.5%, 97.7%로 관찰되었다. 원형질체 융합은 polyethylene glycol 4,000을 이용하여 실시하였고, 이때 원형질체 융합율은 $1.79{\times}10^{-4}$으로 관찰되었다. 원형질체융합과정을 통해 총 1,000주의 융합체를 획득하였고, 획득한 융합체의 전분분해능과 알코올발효능을 비교하여 최종적으로 전분배지 YPS24에서 알코올생성능이 가장 우수한 효모융합체인 FA 776을 최종 선발하였다 최종 선발된 효모융합체 FA 776은 10세대 계대배양 후의 revertent 복귀율이 4.64%로 유전적 안정성을 확인하였으며, 효모융합체의 total DNA 함량을 비교한 결과, Saccharomyces cerevisiae 균주로부터 분리된 단수체 효모 KH-12는 26.56 fg/cell, Saccharomyces diastaticus KCTC 1804 균주는 25.40 fg/cell이었고, 이들을 사용해서 얻어진 융합체 FA 776의 DNA함량은 42.67 fg/cell이었다. 전분배지 YPS24배지에서 60시간 발효하였을때 Saccharomyces cerevisiae KOY-1 균주는 알코올 생성하지 못하였지만 효모융합체 FA 776은 13 mg/mL의 알코올을 생성하였다. 이는 전분이용성 효모인 Saccharomyces diastaticus KCTC 1804 균주에 비해 1.86배나 향상된 알코올 발효능을 나타냈다.

Keywords

References

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