Saccharomyces cerevisiae에서 발현된 Pseudomonas aurantiaca Levansucrase의 분비국재성

Secretion and Localization of Pseudomonas auratiaca Levansucrase Expressed in Saccharomyces cerevisiae

Pseudomonas aurantiaca 유래 levansucrase 유전자(lscA)를 GAL1 promoter 하류에 연결시킨 pYES-lscA와 CAL10 promoter와 Kluyveromyces marxianus exoinulinase의 분비 신호서열(INU1 ss)하류에 연결시킨 pYInu-lscA를 각각 구축하였다. 이들 plasmid를 invertase 결손 변이주(suc2-$\Delta$9)인 S. cerevisiae SEY2102에 형질전환시켜 고활성 형질전환주를 선발하였다. 효모 형질전환주를 galactose 함유 배지로 배양한 결과, pYES-lscA 함유 형질전환주인 경우 levansucrase의 총활성은 8.62 U/ml이고, pYInu-lscA 함유 형질전환주인 경우 5.43 U/ml에 도달하였다. 발현된 levansucrase의 약 80% 정도가 periplasmic space와 cytopla느에 존재하였고, INU1 ss에 의한 분비효율 증가는 관찰할 수 없었다. 또한, 효모에서 발현된 재조합 levansucrase는 과당쇄화된 형으로 생산되는 것으로 보여진다.

Levansucrase gene(lscA) from Pseudomonas aurantiaca was subcloned downstream of GAL1 promoter in pYES 2.0 and pYInu-AT [GAL10 promoter ＋ exoinulinase signal sequence of Kluyveromyces marxianus], resulting pYES-lscA and p YInu-lscA, respectively. The two expression plasmids were introduced into an invertase-deficient strain, Sacchromayces cerevisiae SEY2102, and transformants with high activity of levansucrase were selected. When each yeast transform ants was cultivated in medium containing galactose, the extracellular and intracellular activities of levansucrase reached about 8.62 U/ml with the strain harboring pYES-lscA and 5.43 U/ml with the strain harboring pYInu-lscA. The levansucrase activity of 80% was detected in the periplasmic space and cytoplasm. The levansucrase activity in the medium of SEY2102/pYInu-lscA was 0.87 U/ml whereas that of SEY2102/pYES-lscA was 0.47 U/ml, which implying the exoinulinase signal sequence didn't enhance the secretion efficiency of levansucrase. Furthermore, the recombinant levansucrase expressed in yeast seems to be produced as a hyper-glycosylated form.