Throw Spore Yeast Keto Reductase (kr) Expression In Pichia Pastoris And E. Coli

The Keto Reductase (KR) gene was cloned from Sporobolomycessaimonicoior , and expression of KR gene in E. coil and Pichia pastoris expressionsystems were studied. A preliminary study on optical conversion from ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)4-chloro-3-hydroxybutanoate (R-CHBE) wasalso conducted.Using RT-PCR, the KR gene was cloned from Sporoboiomyces saimonicoior,then the KR gene was ligated to expression vector, PBV22O, and expressed it viatemperature controlled induction in E. coli. The expression amount of KR reached40% against total protein, KR抯 solubility was up to 25%.After subcloning KR gene into extracellular expression vector - pPIC9K andinto intracelluar expression vector - pPLC3 .5K respectively, KR gene was integratedinto GS 115 through linearization and electronic transformation. Transformantsharboring KR gene were obtained in the YPD-041 8 selection plates. The expressionlevels of KR were induced to 30% of total supernant protein by methanol for GS 115harboring pPIC9K, and to 40% of total cell protein for GS115 harboring pPIC3.5K.Conversion experiments using native KR and recombinant KR were performed,the results showed: the conversion yield of COBE for native KR fromSporoboiomyces saimonicolor, recombinant KR from BL2 1 and (IS 115 was 42%,88%, 92% respectively; the CHBE purity was up to 33.2%, 91%, 98% respectively.