Saccharomy cescerevisiae氨基转移酶基因ARO8克隆及对3甲硫基丙醇合成的影响

酿酒酵母可通过艾希利（Ehrlich）途径将L-蛋氨酸转化为3-甲硫基丙醇等食品风味成分,本文研究了氨基转移酶及其基因在Ehrlich途径及3-甲硫基丙醇合成代谢的调控作用。从Saccharomyces cerevisiae S288C克隆氨基转移酶ARO8基因,并基于酿酒酵母-大肠杆菌穿梭型表达载体pYES-pgk,构建重组质粒表达载体pYES-pgk-ARO8,PEG/LiAc转化法将其导入S.cerevisiae S288C。结果表明,克隆的ARO8基因全长1503bp,与NCBI GenBank中酿酒酵母芳香族氨基转移酶Ⅰ编码基因ARO8的序列相似度为100%;构建的ARO8基因过表达工程菌S.cerevisiae AR8,其氨基转移酶活力为187U/mL,较野生型S288C菌株的酶活性提高1.63倍;工程菌AR8的摇瓶发酵3-甲硫基丙醇产量为0.76g/L,较野生型S288C提高28.8%。表明氨基转移酶Aro8p及其ARO8基因在S.cerevisiae 3-甲硫基丙醇合成代谢中发挥重要作用,增强其ARO8基因表达,有助于提高3-甲硫基丙醇的产量。 

Cloning of the aminotransferase gene ARO8 and its influence on the production of methionol in Saccharomyces cerevisiae

Methionol is an important volatile sulphur flavor compound.It can be produced in Saccharomyces cerevisiae using L- methionine by the Ehrlich pathway.To verify aminotransferase'effect, gene ARO8 encoding aminotransferase was cloned from S.cerevisiae S288C, and the gene ARO8 was inserted into the S.cerevisiaeE.coli shuttle constitutive expression vector pYES- pgk, generating a recombinant vector pYES- pgk- ARO8.The vector pYES- pgk- ARO8 was then transformed into S.cerevisiae S288C.The results indicated that the encoding gene shared 100% homology with the aromatic aminotransferase Ⅰ gene ARO8 reported in NCBI GenBank.A recombinant strain S.cerevisiae AR8 was constructed and screened successfully.The aminotransferase activity of S.cerevisiae AR8 was 187U /mL, which was increased by 28.8% compared to what in S.cerevisiae S288C.Methionol yield of AR8 in shaking flask reached 0.76g /L and it was increased by 28.8%.These results showed that aminotransferase Aro8p and gene ARO8 of S.cerevisiae played an important role in methionol anabolism, and the gene ARO8 over-expression was help to methionol yield.