蒎烯是一种应用广泛的天然单萜类化合物,特别是具有高能值的蒎烯二聚体在燃料领域有着极大的应用前景,蒎烯合酶活性是限制酿酒酵母高效生产蒎烯的重要因素。该研究希望获得在酿酒酵母中表现更优的蒎烯合酶突变体,为蒎烯在酿酒酵母中的高效合成提供借鉴。该研究在甲羟戊酸(mevalonate, MVA)通路强化的酿酒酵母中,表达了蒎烯合酶Pt30的Q456位点的饱和突变体,对工程菌株进行两相摇瓶发酵,检测最终蒎烯的产量。结果显示,在构建的蒎烯合酶Pt30的Q456位点饱和突变体中,Pt30(Q456K)和Pt30(Q456I)突变体将蒎烯从不可测定提高到了0.017、0.041 mg/L。蒎烯合酶Pt30的Q456K和Q456I突变体,相对于Pt30野生型酶提高了蒎烯在酿酒酵母中的产量,这也证明了Q456这一位点对蒎烯合酶Pt30的重要性,也为其他蒎烯合酶的改造提供了参考。
Pinene is a natural monoterpene with a wide range of applications. Especially, its dimers have high energy value, thus it has great application prospects in the fuel field. Previous studies had shown that the activity of pinene synthase was the rate-limiting step for efficient synthesis of pinene in Saccharomyces cerevisiae. In this study, we hoped to obtain pinene synthase mutants with high catalytic efficiency in S. cerevisiae, which was proposed to provide a reference for efficient synthesis of pinene in yeast. Saturation mutants of the Q456 site of pinene synthase Pt30 were expressed in S. cerevisiae with enhanced mevalonate (MVA) pathway. And the ability of the recombinant strain to produce pinene was analyzed by two-phase shake flask fermentation and gas chromatographic detection. The results showed that among these mutants, Pt30 (Q456K) and Pt30 (Q456I) increased the pinene titer from non-detectable to 0.017 and 0.041 mg/L, respectively. In conclusion, the mutants Pt30 (Q456K) and Pt30 (Q456I) promoted pinene synthesis in S. cerevisiae. This indicated that Q456 was essential for Pt30 and provided a reference for rational modifications of other pinene synthases.
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