Abstract With special fragrance and various biological properties, linalool has wide applications in food, pharmaceuticals and cosmetics industries. Microbial biosynthesis has become a promising approach to linalool production, however its efficiency is limited by the limited precursor supply and poor efficiency of linalool synthase. With the goal of enhancing heterologous linalool production in Saccharomyces cerevisiae, protein engineering and metabolic engineering were simultaneously employed to solve these bottlenecks. Linalool-producing yeast was first constructed by introduction of t67OMcLIS selected out from a collection of linalool synthases. The efficiency of linalool biosynthesis was then improved by strengthening the supply of geranyl pyrophosphate (GPP) as precursor via overexpression of the complete mevalonate (MVA) pathway and a GPP synthase variant. To accelerate the conversion of the accumulated GPP to linalool, t67OMcLIS was engineered by means of directed evolution after development of a competition-based color-indicated high-throughput screening method. Expression of the t67OMcLIS variant in the engineered S. cerevisiae with enhanced precursor supply yielded 53.14 mg/L of linalool in biphasic shake-flask culture. This study delivered an efficient linalool-producing yeast cell factory and meanwhile provided efficient strategies for biosynthesis of other valuable monoterpenes.

中文翻译：

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通过将芳樟醇合酶的定向进化与完整甲羟戊酸途径的过度表达相结合，提高酿酒酵母中芳樟醇的产量

摘要 芳樟醇具有特殊的香味和多种生物学特性，在食品、医药和化妆品行业有着广泛的应用。微生物生物合成已成为生产芳樟醇的一种有前景的方法，但其效率受到有限的前体供应和芳樟醇合成酶效率低下的限制。为了提高酿酒酵母中异源芳樟醇的产量，同时采用蛋白质工程和代谢工程来解决这些瓶颈。生产芳樟醇的酵母首先是通过引入从一系列芳樟醇合酶中选出的 t67OMcLIS 构建的。然后，通过完全甲羟戊酸 (MVA) 途径和 GPP 合酶变体的过度表达，加强香叶基焦磷酸 (GPP) 作为前体的供应，从而提高芳樟醇生物合成的效率。为了加速累积的 GPP 向芳樟醇的转化，t67OMcLIS 在开发了一种基于竞争的颜色指示高通量筛选方法后，通过定向进化进行了工程设计。t67OMcLIS 变体在具有增强前体供应的工程酿酒酵母中的表达在双相摇瓶培养中产生了 53.14 mg/L 的芳樟醇。该研究提供了一种高效的生产芳樟醇的酵母细胞工厂，同时为其他有价值的单萜的生物合成提供了有效的策略。t67OMcLIS 是在开发基于竞争的颜色指示高通量筛选方法后通过定向进化设计的。t67OMcLIS 变体在具有增强前体供应的工程酿酒酵母中的表达在双相摇瓶培养中产生了 53.14 mg/L 的芳樟醇。该研究提供了一种高效的生产芳樟醇的酵母细胞工厂，同时为其他有价值的单萜的生物合成提供了有效的策略。t67OMcLIS 是在开发基于竞争的颜色指示高通量筛选方法后通过定向进化设计的。t67OMcLIS 变体在具有增强前体供应的工程酿酒酵母中的表达在双相摇瓶培养中产生了 53.14 mg/L 的芳樟醇。该研究提供了一种高效的生产芳樟醇的酵母细胞工厂，同时为其他有价值的单萜的生物合成提供了有效的策略。