Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction.
Metabolic engineering of oleaginous yeast Yarrowia lipolytica for limonene overproduction.
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DOI：
10.1186/s13068-016-0626-7
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发表时间：
2016
影响因子：
6.3
通讯作者：
Hua Q
中科院分区：
文献类型：
--
作者：
Cao X;Lv YB;Chen J;Imanaka T;Wei LJ;Hua Q
柠檬烯是一种单环单萜类化合物，是许多调味品、药物和生物柴油产品的重要前体。目前，d-柠檬烯的生产方法主要是从精油中分离得到，或作为橙子汁生产的副产品，但随着柠檬烯需求量的不断增加，以及柑橘产业中柠檬烯中可能含有一定量的农药，需要探索其他方法来生产柠檬烯。为了构建解脂耶氏酵母柠檬烯合成途径，对编码橙花基二磷酸合成酶1（NDPS 1）和柠檬烯合成酶（LS）的基因进行密码子优化，并在耶氏酵母中异源表达。解脂此外，为了使柠檬烯产量最大化，在MVA途径中涉及的几个基因以相同基因的不同拷贝或组合的形式过表达。在优化的柠檬酸和十二烷浓度条件下，工程菌Po 1f-LN-051的柠檬烯产量和含量分别达到23.56 mg/L和1.36 mg/g DCW，比初始产量0.006 mg/g DCW提高了约226倍。这是首次报道产油酵母Y.通过密码子优化的tLS和tNDPS 1基因的异源表达，在解脂菌中表达。据我们所知，柠檬烯产量为23.56 mg/L，是酵母中报道的最高柠檬烯产量水平。总之，我们证明了Y. Lipolytica为柠檬烯衍生物甚至其它单萜的过量生产提供了一个引人注目的平台。本文的在线版本（doi：10.1186/s13068-016-0626-7）包含补充材料，可供授权用户使用。
Limonene, a monocyclic monoterpene, is known for its using as an important precursor of many flavoring, pharmaceutical, and biodiesel products. Currently, d-limonene has been produced via fractionation from essential oils or as a byproduct of orange juice production, however, considering the increasing need for limonene and a certain amount of pesticides may exist in the limonene obtained from the citrus industry, some other methods should be explored to produce limonene. To construct the limonene synthetic pathway in Yarrowia lipolytica, two genes encoding neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS) were codon-optimized and heterologously expressed in Y. lipolytica. Furthermore, to maximize limonene production, several genes involved in the MVA pathway were overexpressed, either in different copies of the same gene or in combination. Finally with the optimized pyruvic acid and dodecane concentration in flask culture, a maximum limonene titer and content of 23.56 mg/L and 1.36 mg/g DCW were achieved in the final engineered strain Po1f-LN-051, showing approximately 226-fold increase compared with the initial yield 0.006 mg/g DCW. This is the first report on limonene biosynthesis in oleaginous yeast Y. lipolytica by heterologous expression of codon-optimized tLS and tNDPS1 genes. To our knowledge, the limonene production 23.56 mg/L, is the highest limonene production level reported in yeast. In short, we demonstrate that Y. lipolytica provides a compelling platform for the overproduction of limonene derivatives, and even other monoterpenes. The online version of this article (doi:10.1186/s13068-016-0626-7) contains supplementary material, which is available to authorized users.
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